Synthesis and biological evaluation of new podophyllic aldehyde derivatives with cytotoxic and apoptosis-inducing activities

Article abstract of DOI:10.1021/jm901373w

Several series of nonlactonic podophyllic aldehyde analogues were prepared and evaluated against several human tumor cell lines. They had different combinations of aldehyde, imine, amine, ester, and amide functions at C-9 and C-9′ of the cyclolignan skeleton. All the compounds synthesized showed cytotoxicity levels in the μM range and below. Within the new series tested, compounds having an aldehyde or imine at C-9 and an ester at C-9′ were the most potent, with GI₅₀ values in the nM range, some of them being several times more potent against HT-29 and A-549 carcinoma than against MB-231 melanoma cells. Cell cycle studies and analysis of the microtubule-disrupting capacity have demonstrated the existence of two different mechanisms of cell death induction for compounds with closely related structures.

Full text of DOI:10.1021/jm901373w

J. Med. Chem. 2010, 53, 983–993 983
DOI: 10.1021/jm901373w
Synthesis and Biological Evaluation of New Podophyllic Aldehyde Derivatives with Cytotoxic
and Apoptosis-Inducing Activities
a
,†
a
†
M Angeles Castro,* Jose M Miguel del Corral, Pablo A. Garcı
´
a,† Ma Victoria Rojo,† Janis de la Iglesia-Vicente,§
ꢀ
ꢀ
Faustino Mollinedo,§ Carmen Cuevas,‡ and Arturo San Feliciano†
†
´
Departamento de Quımica Farmaceutica, Facultad de Farmacia, Campus Miguel de Unamuno, Universidad de Salamanca, E-37007 Salamanca,
ꢀ
Spain, ‡PharmaMar S.A., Avenida de los Reyes, P.I. La Mina Norte, E-28770 Colmenar Viejo, Madrid, Spain, and Centro de Investigacioꢀn
§
´
ꢀ
del Caꢀncer, Instituto de Biologıa Molecular y Celular del Cancer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno,
E-37007 Salamanca, Spain
Received May 27, 2009
Several series of nonlactonic podophyllic aldehyde analogues were prepared and evaluated against
several human tumor cell lines. They had different combinations of aldehyde, imine, amine, ester, and
amide functions at C-9 and C-90 of the cyclolignan skeleton. All the compounds synthesized showed
cytotoxicity levels in the μM range and below. Within the new series tested, compounds having an
aldehyde or imine at C-9 and an ester at C-90 were the most potent, with GI50 values in the nM range,
some of them being several times more potent against HT-29 and A-549 carcinoma than against MB-231
melanoma cells. Cell cycle studies and analysis of the microtubule-disrupting capacity have demon-
strated the existence of two different mechanisms of cell death induction for compounds with closely
related structures.
Introduction
inhibition of tubulin polymerization, while being only very
weak inhibitors of Topo II in vitro.8 Additionally, it has been
reported that antitubulin lignans (podophyllotoxin,9 deoxy-
podophyllotoxin,10 LL-15,11,12 and other podophyllotoxin
analogues13), as well as related Topo II inhibitors (etopo-
side,14 GL33115), induce apoptotic cell death through inde-
pendent mechanisms, and such an effect would also enhance
their cytotoxicity.
Podophyllotoxin, 1, is a naturally occurring cyclolignan
which is the main component of Podophyllum resin, whose
pharmacological properties have been well-recognized for
centuries.1 A number of biological activities such as cytotoxi-
city, antiviral, cathartic, antirheumatic, insecticidal, etc.,
have been reported for podophyllotoxin and several related
compounds and derivatives.2-5 Among these, the antitumor
activity has been the most attractive, making this cyclolignan
a lead compound for anticancer drug design and development.
Several semisynthetic derivatives are in clinical use for the
treatment of a variety of malignancies6 including lung and testi-
cular carcinoma, lymphoma, nonlymphocytic leukemia, etc.
Such is the case for etoposide, teniposide, and etopophos, a
more soluble prodrug of etoposide (Figure 1). Most interest-
ingly, these semisynthetic derivatives and the parent compound
podophyllotoxin differ substantially in their mechanisms of
action. While podophyllotoxin inhibits the assembly of tubulin
into microtubules, preventing the formation of the achromatic
spindle and arresting cell division in metaphase, etoposide and
analogues inhibit DNA topoisomerase II, preventing religation
of the double-stranded breaks.1,2
Over the years, looking for more potent, less toxic, and
more selective analogues, our group has been engaged in the
design and synthesis of better podophyllotoxin related drugs
and has prepared a large number of cyclolignans through
chemical modification of most rings and positions of the
lignan skeleton.12,16,17 In the course of such studies, we
prepared a potent and selective cytotoxic cyclolignan, the
podophyllic aldehyde 2 (Figure 1) that lacked the trans-
γ-lactone ring, which was previously considered as one of
the most important features needed for the bioactivity of
podophyllotoxin analogues. Consequently, the podophyllic
aldehyde became our lead compound, to be improved by
means of further structure modification. The formation of
imines from 2 was particularly noteworthy because they not
only maintained the cytotoxicity level of the parent aldehyde
but also improved considerably the selectivity against adria-
mycin-resistant HT-29 colon carcinoma.12 Additional assays
demonstrated that both podophyllic aldehyde 2 and its imine
derivatives inhibited tubulin polymerization,11 and notably,
that these molecules were able to induce delayed cellular
apoptosis after 48 h of treatment.12 Those studies confirmed
that the γ-lactone ring is not an essential feature for cytotoxi-
city, whereas the presence of an electrophilic function at C-7/
C-9 seemed important for the antineoplastic potency observed
for this type of cyclolignan.
In a previous paper,7 we made a proposal regarding the
interaction between cyclolignansandbiomolecules thatwould
imply the existence of another, hitherto unknown, mechanism
for antineoplastic cyclolignans on the basis of ring fusion
strengthening and chemical reactivity of trans-lactonic podo-
lignans. This unknown mechanism would explain why
some lignan derivatives had been found to be as cytotoxic
as podophyllotoxin and etoposide, without appreciable
*To whom correspondence should be addressed. Phone: 34 923
294528. Fax: 34 923 294515. E-mail: macg@usal.es.
r
2010 American Chemical Society
Published on Web 01/12/2010
pubs.acs.org/jmc

984 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Castro et al.
Figure 1. Structures of podophyllotoxin and related compounds. (A) Cyclolignans in clinical use. (B) Selective cytotoxic aldehyde.
All the derivatives of 2 prepared previously kept the
β-axially oriented carbomethoxy group at C-90, so we have
planned to investigate the influence of the nature and size of
this molecular fragment on the cytotoxicity and selectivity.
Hence, here we report the preparation and evaluation of new
podophyllic aldehyde derivatives, having longer and structu-
rally varied ester chains at C-90, and also some bioisosteric
amide analogues. There are several bibliographic citations18
dealing with esters and amides at positions C-7 and C-40, and
even with substitution of the γ-lactone by a γ-lactam,19 that
report good cytotoxicity results. However, only a few deriva-
tives20 with such groupings at position C-90 have so far been
described and no report has been found on compounds
containing the aldehyde function at C-9 simultaneously.
Furthermore, although the imines previously prepared by
us12 were more potent than the parent aldehyde, it could be
assumed that they may hydrolyze during the cytotoxicity
assay, thus releasing the aldehyde and the corresponding
amine. Consequently, we have now transformed several
imines into the corresponding amines to analyze the influence
of such stabilization on the activity and selectivity.
potassium carbonate or sodium hydride as bases in the
minimum volume of dry DMF. This aspect was critical
because dilution leads to a relactonization with the forma-
tion of the 80-epimeric lactone, picropodophyllotoxin.
Another series of derivatives involves those compounds
having an amide function at C-90. It is known that the
conversion of carboxylic esters to amides is often a useful
procedure to obtain amides;23 in our case, aldehyde 2,
bearing a methyl ester, could be considered a good starting
substrate to obtain amides at C-90, however, simple esters
like methyl or ethyl esters are not very reactive and require
strongly basic catalysis.23 Looking for an alternative proce-
dure to get the amides, and because we knew, from our
previous studies, that the γ-lactone of 1 is very reactive
toward nucleophiles,7,24 we decided to explore the direct
aminolysis25 of 1 with primary and secondary amines. Thus,
when pyrrolidine and ethylamine were used, as reactants and
solvents, under reflux, the dihydroxyamides 17 and 18 were
obtained, respectively, in good yields. Further Swern oxida-
tion of 17 and 18 afforded the aldehyde-amides 19 and 20,
respectively (Scheme 2).
When aromatic amines, such as aniline, p-anisidine, or
p-toluidine, were used as reagents, the reaction failed and
unchanged 1 or mixtures of 1 and picropodophyllotoxin
were recovered, even if the reaction was attempted under
microwave irradiation or in the presence of potassium tert-
butoxide. In view of the difficulties encountered in getting
N-aryl amides from the lactone, and despite its low reacti-
vity, we reconsidered the possibility of obtaining them by
transformation of the carbomethoxy group present in alde-
hyde 2.23 To avoid the direct reaction between the aldehyde
function of 2 and aniline, the aldehyde group was first
protected as its dithioacetal by treatment with 1,2-ethane-
dithiol. The dithiolane-ester was then tested as reaction
substrate, under reflux or microwave irradiation, without
catalyst or in presence of potassium tert-butoxide or tri-
methylaluminum, but the reaction did not progress appreci-
ably, giving only recovered starting material. After such
disappointing results, we opted for the saponification of
the methyl ester with potassium hydroxide in methanol to
get the free carboxylic acid, which was then successfully
treated with p-anisidine in the presence of DCC and
OHBTZ.26 Final removal of the aldehyde protection with
SeO2 yielded the desired aldehyde-amide 21.
In addition to the results of cytotoxicity evaluation, we also
present here those of cell-cycle arrest studies, which have been
performed with the aim of confirming our previous mecha-
nistic findings for podophyllic aldehyde 2. These results now
give experimental biological support to our previous proposal
that there is a third cytotoxicity mechanism for podophyllo-
toxin related lignans.
Results and Discussion
Chemistry. The natural cyclolignan podophyllotoxin 1,
isolated from Podophyllum resin,21 was the starting material
for all the products presented here. The numbering of the
compounds used in this work (Figure 1) is in accordance with
IUPAC rules for lignans.22
The aldehyde 2 and the imines 22-24 were obtained as
previously described by us.12 Aldehydes 10-16 were pre-
pared by Swern oxidation of the corresponding dihydroxy-
esters 3-9. The latter were prepared, in moderate to good
yields, through esterification of picropodophyllic acid ob-
tained by opening of the lactone ring under basic conditions,
followed by treatment with the corresponding alkyl bromide
(Scheme 1). The ester formation was carried out using

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 985
a
Scheme 1. Preparation of Cyclolignans with Ester Functions at C-90
a (i) (1) KOH/MeOH, (2) HCl/H2O, pH=4, (3) base/DMF, R-Br; (ii) Swern; (iii) R1-NH2, (iv) NaBH4,MeOH/0 °C.
a
Scheme 2. Preparation of Cyclolignans with Amide Functions at C-90
a (i) R1-NH-R2; (ii) Swern oxidation; (iii) (1) KOH/MeOH, Hþ/H2O, pH=4 (2) CH2N2/Et2O; (iv) (1) (CH2SH)2, TMSCl/CH2Cl2, (2) KOH/MeOH,
(3) HCl/H2O; (v) (1) p-anisidine/THF, DCC/HOBTZ, (2) SeO2/AcOH; (vi) (1) R3-NH2, (2) NaBH4, MeOH/0 °C.
The aldehydes 2, 10, 15, and 19 were transformed into the
corresponding imines by condensation with aliphatic and
aromatic primary amines in excess, applying either a classical
method, in solution and with a drying agent, or a solvent-free
method, under microwave irradiation using montmorillonite
K-10 clay as the solid support. In the second case, reaction
times were considerably reduced, but the extraction from the
solid support was not too efficient and a small amount of the
NMR spectra of the extracted crude reaction products. The
condensation could not be monitored by TLC because
hydrolysis occurred during elution, so progress was followed
through the evolution of the aldehyde proton signal in the 1H
NMR spectra until its complete disappearance. Such sensi-
tivity to hydrolysis also made the purification of the imines
difficult and crude reaction products were used in the next
reaction step, without further purification. However, when
the amines used as reagents and solvent were volatile enough
1
parent aldehyde was always present, as deduced from H

986 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Castro et al.
Table 1. Antineoplastic Cytotoxicity of Podophyllic Aldehyde Deriva-
tives (GI50 μM)
notable selectivity, being exceptionally noncytotoxic against
A-549 cells at the maximum concentration tested (10 μg/mL,
17 μM) while displaying GI50 values in the same range as
podophyllotoxin and podophyllic aldehyde against the other
lines tested.
MB-231
A-549
HT-29
a
1
-
0.012
0.061
0.07
0.12
0.19
0.20
0.17
0.024
0.038
0.09
0.08
0.16
0.15
0.18
0.022
0.20
0.02
1.0
2
0.20
0.09
0.11
0.18
0.18
0.16
0.034
0.54
0.15
2.5
In general, the aldehyde-ester derivatives 10-16 main-
tained the cytotoxicity of aldehyde 2, showing GI50 values
below the μM range and being a few times more potent
against HT-29 and A-549 than against MB-231 cells. Com-
pared to dihydroxyesters 3-9, those aldehydes with an
alkyl-chain ester were less potent (10, 11, 12, 16 vs 3, 4, 5,
9, respectively), whereas those having an aromatic ring or an
additional ester function in the chain, turned out to be more
potent than their hydroxylic precursors (13, 14 vs 6, 7).
As happened with previous imines reported by us,12 the
derivatives 22-26 were the most potent analogues, with GI50
values against HT-29 and A-549 cells in the nM range, while
the GI50 values found on MB-231 were several times higher.
Thus, the imino-esters 23 and 26 were around 30 times more
potent against HT-29 and A-549 than against MB-231, with
a significant improvement in the potency of the precursor
aldehydes.
Regarding the C-90 side-chain ester function, it can be
noted that, in a similar way as for the above-mentioned
dihydroxyesters and aldehyde-esters, and particularly for
A-549 and HT-29 cell lines, those imines containing a
benzene ring or an additional ester function were also more
potent than those having an aliphatic alkyl, alkenyl, or
haloalkyl fragment in the ester chain (13, 14, 15 vs 10-12,
16). This comparison can be extended to the imine 25 (ethyl
ester), which turned out to be 1 order of magnitude less
potent than the imine 26 (trimethoxybenzyl ester) against
MB-231 cells and around two orders less potent against
A-549 and HT-29 cells. However, it is interesting to note
that the opposite occurs in the case of amino-esters when
compounds 31 (ethyl ester) and 32 (trimethoxybenzyl ester)
are compared, the latter being around one order less cyto-
toxic against the three cell lines tested.
3
4
5
6
7
9
>17
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
0.18
0.03
1.0
0.13
0.11
0.042
0.17
7.8
0.032
0.013
0.034
0.13
6.8
0.036
0.028
0.020
0.14
7.8
10
9.8
6.6
10
8.2
7.1
7.3
8.0
4.5
1.2
4.6
0.80
0.0022
0.041
0.0094
0.39
0.0043
0.4
-
1.3
0.022
0.041
0.094
0.23
0.0052
0.7
-
1.2
0.13
2.6
5.5
2.4
1.5
1.0
1.0
0.7
1.5
0.9
0.8
0.37
6.4
0.23
1.9
0.27
2.0
>20
>17
>20
15
>20
15
a - = cytotoxicity not tested.
to be removed under vacuum, the resulting crude imines were
technically pure by NMR and suitable for full characterization
and evaluation. This was the case for compounds 25 and 26.
The imines 22-24 were previously characterized by us.12
The reduction of the crude products of imine formation
was done with sodium borohydride in methanol at 0 °C.
Column chromatography of the reduction products afforded
the amines 27-34 in acceptable yields from the correspond-
ing aldehydes (Schemes 1 and 2). In the case of amino-esters
The reduction of the imines to the corresponding amines
27-32 led to a fair decrease of cytotoxicity though retained a
certain degree of selectivity toward HT-29 cells. Finally, the
substitution of the ester moiety by an amide group led to the
less potent compounds, mostly independent of the type of
the amide N-substitution and the function (hydroxyl, alde-
hyde, or amine) located at C-9. Only the anisyl amide 21
showed a GI50 value in the μM range.
27-32, it is necessary to keep in mind that migration of the
0
Δ7 double bond to the Δ8(8 ) position and/or γ-lactam ring
closure could take place27 if temperatures over 30 °C are
attained during reaction workup and solvent removal.
Biological Results. Cytotoxicity. Most cyclolignans
reported here were evaluated in vitro to determine their
cytotoxicities28 against the human tumor cell lines: MB-231
(breast carcinoma), A-549 (lung carcinoma), and HT-29
(colon carcinoma). The results found are shown in Table 1,
and several considerations related to the influence of the
functionalities at positions C-9 and C-90 can be analyzed.
All the compounds tested, considered as analogues of the
podophyllic aldehyde 2, were cytotoxic and some of them
resulted several times more potent against HT-29 and A-459
carcinoma than against MB-231 melanoma cell lines.
The dihydroxyesters 3-9 showed GI50 values in the μM
range or below, with no significant differences between them,
and compared to 2, no significant differences between the
three lines tested were observed. Only compound 9 showed a
Cell Cycle Effects. To gain further insight into the mode of
action of these compounds, we examined the effects of
compounds 11-16, 20, and 21 on cell cycle by flow cytome-
try in HT-29 cells. The results are shown in Table 2 and
Figure 2. The concentrations of the above compounds
required to elicit these effects on cell cycle are consistent
with the corresponding GI50 values for cell proliferation
assays because the concentration of cells used for flow
cytometry analysis exceeded by about 10-20 times that used
in cell proliferation tests. The G2/M arrest was observed for
24 and 48 h, and then this cell cycle arrest led eventually to
cell death, with over 30% apoptosis after 72 h treatment with
1 μM of the above-mentioned compounds (Table 2).
As could be expected due to the close structural similarity
with podophyllotoxin 1 and podophyllic aldehyde 2, which
have been reported to disrupt the microtubule network and
to compete with colchicine in its binding to tubulin,11,12
compounds 11, 13, 14, and 15 induced a nearly total arrest

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 987
Table 2. Effect of Compounds 11-16, 20, and 21 on Cell Cycle and Apoptosis in HT-29 Cellsa
% cells
24 h treatment
48 h treatment
72 h treatment
compd
conc, μM
sub-G1
G0/G1
S
G2/M
sub-G1
G0/G1
S
G2/M
sub-G1
G0/G1
S
G2/M
control
11
12
0.5
0.3
65.0
3.9
8.7
4.5
25.8
91.3
23.2
96.4
97.6
97.5
14.2
13.5
25.2
1.0
1.8
63.8
10.9
25.3
5.3
8.0
3.9
29.6
4.2
3.4
5.1
8.4
3.0
1.8
27.2
83.4
15.9
85.3
85.6
79.5
18.1
2.3
1.8
41.6
82.3
33.7
30.6
32.3
67.7
93.8
52.6
64.3
33.6
16.7
21.1
16.4
18.3
31.6
5.3
9.1
16.8
0.4
24.8
8.0
1
1
1
1
1
1
1
1
19.6
0.4
14.6
1.4
42.6
1.8
29.2
5.2
0.6
13
14
22.0
19.2
12.9
0.3
23.2
33.8
36.5
0.4
0.5
0.5
1.0
0.9
0.9
1.1
6.3
8.6
4.7
6.8
15
16
29.2
25.0
13.6
46.9
51.7
48.0
9.7
9.8
34.6
61.7
45.2
38.9
33.0
50.4
20
21
0.5
8.4
0.4
2.6
13.2
2.6
36.4
a HT-29 cells were incubated with the above compounds at 1 μM for the indicated times, and the proportion of cells in each phase of the cell cycle was
quantitated by flow cytometry. Cells in the sub-G1 region represent apoptotic cells. Untreated control cells were run in parallel. Data shown are
representative of three independent experiments performed.
Figure 3. - Effects of compounds 11, 14, 15, 16, and 20 on the
microtubule network of HT-29 cells. Cells were incubated in the
absence (control) or in the presence of 1 μM of compounds 11, 14,
Figure 2. Effects of compounds 11-16, 20, and 21 on cell cycle in
15, 16, and 20 for 24 h and then fixed and processed for immuno-
HT-29 cells. Cells were incubated with 1 μM of the indicated
fluorescence of microtubules (red fluorescence) and nuclei (blue
compounds for 24 h and stained with propidium iodide (PI). Their
fluorescence) as described in Experimental Section. Bar, 10 μm. The
DNA content was analyzed by fluorescence flow cytometry. The
photomicrographs shown are representative of at least three inde-
positions of the G0/G1 and G2/M peaks are indicated by arrows.
pendent experiments performed.
The experiment shown is representative of three performed.
of cells (higher than 90%) at the G2/M phase after 24 h of
treatment. This was also in agreement with their high
cytotoxicity and the very low GI50 values, ranging between
20 and 36 nM, observed for these compounds in the cell
proliferation assays (Table 1). The G2/M arrest was similarly
observed for HT-29 cells treated with these compounds at
0.2 μM (data not shown).
Most interestingly, the aldehyde-esters 12 and 16 and the
aldehyde-amides 20 and 21 were shown to directly induce
apoptosis, when used at 1 μM for 24-72 h, without any
previous G2/M arrest (Table 2 and Figure 2), suggesting a
different mode of action for these compounds.
By analyzing the effect of the above compounds on the
microtubule network, we found that compounds 11, 13, 14,
and 15 disrupted microtubules (Figure 3, and data not
shown) after 24 h incubation at 1 μM, whereas compounds
12, 16, 20, and 21 had a much lower effect, in some cases
negligible, if any, on microtubules (Figure 3, and data not
shown). Thus, these results on microtubule network square
with those of cell cycle. Compounds 11, 13, 14, and 15
inhibited microtubule polymerization and promoted cell
cycle arrest in G2/M phase, whereas compounds 12, 16, 20,
and 21 seemed to act through a distinct mechanism.
On these grounds, the strong G2/M arrest following
treatment with 11, 13, 14, and 15 appears to be mediated
by microtubule disruption. Interference with the micro-
tubule assembly/disassembly processes leads to apoptosis
through mechanisms that are not completely understood.29
Because those lignans included in this research would have
not any potential anti-Topo II activity, the mechanistic
difference observed between compounds 11, 13, 14, and 15,
which lead to total G2/M arrest before inducing apoptosis,
and their closely related partners 12, 16, 20, and 21, which
promote cell death without any significant cell cycle arrest,
serves as an experimental proof of the existence of a pre-
viously unknown mechanism of action for podophyllotoxin
lignans. Nevertheless, to get further insight into this mecha-
nism, which results in cell death by eventually affecting
microtubule structure or dynamics, an extended chemical
and biological research program must be planned and
executed.
In summary, new nonlactonic cyclolignans derived from
podophyllic aldehyde 2 and having different structural
arrangements at C-9 and C-90 were prepared and evaluated
against several human tumor cell lines. All the compounds
showed GI50 cytotoxicity levels in the μM-nM range.

988 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Castro et al.
In general, those compounds containing an ester function at
C-90 were more potent than the corresponding amides and
the combination of an aldehyde or an imine function at C-9
with an ester at C-90 was found to be the best for potency and
selectivity against HT-29 colon carcinoma. On the other
hand, it was observed that functions conferring chemical
stability have a negative influence on cytotoxicity and that
the presence of a highly electrophilic function at C-9
(aldehyde or imine) is the most important feature for cyto-
toxicity and potency. These findings also support our pre-
vious proposal about the importance of this position, which
seems to constitute a site for interaction with the biological
targets.7,24 Cell cycle studies have revealed the existence of
two different modes of action for these compounds, appar-
ently involving tubulin polymerization, microtubule assem-
bly, or dynamics. The mechanistic difference displayed by
compounds with such similar structures is evidence for a
third mechanism of action for cyclolignans and will provide
the basis for further biological and chemical studies focused
on investigating this subject.
C90-OCH2CH3), 4.25 (d, 1H, J = 8.2 Hz, H70), 4.85 (m, 1H, H7),
5.88 (s, 2H, H10), 6.34 (s, 3H, H3, H20, H60), 6.83 (s, 1H, H6).
13C NMR (CDCl3): δ 14.1 (C90-OCH2CH3), 43.7 (C70), 45.6
(C8), 47.1 (C80), 56.2 (C100, C120), 60.9 (C110, C90-OCH2CH3),
62.6 (C9), 69.8 (C7), 101.1 (C10), 106.4 (C20, C60), 108.4 (C6),
109.4 (C3), 130.1 (C1), 130.9 (C2), 136.7 (C40), 140.0 (C10), 146.8
(C4), 147.7 (C5), 153.1 (C30, C50), 174.4 (C90). IR (film) cm-1
:
3403 (OH), 1725 (COOR), 1125 and 1035 (OH). HRMS: calcd
for C24H28O9Na, 483.1622; found, 483.1611. [R]20 -81.6°
(c 0.9%).
D
Allyl Picropodophyllate (4). From picropodophyllic acid
(80 mg, 0.190 mmol), K2CO3 (105 mg, 0.760 mmol), and allyl
bromide (1 mL, 11 mmol) in dry DMF (2 mL) using method A
during 50 min. The reaction product was chromatographed on
silica gel, eluting with 30% CH2Cl2/EtOAc to give compound 4
1
(17 mg, 38%). H NMR (CDCl3): δ 4.51 (d, 2H, J = 5.5 Hz,
C90-OCH2CHdCH2), 5.16 (d, 1H, J = 10.6 Hz, C90-OCH2-
CHdCH2), 5.14 (d, 1H, J = 16.1 Hz, C90-OCH2CHdCH2),
5.80 (m, 1H, C90-OCH2CHdCH2). 13C NMR (CDCl3): δ 65.5
(C90-OCH2CHdCH2), 118.4 (C90-OCH2CHdCH2), 131.8
(C90-OCH2CHdCH2), 173.9 (C90). IR (film) cm-1: 3442,
1126, 1038 (OH), 1731 (COOR). HRMS: calcd for C25H28-
O9Na, 495.1625; found, 495.1632. [R]20D -77.6° (c 0.8%).
Heptyl Picropodophyllate (5). From picropodophyllic acid
(140 mg, 0.324 mmol), K2CO3 (179 mg, 1.30 mmol), and
1-heptyl bromide (1.0 mL, 6.3 mmol) in dry DMF (1.5 mL)
using method A during 45 min to obtain diol-ester 5 (95 mg,
Experimental Section
Chemistry. NMR spectra were recorded on a Bruker AC 200
at 200 MHz for 1H and 50.3 MHz for 13C in deuterochloroform
with TMS as internal standard. Chemical shift values are
expressed in ppm followed by multiplicity and coupling con-
stants (J) in Hz. The complete NMR signals are given for the
first compound of each series described here, and only char-
acteristic signals are indicated for the remainder. Optical rota-
tions 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 ob-
tained on a Nicolet Impact 410 spectrophotometer, and wave
numbers are given in cm-1. HRMS were run in a VG-TS-250
spectrometer working at 70 eV. Elemental analyses (C, H, N)
were obtained with a LECO CHNS-932 and were within (0.4%
of the theoretical values. Solvents and reagents were purified by
standard procedures as necessary, and the chlorinated solvents,
including deuterochloroform, were filtered through sodium
bicarbonate prior its use in order to eliminate acid traces.
General Methods for the Synthesis of Diols 3-9, 17, and 18.
Method A. A mixture of picropodophyllic acid (obtained from
podophyllotoxin12) and K2CO3 was dissolved in dry DMF and
stirred at room temperature for 30 min. Then the alkylating agent
was added and the mixture stirred at room temperature from
45 min to 1 h. The crude product was diluted with EtOAc
and filtered, and the solutions were washed with satd aq
NaCl, dried over Na2SO4 ,and filtered. The solvent was removed
under vacuum to give the corresponding picropodophyllate deri-
vatives.
Method B. A solution of NaH in dry DMF was cooled to 0 °C
and kept under an argon atmosphere. Picropodophyllic acid
dissolved in DMF was added and the mixture stirred during
15 min. Then the alkylating agent was added and the mixture left
to attain the room temperature and kept stirring for 45 min
more. The reaction was quenched with MeOH, which was then
removed under vacuum and the crude diluted with EtOAc. The
organic phase was washed with satd aq NaCl, dried over
Na2SO4, filtered, and the solvent removed to get the correspond-
ing picropodophyllate derivatives.
Ethyl Picropodophyllate (3). From picropodophyllic acid
(200 mg, 0.460 mmol), K2CO3 (192 mg, 1.39 mmol), and ethyl
bromide (1 mL, 13 mmol) in dry DMF (1.5 mL) using method A
during 1 h to yield compound 3 (185 mg, 85%). 1H NMR
(CDCl3): δ 1.13 (t, 3H, J = 7.1 Hz, C90-OCH2CH3), 2.45 (m, 1H,
H8), 3.33 (dd, 1H, J = 8.2, 3.6 Hz, H80), 3.65 (m, 2H, H9), 3.76 (s,
6H, H100, H120), 3.80 (s, 3H, H110), 4.05 (c, 2H, J = 7.1 Hz,
1
55%). H NMR (CDCl3): δ 0.85 (t, 3H, J = 6.9 Hz, C90-
O-(CH2)6-CH3), 1.15 (m, 8H, C90-O-CH2CH2-(CH2)4-CH3),
1.49 (m, 2H, C90-O-CH2CH2-(CH2)4-CH3), 3.99 (t, 2H, J =
6.6, C90-O-CH2CH2-(CH2)4-CH3). 13C NMR (CDCl3): δ 14.1
(C90-O-(CH2)6-CH3), 22.6, 25.7, 28.5, 28.8 (C90-O-CH2-
CH2-(CH2)4-CH3), 31.7 (C90-O-CH2CH2-(CH2)4-CH3), 65.1
(C90-O-CH2CH2-(CH2)4-CH3), 174.4 (C90). IR (film) cm-1
:
3439, 1127, 1039 (OH), 1728 (COOR). HRMS: calcd for
C29H38O9Na, 553.2908; found, 553.2389.
Cinnamyl Picropodophyllate (6). From picropodophyllic acid
(150 mg, 0.350 mmol), NaH (16 mg, 0.52 mmol), and cinnamyl
bromide (137 mg, 0.690 mmol) in dry DMF (5 mL) using
method B to yield diol-ester 6 (170 mg, 89%). 1H NMR
(CDCl3): δ 4.68 (d, 2H, J = 6.2 Hz, C90-OCH2CHdCH-
C6H5), 6.16 (dt, 1H, J = 16.5, 6.2 Hz, C90-OCH2CHdCH-
C6H5), 6.54 (d, 1H, J = 16.5 Hz, C90-OCH2CHdCH-C6H5),
7.30 (m, 5H, C90-OCH2CHdCH-C6H5). 13C NMR (CDCl3): δ
65.5 (C90-OCH2CHdCH-C6H5), 122.7 (C90-OCH2CHdCH-
C6H5), 126.0 (C), 126.6 (2CH), 128.2 (CH), 128.7 (2CH, C90-
OCH2CHdCH-C6H5), 134.4 (C90-OCH2CH=CH-C6H5),
173.9 (C90). IR (film) cm-1: 3400, 1125, 1036 (OH), 1727
(COOR). HRMS: calcd for C31H32O9Na, 571.1938; found,
571.1973. [R]20 -22.5° (c 0.8%). UV (EtOH) λmax 205 (lg ε
D
4.1), 251 (lg ε 4.2), 359 (lg ε 4.3).
Ethoxycarbonylmethyl Picropodophyllate (7). From picropo-
dophyllic acid (80 mg, 0.18 mmol), NaH (8.3 mg, 0.28 mmol),
and ethyl bromoacetate (41.0 mL, 0.370 mmol) in dry DMF
(2.5 mL) using method B to obtain compound 7 (53 mg, 55%).
1H NMR (CDCl3): δ 1.22 (t, 3H, J = 7.1 Hz, C90-OCH2-
COOCH2CH3), 4.16 (c, 2H, J = 7.1 Hz, C90-OCH2-
COOCH2CH3), 4.37 and 4.75 (AB system, J = 16.0 Hz,
13
C90-OCH2-COOCH2CH3). C NMR (CDCl3): δ 14.1 (C90-
OCH2-COOCH2CH3), 61.8 (C90-OCH2-COOCH2CH3), 62.2
(C90-OCH2-COOCH2CH3), 168.3 (C90-OCH2-COOCH2CH3),
173.2 (C90). IR (film) cm-1: 3430, 1125, 1036 (OH), 1741
(COOR). HRMS: calcd for C26H30O11Na, 541.1680; found,
541.1661.
3,4,5-Trimethoxybenzyl Picropodophyllate (8). From picro-
podophyllic acid (160 mg, 0.370 mmol), K2CO3 (77 mg, 0.55
mmol), and 3,4,5-trimethoxybenzyl bromide (145 mg, 0.55
mmol) in dry DMF (2.5 mL) using method A during 1 h to
1
yield compound 8 (158 mg, 70%). H NMR (CDCl3): δ 3.80
(s, 9H, C90-OCH2-C6H2(OCH3)3), 4.91 and 5.01 (AB system,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 989
J = 10 Hz, C90-OCH2-C6H2(OCH3)3), 6.33 (s, 2H, C90-OCH2-
C6H2(OCH3)3). IR (film) cm-1: 3490, 1125, 1036 (OH), 1728
(COOR).
J = 11.7 Hz, C90-OCH2CHdCH2), 5.16 (d, 1H, J = 16.1 Hz,
C90-OCH2CHdCH2), 5.80 (m, 1H, C90-OCH2CHdCH2), 7.34
(s, 1H, H7), 9.60 (s, 1H, H9). 13C NMR (CDCl3): δ 65.8 (C90-
OCH2CHdCH2), 117.9 (C90-OCH2CHdCH2), 131.7 (C90-
OCH2CHdCH2), 133.1 (C8), 145.7 (C7), 171.4 (C90), 191.3
(C9). IR (film) cm-1: 1732 (COOR), 1670 (CHO). Anal. (C25-
H24O8) C, H, N. HRMS: calcd for C25H24O8Na, 475.1363;
found, 475.1381.
Heptyl 9-Deoxy-9-oxo-r-apopicropodophyllate (12). From
heptyl picropodophyllate 5 (95 mg, 0.18 mmol). The Swern
reaction product was chromatographed on silica gel, eluting
with 5% EtOAc/CH2Cl2 to give compound 12 (41 mg, 44%). 1H
NMR (CDCl3): δ 0.86 (t, 3H, J = 6.6 Hz, C90-O-(CH2)6-CH3),
1.2 (m, 8H, C90-O-CH2CH2-(CH2)4-CH3), 1.5 (m, 2H, C90-O-
CH2CH2-(CH2)4-CH3), 4.01 (t, 2H, J = 6.6 Hz, C90-O-CH2-
CH2-(CH2)4-CH3), 7.33 (s, 1H, H7), 9.60 (s, 1H, H9). 13C NMR
(CDCl3): δ 14.1 (C90-O-(CH2)6-CH3), 22.6, 25.7, 28.4, 28.8 (C90-
O-CH2CH2-(CH2)4-CH3), 31.7 (C90-O-CH2CH2-(CH2)4-CH3),
65.5 (C90-O-CH2CH2-(CH2)4-CH3), 133.4 (C8), 145.4 (C7),
191.3 (C9). IR (film) cm-1: 1728 (COOR), 1672 (CHO). Anal.
(C29H34O8) C, H, N. HRMS: calcd for C29H34O8Na, 533.2146;
found 533.2162. [R]20D -135° (c 1.1%).
3-Bromopropyl Picropodophyllate (9). From picropodophyl-
lic acid (250 mg, 0.58 mmol), K2CO3 (240 mg, 1.74 mmol), and
1,3-dibromopropane (0.177 mL, 1.74 mmol) in dry DMF (1.8 mL)
using method A during 1 h to obtain compound 9 (263 mg, 82%).
1H NMR (CDCl3): δ 2.03 (q, 2H, J = 6.2 Hz, C90-CH2CH2-
CH2Br), 3.20 (t, 2H, J = 6.2 Hz, C90-CH2CH2CH2Br), 4.12
(m, 2H, C90-OCH2CH2CH2Br). 13C NMR (CDCl3): δ 29.1 (C90-
OCH2CH2CH2Br), 31.4 (C90-OCH2CH2CH2Br), 62.5 (C90-
OCH2CH2CH2Br), 174.3 (C90). IR (film) cm-1: 3440, 1125, 1036
(OH), 1728 (COOR). HRMS: calcd for C25H29O9NaBr, 575.0887;
found, 575.0950. [R]20D -64.9° (c 0.9%).
Picropodophyllic Acid Pyrrolidinyl Amide (17). Pyrrolidine
(0.8 mL, 9.6 mmol) was added to a solution of 1 (200 mg, 0.48
mmol) in dry benzene (20 mL), and the mixture was stirred at
50 °C under argon atmosphere during 46 h. Solvent and residual
amine were evaporated in vacuum to give the corresponding
dihydroxyamide 17 in quantitative yield. 1H NMR (CDCl3): δ
1.50-1.90 (m, 4H, C90-N-(CH2)4-), 3.20-3.50 (m, 4H, C90-
N-(CH2)4-). 13C NMR (CDCl3): δ 24.2, 25.9, 46.0, 46.8 (C90-
N-(CH2)4-), 173.9 (C90). IR (film) cm-1: 3380, 1126, 1038
(OH), 1611 (CON-(CH2)4-). HRMS: calcd for C26H31NO8 þ
H, 486.2122; found, 486.2127.
Cinnamyl 9-Deoxy-9-oxo-r-apopicropodophyllate (13). From
cinnamyl picropodophyllate 6 (150 mg, 0.274 mmol). The Swern
reaction product was chromatographed on silica gel, eluting
with 5% EtOAc/CH2Cl2 to give aldehyde 13 (50 mg, 53%). 1H
NMR (CDCl3): δ 4.68 (d, 2H, J = 5.8 Hz, C90-OCH2CHdCH-
C6H5), 6.15 (dt, 1H, J = 16.0, 5.8 Hz, C90-OCH2CHdCH-
C6H5), 6.49 (d, 1H, J = 16.0 Hz, C90-OCH2CHdCH-C6H5),
7.25 (m, 5H, C90-OCH2CHdCH-C6H5), 7.36 (s, 1H, H7), 9.62
N-Ethyl Picropodophyllamide (18). A mixture of 1 (100 mg,
0.241 mmol) and ethylamine (70% in water, 10 mL, 18 mmol)
was stirred and heated under reflux during 1.5 h. The excess of
amine was removed under vacuum to give 18 in quantitative
1
yield. H NMR (CDCl3): δ 0.85 (t, 3H, J = 7.2 Hz, C90-
NHCH2CH3), 2.98 (m, 1H, C90-NHCH2CH3), 3.12 (m, 1H,
C90-NHCH2CH3), 5.75 (m, 1H, NH). 13C NMR (CDCl3): δ 14.3
(C90-NHCH2CH3), 34.4 (C90-NHCH2CH3), 174.9 (C90). IR
(film) cm-1: 3360, 1126, 1038 (NH, OH), 1645 (CONH).
HRMS: calcd for C24H29NO8þH, 460.1966; found, 460.1976.
[R]20D -46.5° (c 0.9%).
13
(s, 1H, H9). C NMR (CDCl3): δ 65.8 (C90-OCH2CHdCH-
C6H5), 122.8 (C90-OCH2CHdCH-C6H5), 125.5, 126.6 (2CH),
128.1, 128.6 (2CH, C90-OCH2CHdCH-C6H5), 133.2 (C8),
133.9 (C90-OCH2CHdCH-C6H5), 145.6 (C7), 191.1 (C9). IR
(film) cm-1: 1730 (COOR), 1669 (CHO). Anal. (C31H28O8) C,
H, N. HRMS: calcd for C31H28O8Na, 551.1676; found
551.1682. [R]20D -131° (c 0.9%).
Swern Oxidation for the Synthesis of r,β-Unsaturated Alde-
hydes. To a precooled (-55 °C) and stirred solution of oxalyl
chloride (3 equiv) in dry CH2C12 (5 mL) was added dropwise a
solution of DMSO (6 equiv) in dry CH2C12 (2 mL). After 5 min
at -55 °C, a solution of the corresponding diol (1 equiv) in dry
CH2C12 was slowly added. The mixture was stirred at the same
temperature for 30 min, and then triethylamine (10 equiv) was
added dropwise. The mixture was warmed to 0 °C over 1 h,
quenched with water, and extracted with CH2C1 or EtOAc. The
organic phase was washed with 2 N HCl and satd aq solutions of
NaHCO3 and NaCl and the solvent was evaporated off. Column
chromatography of the reaction products with mixtures of
EtOAc/CH2Cl2 gave the corresponding aldehydes.
Ethoxycarbonylmethyl 9-Deoxy-9-oxo-r-apopicropodophyl-
late (14). From ethoxycarbonylmethyl picropodophyllate 7
(50 mg, 0.10 mmol). The Swern reaction product was chroma-
tographed on silica gel, eluting with 10% EtOAc/CH2Cl2 to give
compound 14 (20 mg, 46%). 1H NMR (CDCl3): δ 1.20 (t, 3H,
J = 7.3 Hz, C90-OCH2-COOCH2CH3), 4.15 (c, 2H, J = 7.3 Hz,
C90-OCH2-COOCH2CH3), 4.53 and 4.59 (AB system, J = 15.9 Hz,
C90-OCH2-COOCH2CH3), 7.39 (s, 1H, H7), 9.61 (s, 1H,
13
H9). C NMR (CDCl3): δ 14.1 (C90-OCH2-COOCH2CH3),
61.3 (C90-O-CH2-COOCH2CH3), 61.3 (C90-OCH2-COOCH2-
CH3), 132.4 (C8), 145.9 (C7), 167.3 (C90-OCH2-COOCH2CH3),
170.9 (C90), 191.1 (C9). IR (film) cm-1: 2720 (CHO), 1743
(COOR), 1670 (CHO). Anal. (C26H26O10) C, H, N. HRMS:
calcd for C26H26O10Na, 521.1418; found 521.1410. [R]20D -110°
(c 0.7%).
Ethyl 9-Deoxy-9-oxo-r-apopicropodophyllate (10). From ethyl
picropodophyllate 3 (90 mg, 0.20 mmol). The Swern reaction
product was chromatographed on silica gel, eluting with 2%
1
EtOAc/CH2Cl2 to give compound 10 (55 mg, 64%). H NMR
3,4,5-Trimethoxybenzyl 9-Deoxy-9-oxo-r-apopicropodophyl-
late (15). From 3,4,5-trimethoxy-benzyl picropodophyllate 8
(220 mg, 0.36 mmol). The reaction product was chromato-
graphed on silica gel, eluting with 20% EtOAc/CH2Cl2 to give
compound 15 (68 mg, 78%). 1H NMR (CDCl3): δ 3.82 (s,
9H, C90-OCH2-C6H2(OCH3)3), 4.99 (s, 2H, C90-OCH2-C6H2-
(OCH3)3), 6.18 (s, 2H, C90-OCH2-C6H2(OCH3)3), 7.35 (s, 1H,
(CDCl3): δ 1.10 (t, 3H, J = 6.9 Hz, C90-OCH2CH3), 3.74 (s, 6H,
H100, H120), 3.78 (s, 3H, H110), 3.97 (d, 1H, J = 4.0Hz, H80), 4.05
(m, 2H, C90-OCH2CH3), 4.59 (d, 1H, J = 3.8 Hz, H70), 5.99 (d,
1H, J = 1.8 Hz, H10a), 6.01 (d, 1H, J = 1.8 Hz, H10b), 6.21 (s,
2H, H20, H60), 6.65 (s, 1H, H3), 6.88 (s, 1H, H6), 7.34 (s,
13
1H, H7), 9.60 (s, 1H, H9). C NMR (CDCl3): δ 14.1 (C90-
13
OCH2CH3), 44.8 (C70), 46.4 (C70), 56.1 (C100, C120), 60.8 (C110),
61.4 (C90-OCH2CH3), 101.8 (C10), 104.9 (C20, C60), 108.9 (C6),
110.1 (C3), 125.2 (C1), 133.4 (C8), 133.8 (C2), 137.1 (C40), 137.3
(C10), 145.5 (C7), 147.4 (C5), 150.5 (C4), 153.2 (C30, C50), 171.7
(C90), 191.3 (C9). IR (film) cm-1: 1726 (COOR), 1671 (CHO).
HRMS: calcd for C24H24O8Na, 463.1363; found, 463.1348.
Allyl 9-Deoxy-9-oxo-r-apopicropodophyllate (11). From allyl
picropodophyllate 4 (85 mg, 0.18 mmol). The Swern reaction
product was chromatographed on silica gel, eluting with 2%
EtOAc/CH2Cl2 to give compound 11 (41 mg, 50%). 1H NMR
(CDCl3): δ 4.52 (m, 2H, C90-OCH2CHdCH2), 5.14 (d, 1H,
H7), 9,61 (s, 1H, H9). C NMR (CDCl3): δ 56.1 (2C, C90-
OCH2-C6H2(OCH3)3), 60.8 (1C, C90-OCH2-C6H2(OCH3)3),
67.1 (1C, C90-OCH2-C6H2(OCH3)3), 104.8 (2CH, C90-OCH2-
C6H2(OCH3)3), 131.3 (1C, C90-OCH2-C6H2(OCH3)3), 133.1
(C8), 137.7 (1C, C90-OCH2-C6H2(OCH3)3), 145.7 (C7), 153.2
(2C, C90-OCH2-C6H2(OCH3)3), 191.2 (C9). IR (film) cm-1
:
1730 (COOR), 1669 (CHO). Anal. (C32H32O11) C, H, N.
HRMS: calcd for C32H32O11Na, 615.1837; found 615.1844.
[R]20D -108° (c 1%).
3-Bromopropyl 9-Deoxy-9-oxo-r-apopicropodophyllate (16).
From 3-bromopropyl picropodophyllate 9 (630 mg, 1.14 mmol)

990 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Castro et al.
to yield aldehyde 16 (520 mg, 86%). 1H NMR (CDCl3): δ 2.05
(m, 2H, C90-CH2CH2CH2Br), 3.26 (t, 2H, J = 6.6 Hz, C90-
OCH2CH2CH2Br), 4.16 (m, 2H, C90-OCH2CH2CH2Br), 7.34
(s, 1H, H7), 9.60 (s, 1H, H9). 13C NMR (CDCl3): δ 29.2 (C90-
OCH2CH2CH2Br), 31.5 (C90-OCH2CH2CH2Br), 62.9 (C90-
General Methods for the Synthesis of Imines. A mixture of
aldehyde (1 equiv), anhydrous MgSO4 (4 equiv), and the
corresponding amine (2 equiv) was dissolved in CH2Cl2
(3 mL) and stirred at room temperature during 3-13 d. The
reaction mixture was diluted with CH2Cl2, filtered, and con-
centrated under vacuum to give the imines in quantitative yields.
Imines were obtained pure enough to carry on complete char-
acterization without being necessary any purification step.
Imines 22-24 have been described previously.12
OCH2CH2CH2Br), 145.7 (C7), 191.2 (C9). IR (film) cm-1
:
1731 (COOR), 1669 (CHO). Anal. (C25H25BrO8) C, H, N.
HRMS: calcd for C25H25BrO8Na, 555.0625; found 555.0635.
9-Deoxy-9-oxo-r-apopicropodophyllic Acid Pyrrolidinyl
Amide (19). From the diol-amide 17 (167 mg, 0.344 mmol).
The reaction product was chromatographed on silica gel, eluting
with 50% acetone/CH2Cl2 to give compound 19 (144 mg, 90%).
1H NMR (CDCl3): δ 1.65-1.98 (m, 4H, C90-N-(CH2)4-), 3.05
(m, 1H, C90-N-(CH2)4-), 3.28 (m, 1H, C90-N-(CH2)4-), 3.43
(m, 1H, C90-N-(CH2)4-), 3.70 (m, 1H, C90-N-(CH2)4-), 7.33
(s, 1H, H7), 9.53 (s, 1H, H9). 13C NMR (CDCl3): δ 24.3, 26.0,
46.0, 47.0 (C90-N-(CH2)4-), 134.4 (C8), 146.8 (C7), 191.9 (C9).
IR (film) cm-1: 1668 (CHO), 1636 (CON). Anal. (C26H27NO7)
C, H, N. HRMS: calcd for C26H27NO7 þ H, 466.1860; found
466.1862. [R]20D -200° (c 0.85%).
Ethyl 9-Deoxy-9-propylimino-r-apopicropodophyllate (25).
From aldehyde 10 (150 mg, 0.340 mmol) and propyl amine
(56 μL, 0.82 mmol) during 2 d to yield imine 25 (135 mg, 83%).
1H NMR (CDCl3): δ 0.82 (t, 3H, J = 7.3, NCH2CH2CH3), 1.11
(t, 3H, J = 7.2, C90-OCH2CH3), 1.58 (m, 2H, NCH2CH2CH3),
3.40 (m, 2H, NCH2CH2CH3), 4.05 (m, 2H, C90-OCH2CH3),
6.78 (s, 1H, H7) 7.94 (s, 1H, H9). 13C NMR (CDCl3): δ 11.7
(NCH2CH2CH3), 14.1 (C90-OCH2CH3), 24.1 (NCH2CH2CH3),
60.9 (C90-OCH2CH3), 63.1 (NCH2CH2CH3), 131.7 (C8), 135.1
(C7), 161.2 (C9), 172.6 (C90). IR (film) cm-1: 1726 (COOCH3).
HRMS: calcd for C27H21NO7 þ H, 482.2173; found 482.2170.
3,4,5-Trimethoxybenzyl 9-Deoxy-9-propylimine-r-apopicro-
podophyllate (26). From aldehyde 15 (90 mg, 0.15 mmol) and
propylamine (25 μL, 0.30 mmol) during 3 d. 1H NMR (CDCl3):
δ 0.76 (t, 3H, J = 7.2, NCH2CH2CH3), 1.50 (m, 2H, NCH2-
CH2CH3), 3.40 (m, 2H, NCH2CH2CH3), 3.78 (s, 6H, C90-
OCH2-C6H2(OCH3)3), 3.81 (s, 3H, C90-OCH2-C6H2(OCH3)3),
4.96 (s, 2H, C90-OCH2-C6H2(OCH3)3), 6.41 (s, 2H, C90-OCH2-
C6H2(OCH3)3), 6.80 (s, 1H, H7), 7.94 (s, 1H, H9). 13C NMR
(CDCl3): δ 11.7 (NCH2CH2CH3), 24.1 (NCH2CH2CH3), 56.1
(2C, C90-OCH2-C6H2(OCH3)3), 60.9 (1C, C90-OCH2-C6H2-
(OCH3)3), 63.1 (NCH2CH2CH3), 66.9 (C90-OCH2-C6H2-
(OCH3)3), 105.1 (2CH, C90-OCH2-C6H2(OCH3)3), 131.5 (1C,
C90-OCH2-C6H2(OCH3)3), 131.6 (C8), 135.2 (C7), 137.7
(1C, C90-OCH2-C6H2(OCH3)3), 153.2 (2C, C90-OCH2-C6H2-
(OCH3)3), 161.1 (C9), 172.4 (C90). IR (film) cm-1: 1726
(COOR). HRMS: calcd for C35H40NO10 þ H, 634.2647; found,
634.2658.
N-Ethyl 9-Deoxy-9-oxo-r-apopicropodophyllamide (20).
From the diol-amide 18 (80 mg, 0.17 mmol). The reaction
product was chromatographed on silica gel, eluting with 50%
EtOAc/CH2Cl2 to give compound 20 (26 mg, 35%). 1H NMR
(CDCl3): δ 1.04 (t, 3H, J = 7.2 Hz, C90-NHCH2CH3), 3.17
(m, 2H, C90-NHCH2CH3), 7.35 (s, 1H, H7), 9.57 (s, 1H, H9).
13C NMR (CDCl3): δ 14.7 (C90-NHCH2CH3), 34.5 (C90-NH-
CH2CH3), 132.9 (C8), 148.1 (C7), 193.5 (C9). IR (film) cm-1
:
3367 (NH), 1667 (CHO), 1640 (CONH). Anal. (C24H25NO7) C,
H, N. HRMS: calcd for C24H25NO7 þ H, 440.1704; found
440.1713. [R]20D -190° (c 0.31%).
N-(4-Methoxyphenyl) 9-Deoxy-9-oxo-r-apopicropodophyll-
amide (21). A mixture of aldehyde 2 (100 mg, 0.235 mmol),
1,2-ethanedithiol (0.040 mL, 0.47 mmol), and chlorotrimethyl-
silane (7 μL, 0.05 mmol) in dry CH2Cl2 (10 mL) was stirred
at room temperature for 24 h. The crude reaction product
was diluted with CH2Cl2, washed with 4% NaOH and brine,
dried over Na2SO4, filtered, and concentrated in vacuum to
yield the protected aldehyde-ester (100 mg, 84%) that was
dissolved in MeOH (10 mL), treated with 5% KOH/MeOH
(20 mL), and stirred for 22 h. Then 0.2 M NaH2PO4 (200 mL)
was added to the reaction mixture and MeOH removed in
vacuum and then acidified to pH = 4 by adding 2N HCl
and extracted with EtOAc. The organic phase was washed
with brine, dried, filtered, and evaporated off to give the free
acid.
General Methods for the Synthesis of Amines. Method C. The
solution of the imine, obtained as described above, in MeOH
(20 mL) was treated with excess of NaBH4 at 0 °C for 1 h. Water
was added to the reaction mixture, and MeOH was partially
removed in vacuum to give an aqueous solution that was
adjusted with satd Na2CO3 to pH > 7 and then extracted with
EtOAc. The organic phase was washed with brine, dried over
Na2SO4, and finally filtered and solvent removed to give the
corresponding amine.
A solution of the dithiolane-acid (69 mg) in dry THF (4 mL)
was treated with p-anisidine (280 mg, 2.28 mmol), dicyclohex-
ylcarbodiimide (29 mg, 0.14 mmol), and 1-hydroxybenzotriazol
(19 mg, 0.14 mmol) at room temperature for 21 h. The reaction
mixture was diluted with EtOAc, washed with 2 N HCl, satd aq
Na2CO3, and brine. The organic phase was dried, filtered, and
concentrated to yield the protected amide. A solution of this
amide (70 mg, 0.12 mmol) in acetic acid (4.0 mL) was treated
with SeO2 (65 mg, 0.59 mmol), and the mixture was stirred at
room temperature for 24 h. The crude was extracted with
EtOAc, and the organic phase was washed with satd aq NaH-
CO3 and NaCl, dried over Na2SO4, and evaporated off. The
reaction product was chromatographed on silica gel, eluting
with 10% EtOAc/CH2Cl2 to give compound 21 (24 mg, 20%
Method D. The aldehyde and the corresponding amine were
dissolved in CH2Cl2, and montmorillonite K-10 was added and
well mixed. The removal of the solvent gave a solid mixture,
which was subjected to microwave irradiations during 1-2 min.
The mixture was extracted with EtOAc and the organic phase
was dried over Na2SO4, filtered, and evaporated off to get a
reaction product that was reduced with NaBH4, as described
above for method C.
Methyl 9-Ethylamino-9-deoxy-r-apopicropodophyllate (27).
From imine 22 (92 mg, 0.21 mmol) and NaBH4 (92 mg, 2.4
mmol) using method C. The reaction product was chromato-
graphed on silica gel, eluting with 50% EtOAc/MeOH to give
compound 27 (42 mg, 40%). 1H NMR (CDCl3): δ 0.93 (t, 3H,
J = 6.9, NHCH2CH3), 2.30 (m, 2H, NHCH2CH3), 3.30 and
3.48 (AB system, J = 14.4, H9), 3.61 (m, 1H, H80), 3.62 (s, 3H,
C90-OCH3), 3.74 (s, 6H, H100, H120), 3.77 (s, 3H, H110), 4.48
(d, 1H, J = 3.7, H70), 5.90 (d, 1H, J = 1.6, H10a), 5.92 (d, 1H,
J = 1.6, H10b), 6.27 (s, 2H, H20, H60), 6.44 (s, 1H, H7), 6.56
(s, 1H, H3), 6.65 (s, 1H, H6). 13C NMR (CDCl3): δ 14.5
(NHCH2CH3), 42.6 (NHCH2CH3), 48.6 (C70), 49.7 (C80), 52.1
(C90-OCH3), 53.4 (C9), 56.2 (C100, C120), 60.7 (C110), 101.0
(C10), 105.4 (C20, C60), 106.9 (C6), 109.5 (C3), 126.4 (C7), 127.2
(C1), 129.1 (C2), 131.0 (C8), 137.1 (C40), 138.3 (C10), 146.9 (C5),
147.2 (C4), 153.1 (C30, C50), 172.9 (C90). IR (film) cm-1: 3400
1
from aldehyde 2). H NMR (CDCl3): δ 3.76 (s, 3H, C90-NH-
C6H4-OCH3), 6.78 and 7.39 (AB system, J = 9.0 Hz, C90-NH-
C6H4-OCH3), 7.40 (s, 1H, H7), 8.27 (s, 1H, NH), 9,60 (s, 1H,
H9). 13C NMR (CDCl3): δ 55.5 (C90-NH-(C6H4)-OCH3), 114.1
(2CH, C90-NH-(C6H4)-OCH3), 121.1 (2CH, C90-NH-(C6H4)-
OCH3), 131.2 (C, C90-NH-(C6H4)-OCH3), 131.9 (C8), 148.6
(C7), 156.3 (C, C90-NH-(C6H4)-OCH3), 168.4 (C90), 193.9 (C9).
IR (film) cm-1: 3328 (NH), 1668 (CHO), 1640 (CONH). Anal.
(C29H27NO8) C, H, N. HRMS: calcd for C29H27NO8 þ H,
518.1809; found 518.1827.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 991
(NH), 1732 (COOR). HRMS: calcd for C25H29NO7 þ H,
456.2017; found, 456.2003.
1.11 (t, 3H, J = 7.1, NHCH2CH3), 2.65 (m, 2H, NHCH2CH3),
C
3.33 and 3.44 (AB system, J = 11.0, H9), 6.48 (s, 1H, H7). 13
NMR (CDCl3): δ 14.2 (NHCH2CH3), 43.0 (NHCH2CH3), 52.7
(C9), 128.1 (C7), 131.8 (C8). IR (film) cm-1: 3300 (NH), 1629
(CON). HRMS: calcd for C28H34N2O6 þ H, 495.2490; found,
495.2508.
Methyl 9-Propylamino-9-deoxy-r-apopicropodophyllate (28).
From imine 23 (155 mg, 0.33 mmol) and NaBH4 (155 mg, 4.09
mmol) using method C. The reaction product was chromato-
graphed on silica gel, eluting with 5% EtOH/CH2Cl2 to give
compound 28 (100 mg, 64%). 1H NMR (CDCl3): δ 0.77 (t, 3H,
J = 7.5, NHCH2CH2CH3), 1.30 (m, 2H, NHCH2CH2CH3),
2.25 (m, 2H, NHCH2CH2CH3), 3.29 and 3.48 (AB system, J =
14.4, H9), 6.45 (s, 1H, H7). 13C NMR (CDCl3): δ 11.6 (NHCH2-
CH2CH3), 22.5 (NHCH2CH2CH3), 50.0 (NHCH2CH2CH3),
53.6 (C9), 126.5 (C7), 131.1 (C8). IR (film) cm-1: 3396 (NH),
1729 (COOCH3). HRMS: calcd for C26H31NO7 þ H, 470.2173;
found, 479.2183.
9-(4-Methoxyphenyl)amino-9-deoxy-r-apopicropodophyllic
Acid Pyrrolidinyl Amide (34). The corresponding imine, ob-
tained from aldehyde 19 (80 mg, 0.17 mmol) and p-anisidine
(847 mg, 6.88 mmol) during 12 d, was directly reduced using
method C. The reaction product was chromatographed on silica
gel, eluting with 50% acetone/CH2Cl2 to give compound 34
1
(70 mg, 71%). H NMR (CDCl3): δ 3.72 (s, 3H, NH-(C6H4)-
OCH3), 3.73 and 3.95 (AB system, J = 15.5, H9), 6.53 (s, 1H,
H7), 6.54 (d, 2H, J = 9.1, NH-(C6H4)-OCH3), 6.73 (d, 2H, J =
9.1, NH-(C6H4)-OCH3). 13C NMR (CDCl3): δ 49.1 (C9), 55.8
(NH-(C6H4)-OCH3), 114.0 (2CH, NH-(C6H4)-OCH3), 114.8
(2CH, NH-(C6H4)-OCH3), 125.8 (C7), 134.3 (C8), 142.6
(C, NH-(C6H4)-OCH3), 152.3 (C, NH-(C6H4)-OCH3). IR
(film) cm-1: 3341 (NH), 1625 (CON). HRMS: calcd for C33H36-
N2O7 þ H, 573.2595; found, 573.2600.
Methyl 9-(4-Methoxyphenyl)amino-9-deoxy-r-apopicropodo-
phyllate (29). From aldehyde 2 (100 mg, 0.230 mmol) and
p-ansidine (296 mg, 2.35 mmol) on K-10 montmorillonite
(1.2 g) using method D during 2 min. The reaction product
was chromatographed on silica gel, eluting with 10% EtOAc/
CH2Cl2 to give compound 29 (50 mg, 40%). 1H NMR (CDCl3):
δ 3.72 (s, 3H, NH-(C6H4)-OCH3), 3.75 (m, 2H, H9a, H9b), 6.50
(s, 1H, H7), 6.40 and 6.69 (AB system J = 9.0, NH-(C6H4)-
OCH3). 13C NMR (CDCl3): δ 49.7 (C9), 55.6 (NH-(C6H4)-
OCH3), 113.8 (2CH, NH-(C6H4)-OCH3), 114.7 (2CH,
NH-(C6H4)-OCH3), 124.8 (C7), 131.2 (C8), 141.9 (C, NH-
Cell Growth Inhibition Assays. A colorimetric assay using
sulforhodamine B (SRB) was adapted for a quantitative mea-
surement of cell growth and viability, following a previously
described method.28 Cells were seeded in 96-well microtiter
plates, at 5 ꢀ 103 cells per well in aliquots of 195 μL of RPMI
medium, and they were allowed to attach to the plate surface by
growing in a drug-free medium for 18 h. Afterward, samples
were added in aliquots of 5 μL (dissolved in DMSO/H2O, 3:7).
After 72 h exposure, the antitumor effect was measured by the
SRB methodology: cells were fixed by adding 50 μL of cold 50%
(wt/vol) trichloroacetic acid (TCA) and incubating for 60 min at
4 °C. Plates were washed with deionized water and dried; 100 μL
of SBR solution (0.4 wt %/vol in 1% acetic acid) was added to
each microtiter well and incubated for 10 min at room tempera-
ture. Unbound SRB was removed by washing with 1% acetic
acid. Plates were air-dried, and bound stain was solubilized with
Tris buffer. Optical densities were read on an automated spec-
trophotometer plate reader at single wavelength of 490 nm.
Data analyses were generated automatically by the LIMS im-
plementation. Using control OD values (C), test OD values (T)
and time zero OD values (To), the drug concentration that
caused a 50% growth inhibition (GI50 value) was calculated
from the equation: 100[(T - To)/(C - To)] = 50. Each value
represents the mean from triplicate determinations.
(C6H4)-OCH3), 152.0 (C, NH-(C6H4)-OCH3). IR (film) cm-1
:
3400 (NH), 1728 (COOCH3). HRMS: calcd for C30H31NO8 þ
H, 534.2122; found, 534.2137.
Methyl 9-(4-Methylphenyl)amino-9-deoxy-r-apopicropodo-
phyllate (30). From aldehyde 2 (100 mg, 0.235 mmol) and
p-toluidine (251 mg, 2.35 mmol) using method D during
1 min. The reaction product was chromatographed on silica
gel, eluting with 5% EtOAc/CH2Cl2 to give compound 30
1
(80 mg, 71%). H NMR (CDCl3): δ 2.20 (s, 3H, NH-(C6H4)-
CH3), 3.75 (m, 2H, H9), 6.50 (s, 1H, H7), 6.35 and 6.89 (AB
system, J = 8.1, NH-(C6H4)-CH3). 13C NMR (CDCl3): δ 20.3
(NH-(C6H4)-CH3), 49.1 (C9), 112.8 (2CH, NH-(C6H4)-CH3),
124.8 (C7), 126.6 (C, NH-(C6H4)-CH3), 129.6 (2CH, NH-
(C6H4)-CH3), 131.2 (C8), 145.6 (C, NH-(C6H4)-CH3). IR
(film) cm-1: 3407 (NH), 1729 (COOCH3). HRMS: calcd for
C30H31NO7 þ H, 518.2173; found, 518.2200.
Ethyl 9-Propylamino-9-deoxy-r-apopicropodophyllate (31).
From imine 25 (135 mg, 0.28 mmol) and NaBH4 (135 mg, 3.57
mmol) using method C to yield amine 31 (100 mg, 74%). H
1
NMR (CDCl3): δ 0.76 (t, 3H, J = 7.3, NHCH2CH2CH3), 1.30
(m, 2H, NHCH2CH2CH3), 2.20 (m, 2H, NHCH2CH2CH3),
3.25 (d, 1H, J = 14.3, H9a), 3.47 (d, 1H, J = 14.3, H9b), 6.40
(s, 1H, H7). 13C NMR (CDCl3): δ 11.7 (NHCH2CH2CH3), 23.1
(NHCH2CH2CH3), 50.7 (NHCH2CH2CH3), 53.9 (C9), 125.3
(C7), 132.6 (C8). IR (film) cm-1: 3350 (NH), 1725 (COOR).
Anal. (C27H33NO7) C, H, N. HRMS: calcd for C27H33NO7 þ H,
484.2330; found, 484.2338.
Cell Cycle Analysis. For cell cycle analysis, we used the human
colon adenocarcinoma HT-29 cell line grown in RPMI supple-
mented with 10% (v/v) heat-inactivated fetal calf serum,
2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL
streptomycin at 37 °C in a humidified atmosphere of 5% CO2
and 95% air. Cells were periodically tested for Mycoplasma
infection and found to be negative. Untreated and drug-treated
cells ((3-5) ꢀ 105) were centrifuged and fixed overnight in 70%
ethanol at 4 °C. Then cells were washed three times with PBS,
incubated for 1 h with 1 mg/mL RNase A and 20 μg/mL
propidium iodide at room temperature, and analyzed with a
Becton Dickinson FACSCalibur flow cytometer (San Jose, CA)
as described previously.30,31 Quantification of apoptotic cells
was calculated as the percentage of cells in the sub-G1 peak in
cell cycle analysis.
Confocal Microscopy. HT-29 cells were grown on poly-
L-lysine coated coverslips, and after drug treatment coverslips
were washed three times with HPEM (25 mM HEPES, 60 mM
PIPES, 10 mM EGTA, 3 mM MgCl2, pH6.6), fixed with 4%
paraformaldehyde in HPEM buffer for 15 min, and permeabi-
lized with 0.5% Triton X-100 as previously described.32 Cover-
slips were incubated with a specific Ab-1 antialpha-tubulin
mouse monoclonal antibody (diluted 1:150 in PBS)
(Calbiochem) for 1 h, washed four times with PBS, and
then incubated with CY3-conjugated sheep antimouse IgG
3,4,5-Trimethoxybenzyl 9-Propylamino-9-deoxy-r-apopicro-
podophyllate (32). From imine 26 (90 mg, 0.14 mmol) and
NaBH4 (135 mg, 3.57 mmol) using method C. The reaction
product was chromatographed on silica gel, eluting with 7%
1
EtOH/CH2Cl2 to give compound 32 (44 mg, 45%). H NMR
(CDCl3): δ 0.74 (t, 3H, J = 7.3, NHCH2CH2CH3), 1.25 (m, 2H,
NHCH2CH2CH3), 2.17 (m, 2H, NHCH2CH2CH3), 3.26 and
3.45 (AB system, J = 14.6, H9), 6.44 (s, 1H, H7). 13C NMR
(CDCl3): δ 11.6 (NHCH2CH2CH3), 23.0 (NHCH2CH2CH3),
50.6 (NHCH2CH2CH3), 54.0 (C9), 125.7 (C7), 132.0 (C8). IR
(film) cm-1: 3345 (NH), 1726 (COOR). HRMS: calcd for
C35H41NO10 þ H, 636.2803; found, 636.2831.
9-Ethylamino-9-deoxy-r-apopicropodophyllic Aacid Pyrroli-
dinyl Amide (33). Following the general method for the prepara-
tion of imines, aldehyde 19 (80 mg, 0.17 mmol) reacted with
ethyl amine (70% in water, 0.390 mL, 6.88 mmol) during 12 d to
yield the corresponding imine, which was directly reduced to the
amine 33 using method C (50 mg, 68%). 1H NMR (CDCl3): δ

992 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Castro et al.
´ ´
(11) Garcıa-Rocha, M.; Garcıa-Gravalos, M. D.; Avila, J. Character-
ꢀ
(Jackson ImmunoResearch) for 1 h at 4 °C. After washing four
times with PBS, cell nuclei were stained with DAPI (Sigma) for
5-10 min, washed with PBS, and then samples were analyzed by
confocal microscopy using a ZeissLSM310 laser scan confocal
microscope. A drop of SlowFade light antifading reagent
(Molecular Probes) was added to preserve fluorescence. Nega-
tive controls, lacking the primary antibody or using an irrele-
vant antibody, showed no staining.
isation of Antimitotic Products from Marine Organisms that
Disorganise the Microtubule Network: Ecteinascidin 743, Isoho-
mohalichondrin and LL-15. Br. J. Cancer 1996, 73, 875–883.
(12) Castro, M. A.; Miguel del Corral, J. M.; Gordaliza, M.; Garcı
ꢀ
´
a,
ꢀ
P. A.; Gomez-Zurita, M. A.; Garcı
´
a-Gravalos, M. D.; Iglesia-
Vicente, J.; Gajate, C.; An, F.; Mollinedo, F.; San Feliciano, A.
Synthesis and Biological Evaluation of New Selective Cytotoxic
Cyclolignans Derived from Podophyllotoxin. J. Med. Chem. 2004,
47, 1214–1222.
(13) Magedov, I. V.;Manpadi, M.;VanSlambrouck, S.;Steelant, W. F. A.;
Rozhkova, E.; Przheval’skii, N. M.; Rogelj, S.; Kornienko, A. Dis-
covery and Investigation of Antiproliferative and Apoptosis-Inducing
Properties of new Heterocyclic Podophyllotoxin Analogues Accessible
by a One-Step Multicomponent Synthesis. J. Med. Chem. 2007, 50,
5183–5192.
(14) (a) Dona, F.; Ennio, P.; Savio, M.; Coppa, T.; Scovassi, A.;
Mondello, C. Loss of Histone H2AX Increases Sensitivity of
Immortalized Mouse Fibroblasts to the Topoisomerase II Inhibi-
tor Etoposide. Int. J. Oncol. 2008, 33, 613–621. (b) Sordet, O.;
Goldman, A.; Pommier, Y. Topoisomerase II and Tubulin Inhibitors
both Induce the Formation of Apoptotic Topoisomerase I Cleavage
Complexes. Mol. Cancer Ther. 2006, 5, 3139–3144. (c) Chiu, C. C.;
Li, C. H.; Ung, M. W.; Fuh, T. S.; Chen, W. L.; Fang, K. Etoposide
(VP-16) Elicits Apoptosis Following Prolonged G2-M Cell Arrest in
p53-Mutated Human Non-Small Cell Lung Cancer Cells. Cancer Lett.
2005, 223, 249–258.
(15) (a) Chen, Y.; Su, Y. H.; Wang, C. H.; Wu, J. M.; Chen, J. C.; Tseng,
S. H. Induction of Apoptosis and Cell Cycle Arrest in Glioma Cells
by GL331 (a Topoisimerase II Inhibitor). Anticancer Res. 2005, 25,
4203–4208. (b) Huang, T. S.; Shu, C. H.; Lee, C. C.; Chen, L. T.;
Whang-Peng, J. In vitro Evaluation of GL3310s Cancer Cell Killing and
Apoptosis-inducing Activity in Combination with other Chemother-
apeutic Agents. Apoptosis 2000, 5, 79–85. (c) Huang, T. S.; Lee, C. C.;
Chao, Y.; Shu, C. H.; Chen, L. T.; Chen, L. L.; Chen, M. H.; Yuan, C. C.;
Whang-Peng, J. A Novel Podophyllotoxin-Derived Compound GL331
is more Potent than its Congener VP-16 in Killing Refractory Cancer
Cells. Pharm. Res. 1999, 16, 997–1002.
Acknowledgment. Financial support came from Spanish
Ministerio de Ciencia y Tecnologıa (CTQ2008-02899,
´
SAF2008-02251), Red Tematica de Investigacion Cooperativa
ꢀ
en Cancer, Instituto de Salud Carlos III (RD06/0020/1037),
ꢀ
ꢀ
and JuntadeCastilla y Leon (SA005A08 andfellowship toM.
ꢀ
V.R.). We thank Dr. H. B. Broughton for his help with
proofreading.
Supporting Information Available: Complete 1H and 13C
NMR data for all synthesized analogues. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Ayres, D. C.; Loike, J. D. Lignans: Chemical, Biological and
Clinical Properties. Cambridge University Press: London, 1990.
(2) (a) Xu, H.; Lu, M.; Tian, X. A Review on Hemisynhesis, Biosynthe-
sis, Biological Activities, Mode of Action,and Structure-Activity
Relationship of Podophyllotoxins: 2003-2007. Curr. Med. Chem.
2009, 16, 327–349. (b) Liu, Y.-Q.; Yang, L.; Tian, X. Podophyllotoxin:
Current Perspectives. Curr. Bioact. Compd. 2007, 3, 37–66. (c) Lee,
K.-H.; Xiao, Z. Podophyllotoxins and Analogs. In Anticancer Agents
from Natural Products; Cragg, G, Kindston, D. G. I., Newman, D. J.,
Eds.; CRC Press: New York, 2005; pp 71-87.
(3) Saitoh, T.; Kuramochi, K.; Imai, T.; Takata, K.-I.; Takehara, M.;
Kobayashi, S.; Sakaguchi, K.; Sugawara, F. Podophyllotoxin
Directly Binds a Hinge Domain in E2 of HPV and Inhibits
an E2/E7 Interaction in vitro. Bioorg. Med. Chem. 2008, 16,
5815–5825.
(16) (a) Gordaliza, M.; Garcıa, P. A.; Miguel del Corral, J. M.; Castro,
´
ꢀ
M. A.; Gomez-Zurita, M. A. Podophyllotoxin: Distribution,
Sources, Applications and new Cytotoxic Derivatives. Toxicon
2004, 44, 441–459. (b) Castro, M. A.; Miguel del Corral, J. M.;
ꢀ
Gordaliza, M.; Gomez-Zurita, M. A.; García, P. A.; San Feliciano, A.
Chemoinduction of Cytotoxic Selectivity in Podophyllotoxin-Related
(4) (a) Carlstrom, K.; Hedin, P. J.; Jonsson, l.; Lerndal, T.; Lien, J.;
Weitoft, T.; Axelson, M. Endocrine Effects of the Podophyllotoxin
Derivative Drug CPH82 (Reumacon) in Patients with Rheumatoid
Arthritis. Scand. J. Rheumatol. 2000, 29, 89–94. (b) Rantapaa, D. S.;
Landberg, G.; Roos, G.; Norberg, B. Cell Cycle Effects of the Anti-
rheumatic Agent CPH82. Br. J. Rheumatol. 1994, 33, 327–331.
(5) (a) Frackenpohl, J.; Adelt, I.; Antonicek, H.; Arnold, C.;
Behrmann, P.; Blaha, N.; Bohmer, J.; Gutbrod, O.; Hanke, R.;
Hohmann, S.; Van Houtdreve, M.; Losel, P.; Malsam, O.;
Melchers, M.; Neufert, V.; Peschel, E.; Reckmann, U.; Schenke,
T.; Thiesen, H. P.; Velten, R.; Vogelsang, K.; Weiss, H. C.
Insecticidal Heterolignans-Tubuline Polymerization Inhibitors
with Activity against Chewing Pests. Bioorg. Med. Chem. 2009,
17, 4160–4184. (b) Liu, Y.-Q.; Liu, Y.; Xiao, H.; Gao, R.; Tian, X.
Synthesis and Insecticidal Activities of Novel Derivatives of Podophyl-
lotoxin: Part XII. Pestic. Biochem. Physiol. 2008, 91, 116–121.
(6) (a) Lee, K.-H.; Zhiyan, X. Lignans in Treatment of Cancer and
other Diseases. Phytochem. Rev. 2004, 2, 341–362. (b) Bohlin, L.;
Rosen, B. Podophyllotoxin Derivatives: Drug Discovery and Develop-
ment. Drug Discovery Today 1996, 1, 343–351.
Lignans. Phytochem. Rev. 2003, 2, 219–233.
(17) Castro, M. A.; Miguel del Corral, J. M.; Gordaliza, M.; Garcıa,
´
ꢀ
P. A.; Gomez-Zurita, M. A.; San Feliciano, A. Synthesis and
Cytotoxic Evaluation of C-9 Oxidized Podophyllotoxin Deriva-
tives. Bioorg. Med. Chem. 2007, 15, 1670–1678.
(18) (a) You, Y-J-.; Kim, Y.; Nam, N.-H.; Bang, S.-C.; Ahn, B.-Z. Alkyl
and Carboxylalkyl Esters of 40-Demethyl-4-deoxypodophylloto-
xin: Synthesis, Cytotoxic and Antitumor Activity. Eur. J. Med.
Chem. 2004, 39, 189–193. (b) Xiao, Z.; Vance, J. R.; Bastow, K. F.;
Brossi, A.; Wang, H.-K.; Lee, K.-H. Antitumor Agents. Part 235: Novel
40-ester etoposide analogues as Potent DNATopoisomerase II Inhibitors
with Improved Therapeutic Potential. Bioorg. Med. Chem. 2004, 12,
3363–3369. (c) Guianvarch, D.; Duca, M.; Boukarim, C.; Kraus-
ꢀ
ꢀ
Berthier, L.; Leonce, S.; Pierre, A.; Pfeiffer, B.; Renard, P.; Arimondo,
P. B.; Monneret, C.; Dauzonne, D. Synthesis and Biological Activity of
Sulfonamide Derivatives of Epipodophyllotoxin. J. Med. Chem. 2004,
47, 2365–2374. (d) Zhang, F.-M.; Yao, X.-J.; Tian, X.; Tu, Y.-Q.
Synthesis and Biological Evaluation of New 4β-5-Fu-substituted
40-Demethylepipodophyllotoxin Derivatives. Molecules 2006, 11,
ꢀ
ꢀ
849–857. (e) Duca, M.; Arimondo, P. B.; Leonce, S.; Pierre, A.; Pfeiffer,
B.; Monneret, C.; Dauzonne, D. Novel Carbamate Derivatives of
4-β-Amino-40-O-demethyl-4-deoxypodophyllotoxin as Inhibitors of
Topoisomerase II: Synthesis and Biological Evaluation. Org. Biomol.
Chem. 2005, 3, 1074–1080. (f) Xiao, Z.; Bastow, K. F.; Vance, J. R.;
Lee, K.-H. Antitumor Agents. Part 227: Studies on Novel 40-O-
demethyl-epipodophyllotoxins as Antitumor Agents targeting Topoi-
somerase II. Bioorg. Med. Chem. 2004, 12, 3339–3344.
ꢀ
(7) Gordaliza, M.; Miguel del Corral, J. M.; Castro, M. A.; Lopez-
ꢀ
ꢀ
Vazquez, M. L.; San Feliciano, A.; Garcıa-Gravalos, M. D.;
´
Carpy, A. Synthesis and Evaluation of Pyrazolignans. A New
Class of Cytotoxic Agents. Bioorg. Med. Chem. 1995, 3, 1203–1210.
(8) Cho, S. J.; Kashiwada, Y.; Bastow, K. F.; Cheng, Y. C.; Lee, K. H.
Antitumor Agents. 164. Podophenazine, 200,300-Dichloropodophe-
nazine, Benzopodophenazine and their 4β-p-Nitroaniline Deriva-
tives as Novel DNA Topoisomerase II Inhibitors. J. Med. Chem.
1996, 39, 1396–1402.
(9) Tseng, C. J.; Wang, Y. J.; Liang, Y. C.; Jeng, J. H.; Lee, W. S.; Lin,
J. K.; Chen, C. H.; Liu, I. C.; Ho, Y. S. Microtubule Damaging
Agents Induce Apoptosis in HL-60 Cells and G2/M Cell Cycle
Arrest in HT-29. Toxicology 2002, 175, 123–142.
(10) Masuda, T.; Oyama, Y.; Yonemori, S.; Takeda, Y.; Yamazaki, Y.;
Mizuguchi, S.; Nakata, M.; Tanaka, T.; Chikahisa, L.; Inaba, Y.;
Okada, Y. Flow Cytometric Estimation on Cytotoxic Activity of
Leaf Extracts from Seashore Plants in Subtropical Japan: Isola-
tion, Quantification and Cytotoxic Action of (-)-Deoxypodophyl-
lotoxin. Phytother. Res. 2002, 16, 353–358.
(19) Dorbec, M.; Florent, J. C.;Monneret, C.; Rager, M. N.; Bertounesque,
E. 1-Aryltetralin Privileged Structure-Based Libraries: Parallel Synthe-
sis of N-Aryl and N-Biaryl γ-lactam lignans. Tetrahedron 2006, 62,
11766–11781.
(20) (a) Wang, J.; Tian, X.; Tsumura, H.; Shimura, K.; Ito, H. Anti-
tumor Activity of a New Low Immunosuppressive Derivative of
Podophyllotoxin (GP-11) and its Mechanisms. Anti-Cancer Drug
Des. 1993, 8, 193–202. (b) Subrahmanyam, D.; Renuka, B.; Laxmana
Rao, C. V.; Sagar, P. S.; Deevi, D. S.; Babu, J. M.; Vyas, K. Novel
D-Ring Analogues of Podophyllotoxin as Potent Anti-Cancer Agents.
Bioorg. Med. Chem. Lett. 1998, 8, 1391–1396.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 993
(21) Castro, M. A.; Miguel del Corral, J. M.; Gordaliza, M.; Grande,
ꢀ
A. Synthesis and Cytotoxicity of Podophyllotoxin Analogues
Modified in the A-Ring. Eur. J. Med. Chem. 2003, 38, 65–74.
(22) Moss, G. P. Nomenclature of Lignans and Neolignans. Pure Appl.
Chem. 2000, 72, 1493–1523.
(28) (a) Faircloth, G. T.; Stewart, D.; Clement, J. J. A Simple Screening
Procedure for the Quantitative Measurement of Cytotoxicity
Assay. J. Tissue Cult. Methods 1988, 11, 201–205. (b) Skehan, P.;
Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Waren,
J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New colorimetric
cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst.
1990, 82, 1107–1112.
ꢀ
C.; Gomez-Zurita, M. A.; Garcıa-Gravalos, M. D.; San Feliciano,
´
(23) Smith, M. B.; March, J. March’s Advanced Organic Chemistry,
5th ed.; John Wiley & Sons: New York, 2001; pp 510-511.
(29) Mollinedo, F; Gajate, C. Microtubules, microtubule-interfering
ꢀ
(24) Miguel del Corral, J. M.; Gordaliza, M.; Castro, M. A.; Lopez-
a-Gravalos, M. D.; Broughton, H. B.; San
agents and apoptosis. Apoptosis 2003, 8, 413–450.
ꢀ
ꢀ
Vazquez, M. L.; Garcı
´
(30) Gajate, C.; Barasoain, I.; Andreu, J. M.; Mollinedo, F. Induction
of apoptosis in leukemic cells by the reversible microtubule-
disrupting agent 2-methoxy-5-(20,30,40-trimethoxyphenyl)-2,4,6-
cycloheptatrien-1-one: protection by Bcl-2 and Bcl-XL and cell
cycle arrest. Cancer Res. 2000, 60, 2651–2659.
Feliciano, A. Bioactive Isoxazoline and Oxime Derivatives from
7-Ketolignans. Tetrahedron 1997, 53, 6555–6564.
(25) Blay, G.; Cardona, l.; Garcıa, B.; Garcıa, C. L.; Pedro, J. R. A Non-
´ ´
Catalyzed Ring-Opening Aminolysis Reaction of Sesquiterpene
Lactones. Tetrahedron Lett. 1994, 35, 931–934.
(31) Gajate, C.; Santos-Beneit, A. M.; Macho, A.; Lazaro, M.;
Hernandez-, A.; Modolell, M.; Munoz, E.; Mollinedo, F. Involve-
ment of mitochondria and caspase-3 in ET-18-OCH3-induced
apoptosis of human leukemic cells. Int. J. Cancer 2000, 86, 208–218.
(26) Suzuki, T.; Nagaoka, M.; Kondo, Y.; Takahashi, T.; Takeuchi, M.;
Hara, H.; Saito, M.; Yamada, T.; Tomioka, K.; Hamada, M.; Mase,
T. 2-(3-Pyridyl)thiazolidine-4-carboxamide Derivatives. II. Struc-
ture-Activity Relationship and Active Configuration of 2-(3-Pyr-
idyl)thiazolidine-4-carboxamides as Platelet-Activating Factor
Receptor Antagonists. Chem. Pharm. Bull. 1998, 46, 1468–1473.
ꢀ
(32) Maya, A. B. S.; Perez-Melero, C.; Mateo, C.; Alonso, D.;
ꢀ
ꢀ
Fernandez, J. L.; Gajate, C.; Mollinedo, F.; Pelaez, R.; Caballero,
E.; Medarde, M. Further naphthylcombretastatins. An investiga-
tion on the role of the naphthalene moiety. J. Med. Chem. 2005, 48,
556–568.
ꢀ
(27) Garcı
´
a, P. A. Modificacion Molecular de Ciclolignanos Antineo-
ꢀ
plasicos Selectivos. Ph.D. Dissertation , Salamanca University, 2000.