Novel carbamate derivatives of 4-β-amino-4′-O-demethyl-4- desoxypodophyllotoxin as inhibitors of topoisomerase II: Synthesis and biological evaluation

Article abstract of DOI:10.1039/b416862c

A novel series of carbamate derivatives of 4-β-amino-4′-O- demethyl-4-desoxypodophyllotoxin were synthesized. Their effect on human DNA topoisomerase II and antiproliferative activity was evaluated. Compounds 4a-c, 4g, 4j and 4k are topoisomerase II poiso

Full text of DOI:10.1039/b416862c

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A R T I C L E
Novel carbamate derivatives of 4-b-amino-4ꢀ-O-demethyl-4-
desoxypodophyllotoxin as inhibitors of topoisomerase II: synthesis
and biological evaluation
Maria Duca,a,b Paola B. Arimondo,a Ste´phane Le´once,c Alain Pierre´,c Bruno Pfeiffer,c
Claude Monneretb and Daniel Dauzonne*b
a Laboratoire de Biophysique, UMR 5153 CNRS-Muse´um National d’Histoire Naturelle USM
0503, INSERM UR565, 43 rue Cuvier, 75231, Paris cedex 05, France
b UMR 176 CNRS, Institut Curie, Section de Recherche, 26 rue d’Ulm, 75248, Paris cedex 05,
France
c Institut de Recherches Servier, Division Recherche Cance´rologie, 125 Chemin de Ronde, 78290,
Croissy sur Seine, France
Received 4th November 2004, Accepted 12th January 2005
First published as an Advance Article on the web 18th February 2005
A novel series of carbamate derivatives of 4-b-amino-4ꢀ-O-demethyl-4-desoxypodophyllotoxin were synthesized.
Their effect on human DNA topoisomerase II and antiproliferative activity was evaluated. Compounds 4a–c, 4g, 4j
and 4k are topoisomerase II poisons that induce double-stranded breaks in DNA and exhibit increased cytotoxicity
compared to etoposide.
Introduction
Etoposide (VP16) is a semisynthetic glycoside derivative of
podophyllotoxin (Fig. 1) and is one of the most extensively used
anticancer drugs in the treatment of several types of tumors,
including testicular and small cell lung cancer, lymphoma,
leukemia and Kaposi’s sarcoma.1–3 Despite its extensive use
in cancer chemotherapy, it presents several limitations, such
as poor water solubility, the development of drug resistance,
metabolic inactivation, myelosuppression and toxicity.4 Etopo-
side and the 4ꢀ-demethylepipodophyllotoxin (4ꢀ-DMEP) deriva-
tives in general (Fig. 1) unlike podophyllotoxin, do not inhibit
tubuline polymerisation, but inhibit an ubiquitous and essential
enzyme: human DNA topoisomerase II.5,6 This enzyme controls
DNA topology by transient cleavage of the DNA double helix.7,8
When the concentration of these transient intermediates (called
cleavage complexes) increases, DNA strand breaks occur. 4ꢀ-
DMEP derivatives, such as VP16, kill cells by increasing the
levels of topoisomerase II-mediated DNA cleavage and are thus
called topoisomerase II poisons.5,9,10
In order to overcome the limitations cited above, and
to develop more active and more potent analogues, several
chemical modifications have been carried out on the 4ꢀ-
Fig. 1 Chemical structures of podophyllotoxin, 4ꢀ-DMEP, VP16 and
demethylepipodophyllotoxin structure. QSAR studies suggested
the 4ꢀ-demethylepipodophyllotoxin 4-aminoalkyl carbamates previously
that the essential structural features for antitopoisomerase II
synthesized.
activity are: the 4ꢀ-hydroxyl group, the 4-b-stereochemistry and
the 4-substitution of 4ꢀ-demethylepipodophyllotoxin.11,12,13 The
latter has been extensively modified and the glycoside moiety of
and inhibition of topoisomerase II, as well as the DNA binding
affinity, were studied. The compounds are more cytotoxic than
etoposide and some are good topoisomerase II inhibitors.
Finally, structure–activity relationships were established for this
new class of compounds.
etoposide has been replaced with non-sugar groups leading to,
in some cases, better activity compared to etoposide.14,15
Recently, we reported that some non glycoside 4ꢀ-demethyle-
pipodophyllotoxin derivatives bearing a carbamate residue
at the 4 position (4ꢀ-demethylepipodophyllotoxin-4-aminoalkyl
carbamates, Fig. 1) strongly inhibited topoisomerase II and
showed antiproliferative effects in vitro and antitumor activity
in vivo.16
This prompted us to further investigate carbamate substi-
tution in this position. We report herein the synthesis of a
novel series of carbamates bearing variable aliphatic or aromatic
side chains obtained by introducing a nitrogen atom in the 4b
position (Scheme 1, 4). The relationship between cytotoxicity
Results and discussion
Chemistry
The synthesis of these novel 4-b-amino-4ꢀ-O-demethyl-4-desoxy-
podophyllotoxin derivatives (4a–m) is depicted in Scheme 1. The
1 0 7 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 7 4 – 1 0 8 0
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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and II. Furthermore, their effect on L1210 cell proliferation and
on the cell cycle were studied.
All compounds resulted inactive for topoisomerase I inhibi-
tion (data not shown).
Topoisomerase II poisoning was evaluated by the amount
(by percentage) of linear DNA mediated by the action of
topoisomerase II on a supercoiled plasmid in the presence of
etoposide or of the 4-b-N-carbamates (Fig. 2). Supercoiled DNA
was treated with human DNA topoisomerase IIain the presence
of an increasing concentration of the tested compounds. Results
obtained with three of the most potent derivatives (4a, 4b, 4c)
are shown in Fig. 2; etoposide was used as a reference. In the
presence of increasing concentrations of these compounds, a
band corresponding to linear DNA can be clearly observed,
attesting that these molecules stabilize the cleavage complex, in
which the double helix is cleaved by the enzyme on both strands.
Compounds 4a and 4b produced much more double-stranded
DNA cleavage than 4c and are almost equivalent to etoposide.
This shows that introducing an aliphatic chain on the carbamate
group is favourable for topoisomerase inhibition and that
increasing the length of this chain leads to a decrease in activity.
These compounds presented a four to six fold better cytotoxicity
on murine L1210 cell lines than VP16. They also exhibited an
important effect on the cell cycle. Most compounds of the series
showed good activity, confirming that the introduction of a
carbamate group in the 4-position, instead of the glycopyranose
group of etoposide, is favourable for antitopoisomerase activity.
Compound 4g, bearing a terminal methoxy group, presented
the same biological properties as 4b while the introduction of
a terminal chlorine (4d) conferred a decrease in topoisomerase
II inhibition activity. Compounds 4e and 4f, bearing a terminal
double and triple bond, induced a strong effect on cytotoxicity
and on the cell cycle, while topoisomerase II activity decreased.
The introduction of an heterocycle, as in the case of compounds
4h or 4i, which present a pyrane or a maleimide substituent,
respectively, conferred an almost complete loss of topoisomerase
II poisoning. Furthermore, while compound 4h presented the
same cytotoxicity, 4i was four times less active. In fact, the degree
of topoisomerase II inhibition does not always correlate with
cytotoxicity. To examine this point, we investigated the ability
of 4e, 4f, 4g and 4h, to inhibit tubulin polymerization, as the
precursor (podophyllotoxin) is a potent antimicrotubule agent.
We observed that only compound 4g presented a weak inhibition
activity of tubulin polymerization/depolymerization (40% at
20 lM, compared to an IC50 = 3 lM for podophyllotoxin). On
the contrary, compounds 4e, 4f and 4h have no effect on tubulin
polymerisation. Furthermore, the weak inhibition of topoiso-
merase II observed with these latter compounds indicates that,
in these cases, this enzyme is probably not the only cellular target.
The perturbation of the cell cycle induced by these compounds
was also studied on the L1210 cell line. All the tested compounds
induced a marked accumulation (>65%) of cells in the G2 + M
phases at a concentration between 0.25 and 5 lM.
Scheme 1 Synthesis of the 4-b-N-carbamates of 4ꢀ-demethylepipodo-
phyllotoxin. Reagents: (i) phosgene, dry CH2Cl2; (ii) ROH, dry CH2Cl2;
(iii) DowexꢀR 50 × 2–200, methanol.
4ꢀ-tert-butyldimethylsilyloxy-4b-amino-4ꢀ-O-demethyl-4-desoxy-
podophyllotoxin (1), required as starting material, was
prepared as previously described.14 The 4-b-isocyanate-4ꢀ-tert-
butyldimethylsilyloxy-4ꢀ -O-demethyl-4-desoxypodophyllotoxin
(2) was prepared by reacting
1 with phosgene in dry
dichloromethane at room temperature. The preparation of
4-b-(alkyloxycarbonyl)amino-4ꢀ-tert-butyldimethylsilyloxy-4ꢀ-O-
demethyl-4-desoxypodophyllotoxins 3a–i, and 4-b-(aryloxy-
carbonyl)amino-4ꢀ-tert-butyldimethylsilyloxy-4ꢀ-O-demethyl-4-
desoxypodophyllotoxins 3j–m, was achieved in situ by adding
the appropriate alcohol to the reaction mixture of the previous
step. It must be noted that, during the course of this reaction,
an important desilylation occurred and only a small amount of
3 was isolated, along with the desired deprotected compound
4. The subsequent complete regenerations of the 4ꢀ-hydroxyl
group from compounds 3a–m were performed in methanol
R
using a DOWEXꢀ 50 × 2–200 ion exchange resin to provide the
desired phenolic compounds 4a–m in good to excellent yields.
Biological evaluation and DNA topoisomerase II inhibition
We investigated drug–DNA interactions by perturbation of
ethidium bromide–DNA complexes in agarose gel at two
concentrations (50 and 100 lM) of carbamate derivatives 4a–m
(data not shown). While under the same conditions, the known
intercalating drug daunorubicin shifts the ethidium bromide
complexed in supercoiled DNA; the carbamate derivatives had
no effect. This result argues for a non-intercalative binding mode
such as already known for etoposide.17,18,19
In order to further investigate structure–activity relationships,
we also prepared aromatic carbamates. We observed that a ben-
zyl group (4j), a 4-fluorobenzyl group (4k) or a 4-fluorophenethyl
group (4l) maintained good activity (Table 1). In particular,
Biological results for the novel 4-b-N-carbamate derivatives
of 4ꢀ-demethylpodophyllotoxin are shown in Table 1. All com-
pounds were tested for their ability to poison topoisomerases I
Fig. 2 Stimulation of the topoisomerase II-mediated DNA double stranded cleavage. Native supercoiled pAT (lane DNA) was incubated for 30 min
at 30 ◦C with 6 units of topoisomerase II in the absence (lane TopoII) or in the presence of increasing concentrations of the drugs at the indicated
concentrations. Reactions were stopped with SDS and treatment with proteinase K. Samples were analyzed by native agarose gel electrophoresis 1%
in Tris–borate–EDTA buffer (TBE 1×) containing ethidium bromide (1 lg mL−1) at room temperature. Gels were washed and photographed under
UV light. The positions of supercoiled (form I), open-circular (form II), linear (form III) and relaxed DNA species are indicated. Etoposide was used
at 5 and 20 lM; 4a–c at 1, 5, 10 and 20 lM.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 7 4 – 1 0 8 0
1 0 7 5

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Table 1 Biological activities
Cytotoxicity
Compound
R
Inhibition of topoisomerase IIa (% linear DNA)
IC50/lMb
Cell cycle effect c
VP-16
4a
—
CH3
CH2CH3
(CH2)3CH3
(CH2)3Cl
50
50
44
40
31
25
29
42
10
0.83
0.18
0.14
0.23
0.15
0.11
0.09
0.14
0.14
76% (2.5 lM)
74% (0.5 lM)
71% (1 lM)
72% (2 lM)
70% (1 lM)
72% (1 lM)
69% (0.25 lM)
N.T.
4b
4c
4d
=
4e
CH2CH CH2
4f
CH2C≡CH
4g
(CH2)2OCH3
4h
N.T.
4i
15
0.44
N.T.
4j
50
45
0.27
0.36
73% (2 lM)
71% (2 lM)
4k
4l
37
4
0.39
0.57
70% (5 lM)
78% (5 lM)
4m
a Each value reported here is a mean value of at least 3 independent experiments at 20 lM of drug. b IC50: concentration of drug required to reduce
L1210 cell growth to 50%. c % of L1210 cells in the G2M phase at the specified drug concentration. N.T. = not tested.
compound 4j was comparable to etoposide for topoisomerase
II inhibition activity. The fluoro atom in the para position (4k)
does not markedly influence the effect on the enzyme. As in the
case of aliphatic chains, the increase in the length of the side
chain caused a decrease of topoisomerase II inhibition activity
(4k vs. 4l), which is completely lost by the introduction of 2,4-
dichlorobenzyl group (4m). All of these compounds displayed a
two-fold lower cytotoxicity than that observed in the aliphatic
series, except for compound 4m, which is four-fold less efficient.
This latter is in correlation with the loss of topoisomerase II
poisoning activity.
starting material to prepare this series of 4-b-N-carbamates
showed no inhibition of topoisomerase II. This lack of ac-
tivity can be compared to that of 4ꢀ-DMEP13 which is, in
contrast, a poison of topoisomerase II. This is in agree-
ment with the observation that the 4-N-carbamates of 4ꢀ-
demethylepipodophyllotoxin here studied showed lower an-
titopoisomerase II activity than the previously studied 4-O-
carbamates.16 Whether there is a structure–activity relationship
concerning 4-b-N and 4-b-O groups is currently under investiga-
tion in our laboratories. Concerning the side chain, methyl and
benzyl groups gave the best results, strongly suggesting that,
in this series, shorter side chains increased the topoisomerase
II inhibition. As in our previously published works,14,16 we
have underlined that the spatial organization is fundamental
for topoisomerase inhibition and even small changes can
compromise this activity.
In agreement with the lower cytotoxicity, the perturbation of
the cell cycle is induced for these aromatic derivatives at higher
concentrations.
Conclusion
All of these results confirm that the replacement of the glycoside
moiety of etoposide with a 4-b-N-carbamate residue is favorable
to topoisomerase II poisoning. As previously reported,14 the
4-b-amino-4ꢀ-O-demethyl-4-desoxypodophyllotoxin used as the
Materials and methods
Solvents and most of the starting materials were purchased from
Acros, Aldrich or Avocado. Melting points were measured on a
1 0 7 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 7 4 – 1 0 8 0

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Ko¨fler hot stage apparatus and are uncorrected. Mass spectra
were obtained with a Nermag–Ribermag R10–10C spectrometer
applying either desorption chemical ionization (CI, operating in
the positive ion mode using ammonia as the reagent gas) or
fast atom bombardment (FAB). Infrared spectra were obtained
with a Perkin-Elmer 1710 spectrophotometer using chloroform
solvent. Specific rotations were measured with a Perkin Elmer
241 polarimeter. The 1H NMR (300 MHz) spectra were recorded
on a Bruker AC 300 spectrometer. Chemical shifts are expressed
as parts per million from tetramethylsilane. Splitting patterns
have been designated as follows: s (singlet), d (doublet), dd
(doublet of doublet), t (triplet), m (multiplet), and br (broad
signal). Coupling constants (J values) are listed in hertz (Hz).
Reactions were monitored by analytical thin-layer chromatog-
raphy and products were visualized by exposure to UV light.
Merck silica gel (230–400 mesh ASTM) was used for column
chromatography. Acetone, methanol, and dichloromethane,
employed as eluents for column chromatography, were distilled
on a rotary evaporator prior to use. All yields reported are
unoptimized. Elemental analysis for most of the new substances
was performed by CNRS Laboratories (Vernaison, France), and
unless noted otherwise, the results obtained are within 0.4% of
the theoretical values.
6.80 (1H, s, 5-H); 6.52 (1H, s, 8-H); 6.23 (2H, s, 2ꢀ,6ꢀ-H); 5.97
(2H, d, J = 4.3 Hz, CH2O2); 4.98–4.09 (1H, m, NH); 4.83–4.78
(1H, m, 4-H); 4.56 (1H, d, J = 4.5 Hz, 1-H); 4.42 (1H, t, J =
7.3 Hz, 11a-H); 4.32 (2H, t, J = 6.2 Hz, CH2a); 3.94 (1H, t,
J = 9.0 Hz, 11b-H); 3.67 (6H, s, 3ꢀ,5ꢀ-OCH3); 3.61 (2H, t, J =
6.5 Hz, CH2c); 2.98–2.89 (2H, m, 2,3-H); 2.12–2.00 (2H, m,
CH2b); 1.00 (9H, s, tBu); 0.10 (6H, s, 2Me); MS (CI) m/z 651
[M + NH4]+.
4-b-(Allyloxycarbonyl)amino-4ꢀ-tert-butyldimethylsilyloxy-4ꢀ-
O-demethyl-4-desoxypodophyllotoxin (3e). Yield 80%; Rf
=
0.55 (CH2Cl2 : acetone, 97 : 3); 1H-NMR (CDCl3) d: 6.81 (1H, s,
5-H); 6.51 (1H, s, 8-H); 6.20 (2H, s, 2ꢀ,6ꢀ-H); 5.99 (2H, d, J =
=
5.1 Hz, CH2O2); 5.95–5.86 (1H, m, CH2CH CH2); 5.34 (2H,
d, J = 7.0 Hz, CH2CH CH2); 5.02–4.94 (1H, m, 4-H); 4.96
=
(1H, d, J = 4.1 Hz, NH); 4.62–4.50 (3H, m, CH2a, 1-H); 4.40
(1H, t, J = 8.1 Hz, 11a-H); 3.94 (1H, t, J = 9.6 Hz, 11b-H);
3.67 (6H, s, 3ꢀ,5ꢀ-OCH3); 2.94–2.86 (2H, m, 2,3-H); 1.00 (9H, s,
tBu); 0.10 (6H, s, 2Me); MS (CI) m/z 615 [M + NH4]+.
4-b-(Propargyloxycarbonyl)amino-4ꢀ-tert-butyldimethylsilyloxy-
4ꢀ-O-demethyl-4-desoxypodophyllotoxin (3f). Yield 67%;Rf =
0.38 (CH2Cl2 : acetone, 97 : 3); 1H-NMR (CDCl3) d: 6.80 (1H, s,
5-H); 6.52 (1H, s, 8-H); 6.23 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J =
5.4 Hz, CH2O2); 5.04–4.92 (2H, m, 4-H, NH); 4.74–4.62 (3H,
m, CH2C≡CH); 4.56 (1H, d, J = 4.0 Hz, 1-H); 4.41 (1H, t,
J = 7.9 Hz, 11a-H); 3.94 (1H, t, J = 9.5 Hz, 11b-H); 3.67 (6H, s,
3ꢀ,5ꢀ-OCH3); 2.98–2.85 (2H, m, 2,3-H); 1.00 (9H, s, tBu); 0.10
(6H, s, Me); MS (CI) m/z 596 [M + H]+, 613 [M + NH4]+.
4-b-(2-Methoxyethoxycarbonyl)amino-4ꢀ-tert-butyldimethyl-
silyloxy-4ꢀ-O-demethyl-4-desoxypodophyllotoxin (3g). Yield
95%; Rf = 0.46 (CH2Cl2 : acetone, 80 : 20); 1H-NMR (CDCl3)
d: 6.80 (1H, s, 5-H); 6.51 (1H, s, 8-H); 6.23 (2H, s, 2ꢀ,6ꢀ-H);
5.97 (2H, d, J = 3.3 Hz, CH2O2); 4.98–4.90 (2H, m, NH, 4-H);
4.57 (1H, d, J = 3.8 Hz, 1-H); 4.42 (1H, t, J = 7.2 Hz, 11a-H);
4.30–4.18 (2H, m, CH2a); 3.98 (1H, t, J = 9.4 Hz, 11b-H);
3.67 (6H, s, 3ꢀ,5ꢀ-OCH3); 3.60–3.52 (2H, m, CH2b); 3.39 (3H, s,
OCH3); 2.97–2.84 (2H, m, 2,3-H); 1.00 (9H, s, tBu); 0.10 (6H, s,
Me); MS (CI) m/z 616 [M + H]+, 633 [M + NH4]+.
4-b-[(Tetrahydropyran-2-yl)methoxycarbonyl]amino-4ꢀ-tert-
butyldimethylsilyloxy-4ꢀ -O-demethyl-4-desoxypodophyllotoxin
(3h). Yield 96%; Rf = 0.34 (CH2Cl2 : acetone, 97 : 3); 1H-NMR
(CDCl3) d: 6.79 (1H, s, 5-H); 6.50 (1H, s, 8-H); 6.22 (2H, s,
2ꢀ,6ꢀ-H); 5.97 (2H, d, J = 4.8 Hz, CH2O2); 5.02–4.85 (2H, m,
NH, 4-H); 4.56 (1H, d, J = 3.7 Hz, 1-H); 4.39 (1H, t, J =
7.5 Hz, 11a-H); 4.08–3.90 (4H, m, CH2a, CHO, 11b-H); 3.67
(6H, s, 3ꢀ,5ꢀ-OCH3); 3.55–3.40 (2H, m, CH2O); 3.00–2.85 (2H,
m, 2,3-H); 1.72–1.45 (6H, m, CH2); 1.00 (9H, s, tBu); 0.10
(6H, s, Me); MS (CI) m/z 656 [M + H]+, 673 [M + NH4]+.
4-b-[(2,5-Dioxopyrrolidin-1-yl)ethoxycarbonyl]amino-4ꢀ-tert-
butyldimethylsilyloxy-4ꢀ -O-demethyl-4-desoxypodophyllotoxin
(3i). Yield 65%; Rf = 0.45 (CH2Cl2 : acetone, 80 : 20);
1H-NMR (CDCl3) d: 6.88 (1H, s, 5-H); 6.51 (1H, s, 8-H); 6.23
(2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J = 3.8 Hz, CH2O2); 4.98–4.90
(1H, m, NH); 4.89–4.82 (1H, m, 4-H); 4.55 (1H, d, J = 3.93 Hz,
1-H); 4.23 (1H, t, J = 5.0 Hz, 11a-H); 4.30–4.20 (2H, m, CH2b);
4.04 (1H, t, J = 9.5 Hz, 11b-H); 3.85–3.78 (2H, m, CH2a); 3.67
(6H, s, 3ꢀ,5ꢀ-OCH3); 2.98–2.85 (2H, m, 2,3-H); 2.73 (4H, t, J =
8.5 Hz, 2CH2); 1.00 (9H, s, tBu); 0.10 (6H, s, Me); MS (CI) m/z
683 [M + H]+, 700 [M + NH4]+.
Synthesis of 4-b-isocyanate-4ꢀ-tert-butyldimethylsilyloxy-4ꢀ-O-
demethyl-4-desoxypodophyllotoxin (2)
To a solution of 4-b-amino-4ꢀ-tert-butyldimethylsilyloxy-4ꢀ-O-
demethyl-4-desoxypodophyllotoxin (1) (108 mg, 0.2 mmol)
in dry dichloromethane (10 mL) were added a solution of
phosgene in toluene 1.93 N (1.2 mL) and triethylamine (30 lL).
The mixture was stirred overnight at room temperature. After
concentration under reduced pressure, the product was purified
by flash chromatography (CH2Cl2 : acetone, 97 : 3) leading to 2.
Yield 90%; Rf 0.8 (CH2Cl2 ; acetone, 97 : 3); 1H-NMR (CDCl3):
d (ppm) 6.83 (1H, s, 5-H); 6.54 (1H, s, 8-H); 6.21 (2H, s, 2ꢀ,6ꢀ-H);
6.00 (2H, d, J = 5.8 Hz, CH2O2); 4.93 (1H, d, J = 3.9 Hz, 4-H);
4.61 (1H, d, J = 5.3 Hz, 1-H); 4.46 (1H, t, J = 8.2 Hz, 11a-H);
4.16 (1H, t, J = 7.1 Hz, 11b-H); 3.66 (6H, s, 3ꢀ,5ꢀ-OCH3); 3.17–
3.05 (2H, m, 2,3-H); 0.97 (9H, s, tBu); 0.1 (6H, s, Me2); MS (CI)
m/z 540 [M + H]+, 557 [M + NH4]+.
General procedure for the synthesis of compounds 3a–m
1.5 eq. of the appropriate alcohol were added (0.3 mmol) to
the reaction mixture obtained from the previous step. The
reaction mixture was further stirred at room temperature for
the reported times (monitored by TLC).
Evaporation of the solvent in vacuo afforded a crude material
chromatographed on silica gel (100 g, eluent: various mixtures
CH2Cl2 : acetone, see Rf ) to provide satisfactory pure com-
pounds with the reported yields. In the case of 3a, 3b, 3j and 3m
the products were simultaneously deprotected after reaction into
4a, 4b, 4j and 4m respectively. Chromatographed compounds 3c–
i and 3k–l were directly employed in the subsequent deprotection
step without further purification.
4-b-(Butyloxycarbonyl)amino-4ꢀ-tert-butyldimethylsilyloxy-4ꢀ-
O-demethyl-4-desoxypodophyllotoxin (3c). Yield 71%; Rf
=
0.6 (CH2Cl2 : acetone, 97 : 3); 1H-NMR (CDCl3) d: 6.81 (1H, s,
5-H); 6.51 (1H, s, 8-H); 6.23 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J =
4.6 Hz, CH2O2); 5.02–4.93 (1H, m, NH); 4.84–4.78 (1H, m,
4-H); 4.42 (1H, t, J = 4.6 Hz, 11a-H); 4.56 (1H, d, J = 3.8 Hz,
1-H); 4.18–4.08 (2H, m, CH2a); 3.98 (1H, t, J = 7.6 Hz, 11b-H);
3.71 (3H, s, OCH3); 3.67 (6H, s, 3ꢀ,5ꢀ-OCH3); 2.94–2.86 (2H,
m, 2,3-H); 1.70–1.58 (2H, m, CH2b); 1.42–1.32 (2H, m, CH2c);
1.00 (9H, s, tBu); 0.98 (3H, t, J = 7.2 Hz, CH3); 0.10 (6H, s,
2Me); MS (CI) m/z 631 [M + NH4]+.
4-b-(4-Fluorobenzyloxycarbonyl)amino-4ꢀ-tert-butyldimethyl-
silyloxy-4ꢀ-O-demethyl-4-desoxypodophyllotoxin (3k). Yield
1
98%; Rf = 0.42 (CH2Cl2 : acetone, 97 : 3); H-NMR (CDCl3)
d: 7.38–7.30 (2H, m, Ph); 7.08–6.98 (2H, m, Ph); 6.79 (1H, s,
5-H); 6.51 (1H, s, 8-H); 6.22 (2H, s, 2ꢀ,6ꢀ-H); 5.96 (2H, d, J =
5.3 Hz, CH2O2); 5.11 (2H, br s, CH2Ph); 5.28–5.10 (1H, m,
NH); 5.18–5.12 (1H, m, 4-H); 4.57 (1H, d, J = 4.8 Hz, 1-H);
4-b-(3-Chloropropyloxycarbonyl)amino-4ꢀ-tert-butyldimethyl-
silyloxy-4ꢀ-O-demethyl-4-desoxypodophyllotoxin (3d). Yield
84%; Rf = 0.7 (CH2Cl2 : acetone, 97 : 3); 1H-NMR (CDCl3) d:
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 7 4 – 1 0 8 0
1 0 7 7

View Article Online
4.42 (1H, t, J = 7.8 Hz, 11a-H); 3.98 (2H, t, J = 9.8 Hz, 11b-H);
3.67 (6H, s, 3ꢀ,5ꢀ-OCH3); 2.98–2.85 (2H, m, 2,3-H); 1.00 (9H, s,
tBu); 0.10 (6H, s, Me); MS (CI) m/z 666 [M + H]+, 683 [M +
NH4]+.
(1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J = 5.7 Hz,
CH2O2); 5.41 (1H, br s, 4ꢀ-OH); 5.01–4.85 (1H, m, 4-H); 4.86
(1H, br d, J = 7.0 Hz, 4-NH); 4.57 (1H, d, J = 4.2 Hz, 1-H);
4.42 (1H, t, J = 7.7 Hz, 11a-H); 4.27 (2H, t, J = 6.0 Hz, CH2a);
3.94 (1H, t, J = 9.5 Hz, 11b-H); 3.78 (6H, s, 3ꢀ,5ꢀ-OCH3); 3.61
(2H, t, J = 6.3 Hz, CH2a); 3.00–2.87 (2H, m, 2,3-H); 2.16–2.04
(2H, m, CH2b); IR (CHCl3): m 3366 (NH), 3020 (aliphatic
4-b-(4-Fluorophenethyloxycarbonyl)amino-4ꢀ -tert-butyldi-
methylsilyloxy-4ꢀ -O-demethyl-4-desoxypodophyllotoxin (3l).
Yield 92%; Rf = 0.45 (CH2Cl2 : acetone, 97 : 3); 1H-NMR
(CDCl3) d: 7.20–7.12 (2H, m, Ph); 7.08–6.98 (2H, m, Ph); 6.76
(1H, s, 5-H); 6.51 (1H, s, 8-H); 6.22 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H,
d, J = 4.4 Hz, CH2O2); 5.00–4.90 (1H, m, NH); 4.82–4.77
(1H, m, 4-H); 4.56 (1H, d, J = 4.5 Hz, 1-H); 4.31 (1H, t, J =
6.4 Hz, 11a-H); 3.09–3.80 (3H, m, 11b-H, CH2a); 3.67 (6H, s,
3ꢀ,5ꢀ-OCH3); 2.98–2.80 (4H, m, 2,3-H, CH2b); 1.00 (9H, s, tBu);
0.10 (6H, s, Me); MS (CI) m/z 680 [M + H]+, 697 [M + NH4]+.
=
=
C–H), 1777 (C O lactone), 1587, 1507, 1485 (aromatic C C);
MS (CI) m/z 537 [M + NH4]+.
4-b-(Allyloxycarbonyl)amino-4ꢀ -O-demethyl-4-desoxypodo-
phyllotoxin (4e). Yield 62%; Rf = 0.60 (CH2Cl2 : acetone, 90 :
◦
10); mp = 130–132 C (white crystals); [a]D20 = −113.5 (c =
1
0.620, CHCl3); H-NMR (CDCl3) d: 6.82 (1H, s, 5-H); 6.50
(1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J = 6.2 Hz,
=
CH2O2); 5.93–5.86 (1H, m, CH2CH CH2); 5.41 (1H, br s,
4 -OH); 5.28 (2H, d, J = 8.0 Hz, CH2CH CH2); 5.15–4.98 (1H,
ꢀ
=
General procedure for the synthesis of compounds 4a–m
m, 4-H); 4.91 (1H, br d, J = 7.1 Hz, 4-NH); 4.68–4.54 (3H, m,
Dowex 50 × 2–200 ion-exchange resin (3 g), previously washed
with water, then MeOH, was added to a solution of 3a–m
(0.6 mmol) in MeOH (65 mL). The mixture was vigorously
stirred for 16 h at room temperature. The resin was removed by
filtration and thoroughly washed with MeOH. Evaporation of
the filtrate in vacuo gave the desired compound in the reported
yields. This was also performed on compounds 3a, 3b, 3j and
3m, which have not been isolated in order to deprotect them
completely.
=
1-H, CH2CH CH2); 4.42 (1H, t, J = 7.0 Hz, 11a-H); 3.95 (1H,
t, J = 9.8 Hz, 11b-H); 3.77 (6H, s, 3ꢀ,5ꢀ-OCH3); 2.95–2.87 (2H,
m, 2,3-H); IR (CHCl3): m 3358 (NH), 3020 (aliphatic C–H),
=
=
1773 (C O lactone), 1559, 1508, 1458 (aromatic C C); MS
(CI) m/z 501 [M + NH4]+.
4-b-(Propargyloxycarbonyl)amino-4ꢀ -O-demethyl-4-desoxy-
podophyllotoxin (4f). Yield 50%; Rf = 0.55 (CH2Cl2 : acetone,
90 : 10); mp = 140–142 ◦C (white crystals); [a]2D0 = −109.4 (c =
1
0.575, CHCl3); H-NMR (CDCl3) d: 6.81 (1H, s, 5-H); 6.50
4-b-(Methyloxycarbonyl)amino-4ꢀ-O-demethyl-4-desoxypodo-
phyllotoxin (4a). Yield 80%; Rf = 0.45 (CH2Cl2 : acetone, 90 :
(1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J = 4.5 Hz,
CH2O2); 5.41 (1H, s, 4ꢀ-OH); 5.02–4.95 (2H, m, NH, 4-H); 4.57
(1H, d, J = 4.2 Hz, 1-H); 4.41 (1H, t, J = 7.7 Hz, 11a-H);
4.35–4.20 (3H, m, CH2C≡CH); 3.96 (1H, t, J = 9.5 Hz, 11b-H);
3.77 (6H, s, 3ꢀ,5ꢀ-OCH3); 2.98–2.85 (2H, m, 2,3-H); IR (CHCl3):
◦
10); mp = 150–152 C (white crystals); [a]D20 = −109.2 (c =
1
0.610, CHCl3); H-NMR (CDCl3) d: 6.81 (1H, s, 5-H); 6.49
(1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.96 (2H, d, J = 7.6 Hz,
CH2O2); 5.41 (1H, br s, 4ꢀ-OH); 4.97 (1H, br d, J = 3.2 Hz,
4-NH); 4.95–4.86 (1H, m, 4-H); 4.55 (1H, d, J = 4.1 Hz, 1-H);
4.41 (1H, t, J = 7.8 Hz, 11a-H); 3.95 (1H, t, J = 9.6 Hz, 11b-H);
3.77 (6H, s, 3ꢀ,5ꢀ-OCH3); 3.71 (3H, s, OCH3); 2.94–2.82 (2H, m,
2,3-H); IR (CHCl3): m 3366 (NH), 3020 (aliphatic C–H), 1777
=
m 3358 (NH), 3020 (aliphatic C–H), 1773 (C O lactone), 1559,
+
=
1508, 1458 (aromatic C C); MS (CI) m/z 482 [M + H] , 499
[M + NH4]+.
4-b-(2-Methoxyethoxycarbonyl)amino-4ꢀ-O-demethyl-4-desoxy-
=
=
(C O lactone), 1587, 1507, 1485 (aromatic C C); MS (CI)
podophyllotoxin (4g). Yield 77%; Rf = 0.62 (CH2Cl2 : acetone,
m/z 475 [M + NH4]+.
◦
90 : 10); mp = 136–138 C (white crystals); [a]D20 = −104.8
1
4-b-(Ethyloxycarbonyl)amino-4ꢀ -O-demethyl-4-desoxypodo-
phyllotoxin (4b). Yield 99%; Rf = 0.57 (CH2Cl2 : acetone, 90 :
(c = 0.425, CHCl3); H-NMR (CDCl3) d: 6.81 (1H, s, 5-H);
6.51 (1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J = 6.1 Hz,
CH2O2); 5.42 (1H, br s, 4ꢀ-OH); 5.15–4.92 (2H, m, NH, 4-H);
4.57 (1H, d, J = 4.2 Hz, 1-H); 4.42 (1H, t, J = 7.9 Hz, 11a-H);
4.45–4.29 (2H, m, CH2a); 3.96 (1H, t, J = 9.6 Hz, 11b-H);
3.77 (6H, s, 3ꢀ,5ꢀ-OCH3); 3.65–3.58 (2H, m, CH2b); 3.39 (3H, s,
OCH3); 2.97–2.84 (2H, m, 2,3-H); IR (CHCl3): m 3358 (NH),
◦
10); mp = 146–148 C (white crystals); [a]D20 = −104.2 (c =
1
0.515, CHCl3); H-NMR (CDCl3) d: 6.82 (1H, s, 5-H); 6.50
(1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J = 6.2 Hz,
CH2O2); 5.41 (1H, br s, 4ꢀ-OH); 4.97 (1H, br d, J = 3.1 Hz,
4-NH); 4.90–4.80 (1H, m, 4-H); 4.57 (1H, d, J = 3.9 Hz, 1-H);
4.42 (1H, t, J = 7.4 Hz, 11a-H); 4.21–4.09 (2H, m, CH2); 3.95
(1H, t, J = 9.2 Hz, 11b-H); 3.77 (6H, s, 3ꢀ,5ꢀ-OCH3); 2.97–2.85
(2H, m, 2,3-H); 1.26 (3H, t, J = 7.0 Hz, CH3); IR (CHCl3): m
=
3020 (aliphatic C–H), 1773 (C O lactone), 1559, 1508, 1458
+
+
=
(aromatic C C); MS (CI) m/z 502 [M + H] , 519 [M + NH4] .
4-b-[(Tetrahydropyran-2-yl)methoxycarbonyl]amino-4ꢀ -O-
demethyl-4-desoxypodophyllotoxin (4h). Yield 55%; Rf = 0.45
(CH2Cl2 : acetone, 90 : 10); mp = 139–141 ◦C (white crystals);
=
3324 (NH), 3020 (aliphatic C–H), 1776 (C O lactone), 1604,
+
=
1485 (aromatic C C); MS (CI) m/z 489 [M + NH4] .
1
[a]D20 = −98.1 (c = 0.615, CHCl3); H-NMR (CDCl3) d: 6.80
4-b-(Butyloxycarbonyl)amino-4ꢀ -O-demethyl-4-desoxypodo-
phyllotoxin (4c). Yield 76%; Rf = 0.48 (CH2Cl2 : acetone, 90 :
10); mp = 126–128 ◦C (white crystals); [a]2D0 = −99.5 (c = 0.485,
(1H, s, 5-H); 6.49 (1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H,
d, J = 4.2 Hz, CH2O2); 5.42 (1H, br s, 4ꢀ-OH); 4.98–4.88 (2H,
m, NH, 4-H); 4.57 (1H, d, J = 4.0 Hz, 1-H); 4.41 (1H, t, J =
7.2 Hz, 11a-H); 4.12–3.95 (2H, m, CH2a, CHO, 11b-H); 3.77
(6H, s, 3ꢀ,5ꢀ-OCH3); 3.50–3.35 (2H, m, CH2O); 3.00–2.85 (2H,
m, 2,3-H); 1.72–1.50 (6H, m, CH2); IR (CHCl3): m 3358 (NH),
1
CHCl3); H-NMR (CDCl3) d: 6.82 (1H, s, 5-H); 6.50 (1H, s,
8-H); 6.29 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J = 5.7 Hz, CH2O2);
5.41 (1H, br s, 4ꢀ-OH); 4.82 (1H, br d, J = 6.9 Hz, 4-NH);
4.90–4.80 (1H, m, 4-H); 4.55 (1H, d, J = 4.1 Hz, 1-H); 4.41 (1H,
t, J = 7.8 Hz, 11a-H); 4.20–4.10 (2H, m, CH2a); 3.95 (1H, t,
J = 9.6 Hz, 11b-H); 3.77 (6H, s, 3ꢀ,5ꢀ-OCH3); 3.71 (3H, s, OCH3);
2.94–2.82 (2H, m, 2,3-H); 1.65–1.55 (2H, m, CH2b); 1.40–1.30
(2H, m, CH2c). IR (CHCl3): m 3367 (NH), 3038 (aliphatic C–H),
=
3020 (aliphatic C–H), 1773 (C O lactone), 1559, 1508, 1458
+
+
=
(aromatic C C); MS (CI) m/z 542 [M + H] , 559 [M + NH4] .
4-b-[(2,5-Dioxopyrrolidin-1-yl)ethoxycarbonyl]amino-4ꢀ -O-
demethyl-4-desoxypodophyllotoxin (4i). Yield 57%; Rf = 0.54
(CH2Cl2 : acetone, 90 : 10); mp = 145–147 ◦C (white crystals);
=
=
1776 (C O lactone), 1622, 1505, 1485 (aromatic C C); MS
(CI) m/z 517 [M + NH4]+.
1
[a]D20 = −80.3 (c = 0.590, CHCl3); H-NMR (CDCl3) d: 6.89
4-b-(3-Chlororopyloxycarbonyl)amino-4ꢀ-O-demethyl-4-desoxy-
podophyllotoxin (4d). Yield 58%; Rf = 0.35 (CH2Cl2 : acetone,
90 : 10); mp = 132–134 ◦C (white crystals); [a]2D0 = −102.6 (c =
(1H, s, 5-H); 6.50 (1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H,
d, J = 5.1 Hz, CH2O2); 5.42 (1H, br s, 4ꢀ-OH); 4.98–4.84 (2H,
m, NH, 4-H); 4.56 (1H, d, J = 4.8 Hz, 1-H); 4.37 (1H, t,
J = 6.3 Hz, 11a-H); 4.28–4.17 (2H, m, CH2b); 4.06 (1H, t,
1
0.425, CHCl3); H-NMR (CDCl3) d: 6.82 (1H, s, 5-H); 6.51
1 0 7 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 7 4 – 1 0 8 0

View Article Online
J = 9.1 Hz, 11b-H); 3.92–3.88 (2H, m, CH2a); 3.77 (6H, s,
Topoisomerase I-mediated DNA cleavage assay
3ꢀ,5ꢀ-OCH3); 2.93–2.82 (2H, m, 2,3-H); 2.75–2.68 (4H, m, CH2);
Each reaction mixture (20 ll total volume) contained 0.1 lg of
pBS supercoiled DNA, the reaction buffer consisting of 35 mM
Tris-HCl, pH 8, 72 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol,
5 mM spermidine and 0.01% BSA. Drugs were added at different
concentrations (from 1 to 20 lM). The reaction was initiated by
the addition 2 units topoisomerase I and allowed to proceed at
30 ◦C for 20 min.
The reaction was stopped by adding 10 × loading buffer
(0.25% bromophenol blue, 50% glycerol) and 10% sarkosyl.
Samples were electrophoresed for 3 h at 100 V in 1% agarose
gel in Tris–borate–EDTA buffer (TBE). The gel was stained with
SYBR Gold 1X (Molecular Probes, USA) in TBE 1x buffer for
20 min and photographed under UV light.
=
IR (CHCl3): m 3358 (NH), 3020 (aliphatic C–H), 1773 (C O
=
lactone), 1559, 1508, 1458 (aromatic C C); MS (CI) m/z 569
[M + H]+, 586 [M + NH4]+.
4-b-(Benzyloxycarbonyl)amino-4ꢀ-O-demethyl-4-desoxypodo-
phyllotoxin (4j). Yield 75%; Rf = 0.48 (CH2Cl2 : acetone, 90 :
10); mp = 128–130 ◦C (white crystals); [a]2D0 = −91.6 (c = 0.470,
1
CHCl3); H-NMR (CDCl3) d: 7.36 (5H, br s, Ph); 6.81 (1H, s,
5-H); 6.50 (1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J =
6.3 Hz, CH2O2); 5.41 (1H, br s, 4ꢀ-OH); 5.15 (2H, s, CH2Ph);
5.05–4.98 (1H, br s, NH); 4.95–4.88 (1H, m, 4-H); 4.56 (1H, d,
J = 4.5 Hz, 1-H); 4.42 (1H, t, J = 7.0 Hz, 11a-H); 3.98 (2H, t,
J = 9.0 Hz, 11b-H); 3.77 (6H, s, 3ꢀ,5ꢀ-OCH3); 2.90–2.82 (2H, m,
2,3-H); IR (CHCl3): m 3358 (NH), 3020 (aliphatic C–H), 1773
Topoisomerase II-mediated DNA cleavage assay
=
=
(C O lactone), 1559, 1508, 1458 (aromatic C C); MS (CI)
m/z 534 [M + H]+, 551 [M + NH4]+.
Supercoiled pBS DNA (0.1 lg) was incubated for 15 min at
◦
30 C, in a 50 mM Tris–HCl buffer, pH 7.5, containing 1 mM
4-b-(4-Fluorobenzyloxycarbonyl)amino-4ꢀ-O-demethyl-4-des-
ATP, 120 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM
EDTA and 30 lg BSA, in the presence of the drug at the desired
concentration (5, 10, 20 or 50 lM, total reaction volume 10 ll).
6 units of human DNA topoisomerase IIa were added to the
duplex, preincubated as described, and incubated for 30 min
at 30 ◦C. The DNA-topoisomerase II cleavage complexes were
dissociated by addition of SDS (GibcoBRL), final concentration
0.5%, and of proteinase K (Sigma) to 500 lg mL−1, followed by
incubation for 30 min at 50 ◦C. DNA samples were then added
to the electrophoresis dye mixture (5 lL) and electrophoresed
(35 V cm−1) in a 1% agarose gel in TBE 1×, containing ethidium
bromide (1 lg mL−1), at room temperature for 2 h. Gels were
washed and photographed under UV light.
oxypodophyllotoxin (4k). Yield 50%; Rf = 0.60 (CH2Cl2
:
=
◦
acetone, 90 : 10); mp = 140–142 C (white crystals); [a]2D0
−88.8 (c = 0.525, CHCl3); 1H-NMR (CDCl3) d: 7.38–7.30 (2H,
m, Ph); 6.80 (1H, s, 5-H); 6.49 (1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H);
5.97 (2H, d, J = 7.7 Hz, CH2O2); 5.41 (1H, br s, 4ꢀ-OH); 5.10
(2H, br s, CH2Ph); 5.04–4.98 (1H, m, NH); 4.95–4.89 (1H,
m, 4-H); 4.56 (1H, d, J = 4.4 Hz, 1-H); 4.41 (1H, t, J =
7.7 Hz, 11a-H); 4.39 (2H, t, J = 9.6 Hz, 11b-H); 3.77 (6H, s,
3ꢀ,5ꢀ-OCH3); 2.98–2.85 (2H, m, 2,3-H); IR (CHCl3): m 3358
=
(NH), 3020 (aliphatic C–H), 1773 (C O lactone), 1559, 1508,
+
=
1458 (aromatic C C); MS (CI) m/z 552 [M + H] , 570 [M +
NH4]+.
4-b-(4-Fluorophenethyloxycarbonyl)amino-4ꢀ -O-demethyl-4-
Cell culture and cytotoxicity assays and cell cycle analysis
Assays were performed as previously described.20,21
desoxypodophyllotoxin (4l). Yield 75%; Rf = 0.58 (CH2Cl2 :
◦
acetone, 80 : 20); mp = 127–129 C (white crystals); [a]2D0
=
1
−94.2 (c = 0.600, CHCl3); H-NMR (CDCl3) d: 7.17 (2H, t,
J = 6.7 Hz, Ph); 7.00 (2H, t, J = 8.6 Hz, Ph); 6.77 (1H, s,
5-H); 6.50 (1H, s, 8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J =
4.8 Hz, CH2O2); 5.42 (1H, br s, 4ꢀ-OH); 4.98–4.92 (1H, m, NH);
4.85–4.78 (1H, m, 4-H); 4.56 (1H, d, J = 4.2 Hz, 1-H); 4.38 (1H,
t, J = 7.8 Hz, 11a-H); 4.31 (2H, t, J = 6.6 Hz, CH2a); 3.87 (1H,
t, J = 9.3 Hz, 11b-H); 3.77 (6H, s, 3ꢀ,5ꢀ-OCH3); 2.98–2.82 (4H,
m, 2,3-H, CH2b); IR (CHCl3): m 3358 (NH), 3020 (aliphatic
Tubuline test
Tubulin polymerization inhibition was determined as previously
reported.22
Acknowledgements
=
=
C–H), 1773 (C O lactone), 1559, 1508, 1458 (aromatic C C);
This work was supported by grants (to P. B. A.) from Ligue
Nationale contre le Cancer and Ministe`re de la Recherche et
de lꢀIndustrie (ACI “Mole´cules et Cibles The´rapeutiques”) and
(to M. D.) from ARC. Thanks are also due to S. Lacombe
for technical assistance and to S. Thoret (ICSN-CNRS-Gif) for
monitoring tubulin assays.
MS (CI) m/z 566 [M + H]+, 583 [M + NH4]+.
4-b-(2,4-Dichlorobenzyloxycarbonyl)amino-4ꢀ-O-demethyl-4-
desoxypodophyllotoxin (4m). Yield 40%; Rf = 0.35 (CH2Cl2 :
◦
acetone, 90 : 10); mp = 242–244 C (white crystals); [a]2D0
=
1
−81.9 (c = 0.430, CHCl3); H-NMR (CDCl3) d: 7.43 (1H, s,
Ph); 7.35 (2H, d, J = 5.9 Hz, Ph); 6.81 (1H, s, 5-H); 6.50 (1H, s,
8-H); 6.28 (2H, s, 2ꢀ,6ꢀ-H); 5.97 (2H, d, J = 6.3 Hz, CH2O2); 5.41
(1H, br s, 4ꢀ-OH); 5.21 (2H, d, J = 6.3 Hz, CH2Ph); 5.05–4.93
(2H, m, NH, 4-H); 4.56 (1H, d, J = 4.0 Hz, 1-H); 4.42 (1H,
t, J = 7.3 Hz, 11a-H); 3.96 (2H, t, J = 9.4 Hz, 11b-H); 3.77
(6H, s, 3ꢀ,5ꢀ-OCH3); 2.90–2.80 (2H, m, 2,3-H); IR (CHCl3): m
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=
3358 (NH), 3020 (aliphatic C–H), 1773 (C O lactone), 1559,
+
=
1508, 1458 (aromatic C C); MS (CI) m/z 534 [M + H] , 551
[M + NH4]+.
DNA and biochemicals
The plasmid pBS was obtained from Stratagene (France).
Purified human topoisomerase I and IIa were purchased from
TopoGEN Inc. (USA) and etoposide from Sigma Chemicals.
Compounds were dissolved in dimethyl sulfoxide at 5 mM, then
diluted to working concentrations in distilled water immediately
before use.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 0 7 4 – 1 0 8 0
1 0 7 9

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