Synthesis and Biological Activity of Sulfonamide Derivatives of Epipodophyllotoxin

Article abstract of DOI:10.1021/jm031117b

A series of novel 4β-substituted sulfonamide derivatives of 4′-O-demethyl-4-desoxypodophyllotoxin has been synthesized. Their effects on human DNA topoisomerase II and, in some cases, on tubulin polymerization were evaluated. Compounds 8a, 8c, 8f, 8g, 8n,

Full text of DOI:10.1021/jm031117b

J . Med. Chem. 2004, 47, 2365-2374
2365
Syn th esis a n d Biologica l Activity of Su lfon a m id e Der iva tives of
Ep ip od op h yllotoxin †
Dominique Guianvarc’h,‡ Maria Duca,‡,§ Chawki Boukarim,§ Laurence Kraus-Berthier,| Ste´phane Le´once,|
Alain Pierre´,| Bruno Pfeiffer, Pierre Renard, Paola B. Arimondo,‡ Claude Monneret,§ and Daniel Dauzonne*,§
Laboratoire de Biophysique, CNRS UMR 5153-MNHN USM 0503, INSERM UR 565, 43 rue Cuvier,
75231 Paris Cedex 05, France, UMR 176 CNRS, Institut Curie, Section de Recherche, 26 rue d’Ulm,
75248 Paris Cedex 05, France, Institut de Recherches Servier, Division Recherche Cance´rologie, 125 Chemin de Ronde,
78290 Croissy sur Seine, France, and Les Laboratoires Servier, 1 rue Carle He´bert, 92415 Courbevoie Cedex, France
Received December 1, 2003
A series of novel 4â-substituted sulfonamide derivatives of 4′-O-demethyl-4-desoxypodophyl-
lotoxin has been synthesized. Their effects on human DNA topoisomerase II and, in some cases,
on tubulin polymerization were evaluated. Compounds 8a , 8c, 8f, 8g, 8n , 8q, 8r , and 8s and
the synthetic precursor 4 are potent topoisomerase II poisons that induce double-stranded
breaks in DNA, with either improved or similar activity compared to etoposide. Only the amino
precursor, compound 5, was slightly active in tubulin polymerization inhibition assays. We
observed that the derivatives bearing an aromatic ring on the 4â-sulfonamide substituent were
either less cytotoxic or equivalent to the parent drug, while the sulfonamides containing an
aliphatic side chain and the amino-sulfonamide derivatives, except 8d and 8g, exhibited
increased cytoxicity compared to etoposide. In vivo, against the P388 leukemia and the A-549
orthotopic model of lung carcinoma, the most promising compounds were the morpholino- and
the piperazino-containing sulfonamides derivatives 8r and 8s.
In tr od u ction
The podophyllotoxin derivative etoposide (VP16, 1) is
a semisynthetic glycoside derivative of podophyllotoxin
first synthesized in 1966, tested clinically as an anti-
cancer agent starting in 1971, and officially approved
for clinical use in 1983.1,2 Eighteen years after its
introduction in medical practice, etoposide remains one
of the most extensively used antitumor agents in clinical
use directed against various types of cancers, including
mainly breast cancer, testicular cancer, small-cell lung
cancer, lymphoma, and chilhood leukemia.3,4 In contrast
to the parent podophyllotoxin, etoposide neither binds
to tubulin nor inhibits microtubule assembly. The
mechanism by which etoposide exerts its antineoplastic
effects was elucidated in the 1980s after the discovery
of the DNA-cleaving enzyme topoisomerase II.5 In fact,
molecules such as etoposide, amsacrine, and mito-
xantrone are topoisomerase II inhibitors that induce cell
death by enhancing the topoisomerase II-mediated DNA
cleavage through the stabilization of the transient DNA/
topoisomerase II cleavage complex. In such a complex,
DNA is cleaved on both strands and covalently linked
to the enzyme; the topoisomerase II poison prevents it
from dissociating.6 Recently, it has been suggested that
etoposide-topoisomerase II interactions mediate cleav-
age complex stabilization, rather than etoposide-DNA,
as is the case with amsacrine, another potent topo-
isomerase II inhibitor.7
Despite its extensive use, etoposide is not devoid of
toxic side effects. Bone-marrow depression is a frequent
dose-limiting toxicity encountered in patients receiving
etoposide, and the efficacy of the drug is calamitously
associated with an increased risk of secondary acute
myelogenous leukemia.8,9 For this reason, the develop-
ment of more active and more potent analogues remains
highly valuable. Extensive structure-activity studies
with etoposide analogues10-12 have suggested that the
activity of the epipodophyllotoxin derivatives is related
to three structurally distinct pharmacophoric domains:
the “DNA-intercalating” moiety (central part of the
molecule), the binding site (southern part), and a
variable substituent region (glycoside moiety).11,12 Nu-
merous glycoside analogues of etoposide have been
synthesized, but only one of them has reached the
preclinical level before the trials were stopped, NK611.13
For our part, we have developed a series of 3-N,N-
dimethylamino-2-deoxy analogues14 that exhibited rel-
evant antitumor activities in mice. The glycoside moiety
of etoposide has thus also been replaced with nonsugar
groups such as pyrrolecarboxamidino elements15 and,
above all, by a p-nitroanilino substituent. This latter
afforded the drug GL-331, which was undergoing a
† Dedicated to the memory of Prof. Claude He´le`ne, deceased on
February 11, 2003.
* Corresponding author. Phone: (33) 1 42 34 66 64. Fax: (33) 1 42
34 66 31. E-mail: daniel.dauzonne@curie.fr.
‡ Laboratoire de Biophysique, CNRS UMR 5153-MNHN USM 0503,
INSERM UR 565.
§ UMR 176 CNRS-Institut Curie.
| Institut de Recherches Servier.
Les Laboratoires Servier.
10.1021/jm031117b CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/30/2004

2366 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Guianvarc’h et al.
Sch em e 1a
phase II clinical trial for treatment of various cancers,16
but the trial was stopped in 2001.
In an ongoing effort to develop new and more potent
anticancer agents that are water-soluble and less toxic,
we synthesized 4â-substituted sulfonamide derivatives
containing alkyl (type A), aromatic (type B), or amino
groups (type C) on the 4′-O-demethyl-4-desoxypodophyl-
lotoxin pharmacophore (Table 1). The choice of the
sulfonamide group was dictated by the fact that the
methanesulfonyl-m-anisidide group plays an important
role in the topoisomerase II inhibitory activity of
amsacrine,17-19 and by the fact that the replacement of
the glycoside moiety of 1 by a methanesulfonyl-m-
anisidide group still maintains the topoisomerase II
inhibition, depending on the length of the linker be-
tween the latter group and the aglycon moiety.20 The
relationship between cytotoxicity and inhibition of to-
poisomerase II, as well as the DNA binding affinity, was
studied.
Ch em istr y
The synthesis of the novel 4â-(sulfonamido)-4′-O-
demethyl-4-desoxypodophyllotoxin (8a-s) studied herein
is depicted in Scheme 1. The 4â-amino-4′-O-demethyl-
4-desoxypodophyllotoxin (5) requisite intermediate was
prepared on a 20-g scale according to known procedures
starting from podophyllotoxin (2) via 4′-O-demethyl-4-
epipodophyllotoxin (3)21 and then 4â-azido-4′-O-dem-
ethyl-4-desoxypodophyllotoxin (4).22 The preparation of
the new silyl-protected derivative 6 was achieved in
excellent yield following a classical method involving
tert-butyldimethylsilyl chloride (TBDMSCl) and imida-
zole in DMF. The 4′-silylated 4â-alkylsulfonamido-4′-
O-demethyl-4-desoxypodophyllotoxin 7a -e, 4â-aryl-
sulfonamido-4′-O-demethyl-4-desoxypodophyllotoxin 7h -
j, 4â-(thiophen-2-yl)sulfonamido-4′-O-demethyl-4-des-
oxypodophyllotoxin 7l, 4â-(thiazol-5-yl)sulfonamido-4′-
O-demethyl-4-desoxypodophyllotoxin 7m , and 4â-amino-
sulfonamido-4′-O-demethyl-4-desoxypodophyllotoxin (7n -
s) were further obtained by reacting 6 and the appropri-
ate sulfonyl chloride in the presence of 1,4-diazabicyclo-
[2.2.2]octane (DABCO) in CH2Cl2. The synthesis of the
alkylazido derivative 7f was performed by treatment of
the ω-chloro compound 7e by NaN3 in dry DMF at 60
°C. It must be noted that during the course of this
reaction, an important desilylation occurred and only a
little 7f was isolated aside from a large amount of the
deprotected compound 8f. The arylamino derivative 7k
was easily prepared by catalytic reduction (Pd/C 10%)
in dioxane of the azido group of 7j. The subsequent
regenerations of the 4′-hydroxyl group from compounds
7a -q were performed, in most cases (7a -l and 7n -r ),
in methanol by using a DOWEX 50 × 2-200 ion-
exchange resin to provide the wanted phenolic com-
pounds 8a -l and 8n -r in good to excellent yields. With
regard to the protected derivatives 7m and 7s, the above
desilylation method gave unsatisfactory yields, probably
because of an important retention of the product on the
resin, and the procedure involving tetrabutylammonium
fluoride (Bu4NF) in THF was successfully employed to
obtain 8m and 8s. The 4â-(3-aminopropyl)sulfonamido-
4′-O-demethyl-4-desoxypodophyllotoxin (8g) and 4â-(4-
aminophenyl)sulfonamido-4′-O-demethyl-4-desoxypodo-
phyllotoxin (8k ) were directly obtained from the cor-
a
Reagents: (a) MeSO3H, d,l-methionine, H2O, acetone; (b)
NaN3, CHCl3, CF3COOH; (c) H2, Pd/C (10%) AcOEt; (d) TBDMSCl,
imidazole, anhydrous DMF; (e) RSO2Cl, DABCO, anhydrous
CH2Cl2; (f) Dowex 50 × 2-200, MeOH for 8a -1 and 8a -r or (g)
Bu4NF, THF for 8m and 8s.
responding already deprotected azido derivative 8f and
8j by catalytic reduction under atmospheric pressure.
Biologica l Resu lts a n d Discu ssion
As illustrated in Table 1, in the sulfonamides of the
type A series, the alkyl side chain was found to have
an important effect on the activity of inhibiting human
DNA topoisomerase II. Indeed, the compound containing
the shortest chain, the methylsulfonamide analogue 8a ,
showed the strongest inhibition value. As the length of
the chain was increased, the inhibition activity de-
creased strongly for the butylsulfonamide derivative 8b
(5-fold less active), and this activity was completely lost
in the case of the compound containing the longest side
chain 8d [R ) (CH2)15CH3]. Surprisingly, the octylsul-
fonamide 8c was found to be more active on topo-
isomerase II inhibition than the shorter analogue 8b.

Sulfonamide Derivatives of Epipodophyllotoxin
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2367
Ta ble 1. Biological Evaluation of 4â-Substituted Sulfonamide Derivatives of 4′-O-Demethyl-4-desoxypodophyllotoxin
a
Each value reported here is a medium value of three independent experiments in the presence of the drug at 50 µM. b IC50: concentration
of drug required to reduce by 50% L1210 cell growth. c Percent of L1210 cells in the G2+M phases at the indicated concentration.

2368 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Guianvarc’h et al.
Ta ble 2. Antiproliferative Effects of 1 (VP16) and 8b against
Various Cell Lines
The most potent topoisomerase II inhibitor 8a was also
more cytotoxic than VP16 on L1210 murine leukemia
cells (IC50 ) 0.07 and 0.83 µM, respectively). On the
other hand, the presence of the butyl chain in compound
8b, while it strongly decreased the topoisomerase II
inhibitory activity, it increased 2-fold the inhibition of
tumor cell growth. In fact, not always the degree of
topoisomerase II inhibition correlates with cytotoxicity.
To further study the cellular target of compound 8b, we
investigated its ability to inhibit tubulin polymerization,
as its precursor (the podophyllotoxin) is a potent anti-
microtubule agent. Only a slight inhibition was observed
with this compound (18% of inhibition of tubulin po-
lymerization at 67 µM, compared to an IC50 ) 3 µM for
podophyllotoxin), as for 8a (TPI ) 3% at 67 µM),
indicating that tubulin is probably not its cellular target.
On the contrary, the synthetic precursor 5 (Scheme 1)
was more active in inhibiting the polymerization of
tubulin (56% at 67 µM), while it was completely inactive
against topoisomerase II and showed a modest cytoxicity
(Table 1). For the other alkyl derivatives 8c and 8d , as
the length of the chain increases, the cytotoxicity
decreases dramatically in correlation with their activity
on topoisomerase II.
IC50 (nM)
1 (VP16) 8b
IC50 (nM)
cell lines 1 (VP16) 8b
cell lines
L1210
201.4
29.8
33.5 IGROV1
16.1 A549
984.5
664.0
61.4
62.6
P388
K-562
A431
DU-145
MDA-MB-231
522.2
591.0
2373.3
600.0
138.6 NCI-H69
141.9 KB-3-1
303.7 SK-N-MC
69.2 HT-29
1192.2
566.5
275.0
316.3
186.1
85.7
2366.7
237.4
the most interesting derivative 8n was tested for tubulin
polymerization inhibition and proved inactive (TPI )
13% at 67 µM).
To have some indications on the drug-DNA interac-
tion, we studied perturbations of ethidium bromide-
DNA complexes in agarose gel by increasing concentra-
tions (10, 50, 100 µM) of sulfonamide derivatives 8a -s.
While, under the same conditions, the known interca-
lating drug daunorubicin shifts the ethidium bromide
in the DNA, the sulfonamide derivatives had no effect.
This result argues for a nonintercalative binding mode
such as known for VP16.
The perturbation of the cell cycle induced by these
compounds was studied on the L1210 cell line. All the
compounds induced a marked accumulation (>70%) of
cells in the G2+M phases at a concentration between
0.1 and 2.5 µM, except alkylsulfonamides 8d , 8g, and
8m , which were less cytotoxic than the others, requiring
a 25, 50, and 10 µM concentration, respectively. Com-
pound 8i was revealed to be very inactive. Noteworthy,
there is no correlation between the arrest in G2+M
phase and topoisomerase II inhibition; the former is
rather correlated to cell growth inhibition.23
Among the most cytotoxic compounds against L1210
cell lines, compound 8b (IC50 ) 34 nM) was further
evaluated on a panel of 12 cell lines. Cell growth
inhibition by 8b was observed on all the cell lines (Table
2), the most sensitive cell lines being P388 leukemia,
L1210 leukemia, and, in a much more moderate way,
human tumor cell lines such as breast MDA-MB-231,
ovarian IGROV1, lung A-549, and neuroblastoma SK-
NMC. A comparison with VP16 shows that 8b is more
cytotoxic for each cell line tested (from 2- to 16-fold).
Compounds 8b, 8l, 8n , 8o, 8q, 8r , and 8s were
subsequently tested in vivo against P388 leukemia and
the orthotopic model of human lung cancer A549,24 with
iv administration at J 1 in the first case and at J 14, J 21,
and J 28 in the second (Table 3). Against P388, the
majority of these compounds were active (T/C > 120%),
except the thiophene derivative 8l (T/C ) 102%) and
the diethylamine derivative 8o (T/C ) 106%). The most
active compounds were the morpholine-containing (8r )
and the piperazine-containing (8s) sulfonamides with
T/C of 235% and 229%, respectively. However, they did
not induce long-term survivors. Comparatively, VP16
exhibits a T/C ) 233%, with 6/42 long-term survivors
(LTS). Against the A-549 orthotopic model of lung
cancer, 8r and 8s were more efficient than etoposide
with T/C ) 155% and 159%, respectively; VP16 present-
ing a T/C ) 122-131%. The cytotoxic and potent
topoisomerase II poison 8n showed next best antitumor
properties comparable to VP16 in both tumor models
(T/C ) 192% in P388 and T/C ) 133% in A549), but
again no long-term survivors were recorded.
We next compared three analogues of compound 8b.
The substitution by a terminal amine on the alkyl chain
of the cytotoxic butylsulfonamide 8b increased by 3-fold
the topoisomerase II inhibitory activity with complete
loss of cytotoxicity for compound 8g. The presence of a
terminal halogen, as in compound 8e, did not modify
either the inhibition of topoisomerase II, the tubuline
polymerization (TPI ) 8% at 67 µM), or the cytotoxicity.
The presence of an azido group as in compound 8f
increased the topoisomerase II activity and maintained
the same cell growth inhibition potency and inactivity
against tubulin polymerization (TPI ) 19% at 67 µM).
The azide precursor 4 inhibited strongly topoisomerase
II but was less cytotoxic.
The poor correlation between topoisomerase II inhibi-
tion and cytotoxicity, for compounds 4 and 8g, could be
related to the drug uptake or metabolism.
Compounds of type B, bearing an aromatic ring on
the 4â-sulfonamide substituent, displayed low topo-
isomerase II inhibition but a cytotoxic potency compa-
rable to or slightly better than that of VP16, except
compounds 8i and 8m .
In group C, the substitution of the 4â-sulfonamide
group by six different amino substituents preserved the
high topoisomerase II inhibition and the cytotoxic
activity of the molecule, except for the dibutyl compound
8p . For this class of derivatives, fairly good correlation
between activity against topoisomerase II and inhibition
of tumor cell proliferation was observed. The most
potent drugs are the N,N-(dimethylamino)-substituted
sulfonamide 8n and the pyrazine analogue 8s (IC50
)
0.037 and 0.048 µM, respectively, with anti-topo-
isomerase II activity comparable to that VP16). Length-
ening of the alkyl chain on the amine decreased by 2-fold
the activity of the N,N-diethylamino-substituted sul-
fonamide 8o compared to its dimethylamino-analogue
8n and drastically so (up to 12-fold) for the dibutyl
analogue 8p . The piperidine 8q and morpholine 8r
analogues showed similar topoisomerase II inhibition
and cytotoxic activities compared to compound 8o. Only

Sulfonamide Derivatives of Epipodophyllotoxin
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2369
Ta ble 3. Antitumor Activity of Compounds 1 (VP16), 8b, 8l, 8n , 8o, 8q, 8r , and 8s
P388 leukemia
(treatment on day 1)
A549 non-small-cell lung carcinoma
(treatment on days 14, 21, and 28)
dose range,
mg/kg
median
T/Cb
dose range,
mg/kg
compd
MTDa
LTS D60c
MTDa
T/Cb
1 (VP16)
8b
8l
8n
8o
8q
8r
6.25-100
6.25-100
3.12-50
12.5-200
1.56-25
12.5-200
12.5-200
1.56-25
100
100
12.5
100
25
200
200
12.5
233
161
102
192
106
121
235
229
6/42
0/6
0/6
0/6
0/6
0/6
0/6
0/6
70-100
25-100
-
6.25-25
3.12-12.5
-
25-100
1.56-6.25
70
50
122-131
105
-
133
120
-
155
NTd
6.25
6.25
NT
50
8s
6.25
126-159
a
b
MTD (mg/kg): maximum tolerated dose; i.e., the dose that does not induce toxic death and/or weight loss higher than 20%. T/C (%):
median survival time of treated animals/median survival time of control animals × 100. c LTS: long-term survivors, scored at day 60.
d
NT: not tested.
mmol),21,22 TBDMSCl (9.8 g, 65 mmol), and imidazole (20.4 g,
Con clu sion s
0.3 mol) in anhydrous DMF (1115 mL) was stirred in a 2 L
We have synthesized a series of new derivatives of
dry round-bottomed flask under inert atmosphere for 17 h. The
4-desoxy-4-amino-4′-demethylepipodophyllotoxin in which
the glucose acetal moiety has been replaced by various
sulfonamide substituents. This led, especially in the case
of morpholine- and piperazine-containing sulfonamide
side chains (8r and 8s) to highly promising new anti-
tumor compounds. This relevant activity correlated with
inhibition activity against topoisomerase II inhibition
and with marked accumulation of cells in the G2+M
phase.
More complete evaluation of the most active com-
pounds is now in progress against tumor models in vivo
to evaluate whether they are worthy of further develop-
ment as anticancer agents.
reaction mixture was poured in a 5 L separating funnel, and
water (2 L) was added. (Ca u tion : The reaction is slightly
exothermic.) The mixture was allowed to cool to room tem-
perature and extracted with Et2O (1 L then 4 × 500 mL). The
combined organic extracts were dried (MgSO4) and then
filtered. Removal of the solvents in vacuo (increasing gradually
the temperature from 30 to 100 °C) gave a crude material
(18.75 g, 99%) directly recrystallized in benzene/heptane to
afford analytically pure 6 (15.16 g, 80%) as white plates. Rf )
0.23 (CH2Cl2/acetone 90:10). Mp ) 227-229 °C. [R]20D ) -62.5
1
(c ) 0.55, CHCl3). H NMR (CDCl3) δ: 6.80 (1H, s, 5-H), 6.50
(1H, s, 8-H), 6.27 (2H, s, 2′,6′-H), 5.95 (2H, d, J ) 6.0 Hz,
CH2O2), 4.55 (1H, d, J ) 5.1 Hz, 1-H), 4.28 (2H, d, J ) 9.3 Hz,
11-H), 4.19 (1H, d, J ) 4.1 Hz, 4-H), 3.67 (6H, s, 3′,5′-OCH3),
3.26 (1H, dd, J ) 5.1, 14.0 Hz, 2-H), 2.89-2.78 (1H, m, 3-H),
1.95-1.60 (2H, m, NH2), 0.98 (9H, s, tBu), 0.10 (6H, s, 2Me).
IR (CHCl3) ν: 3573 (NH2), 2931 (aliphatic C-H), 1775 (CdO
lactone), 1621, 1505, 1465 (aromatic CdC). MS (CI) m/z: 531
[M + NH4]+. Anal. (C27H35NO7Si) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
7a -e, 7h -l, a n d 7n -s. To a solution of 6 (309 mg, 0.6 mmol)
and DABCO (45 mg, 0.4 mmol) in anhydrous CH2Cl2 (10 mL),
under inert atmosphere, was added the appropriate sulfonyl
chloride (1 mmol). The reaction mixture was further stirred
at room temperature for the reported times (monitored by
TLC). The medium was next taken up with CH2Cl2 (50 mL)
and water (20 mL). The aqueous phase was extracted with
CH2Cl2 (3 × 15 mL). The combined organic extracts were dried
(MgSO4) and then filtered. Evaporation of the solvents in vacuo
afforded a crude material chromatographed on silica gel (100
g; eluent, various mixtures CH2Cl2/acetone; see Rf) to provide
satisfactorily pure compounds in the reported yields. In the
cases of 7a -e, 7h -l, 7n , 7p , and 7r , the product was
recrystallized from benzene/hexane. In the cases of 7o, 7q, and
7s, the chromatographed silylated compound was directly
employed in the subsequent deprotection step without further
purification.
Exp er im en ta l Section
Ch em ist r y. Solvents and most of the starting materials
were purchased from Acros, Aldrich, or Avocado. The com-
mercially unavailable diethylsulfamoyl chloride,25 dibutylsul-
famoyl chloride,25 morpholine-4-sulfonyl chloride,26 piperidine-
1-sulfonyl chloride,27 and 4-methylpiperazine-1-sulfonyl
chloride28 where prepared according to previously reported
procedures. Reference etoposide (1) was obtained from Sigma
Chemical Co. Melting points were measured on a 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 for
chloroform solutions or KBr disks. Specific rotations were
1
measured with a Perkin-Elmer 241 polarimeter. The H 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), dt (double triplet), m (multiplet), and br
(broad signal). Coupling constants (J values) are listed in hertz
(Hz). Reactions were monitored by analytical thin-layer chro-
matography and products were visualized by exposure to UV
light. Merck silica gel (230-400 mesh ASTM) was used for
column chromatography. Acetone, methanol, and dichlo-
romethane employed as eluents for column chromatography
were distilled on a rotary evaporator prior to use. Anhydrous
DMF was obtained by prolonged contact with activated Linde-
type 4 Å molecular sieves. Dry THF was prepared by distil-
lation from benzophenone/sodium. All yields reported are
unoptimized. Elemental analysis for most of the new sub-
stances was performed by CNRS Laboratories (Vernaison,
France), and unless noted otherwise, the results obtained are
within 0.4% of the theoretical values.
4 â-Meth ylsu lfon a m id o-4′-ter t-bu tyld im eth ylsilyloxy-
4′-O-d em et h yl-4-d esoxyp od op h yllot oxin (7a ). Reaction
time: 16 h. Yield: 91%. Rf ) 0.39 (CH2Cl2/acetone 96:4). Mp
) 206-208 °C (white crystals). [R]20 ) -79.0 (c ) 0.345,
D
CHCl3). 1H NMR (CDCl3) δ: 6.84 (1H, s, 5-H), 6.53 (1H, s, 8-H),
6.20 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 7.0 Hz, CH2O2), 4.84-
4.78 (1H, m, 11a-H), 4.56 (1H, d, J ) 4.1 Hz, 1-H), 4.57-4.32
(3H, m, 11b,4-H, NH), 3.67 (6H, s, 3′,5′-OCH3), 3.15 (3H, s,
CH3SO2), 2.92-2.89 (2H, m, 2,3-H), 0.98 (9H, s, tBu), 0.10 (6H,
s, 2Me). IR (CHCl3) ν: 3380 (NH), 2934 (aliphatic C-H), 1776
(CdO lactone), 1587, 1507, 1485 (aromatic CdC), 1389, 1132
(SO2). MS (CI) m/z: 609 [M + NH4]+. Anal. (C28H37NO9SSi)
C, H, N.
4â-Bu tylsu lfon a m id o-4′-ter t-bu tyld im eth ylsilyloxy-4′-
O-d em eth yl-4-d esoxyp od op h yllotoxin (7b). Reaction
time: 18 h. Yield: 90%. Rf ) 0.59 (CH2Cl2/acetone 96:4). mp
4â-Am in o-4′-ter t-bu tyld im eth ylsilyloxy-4′-O-d em eth yl-
4-d esoxyp od op h yllotoxin (6). A solution of 5 (14.73 g, 36.9
) 127-129 °C (white crystals). [R]20 ) -61.0 (c ) 0.535,
D

2370 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Guianvarc’h et al.
CHCl3). 1H NMR (CDCl3) δ: 6.83 (1H, s, 5-H), 6.52 (1H, s, 8-H),
6.20 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 5.1 Hz, CH2O2), 4.82-
4.76 (1H, m, 11a-H), 4.56 (1H, d, J ) 3.8 Hz, 1-H), 4.42-4.23
(3H, m, 11b, 4-H, NH), 3.67 (6H, s, 3′,5′-OCH3), 3.22-3.10 (2H,
m, CH2SO2), 2.96-2.88 (2H, m, 2,3-H), 1.96-1.78 (2H, m,
CH2â), 1.61-1.42 (2H, m, CH2γ), 1.07-0.91 (12H, m, CH3,
tBu), 0.10 (6H, s, 2Me). IR (CHCl3) ν: 3395 (NH), 2934
(aliphatic C-H), 1775 (CdO lactone), 1586, 1507, 1485 (aro-
matic CdC), 1335, 1132 (SO2). MS (CI) m/z: 651 [M + NH4]+.
Anal. (C31H43NO9SSi) C, H, N.
J ) 9.8 Hz, 11b-H), 3.67 (6H, s, 3′,5′-OCH3), 3.02-2.90 (2H,
m, 2,3-H), 0.98 (9H, s, tBu), 0.10 (6H, s, Me). IR (CHCl3) ν:
3369 (NH), 2931 (aliphatic C-H), 1779 (CdO lactone), 1520,
1500, 1485 (aromatic CdC), 1337, 1132 (SO2). MS (CI) m/z:
761 [M + NH4]+
4â-(4-Azid op h en yl)su lfon a m id o-4′-ter t-bu tyld im eth yl-
silyloxy-4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7j). Re-
action time: 18 h. Yield: 94%. Rf ) 0.61 (CH2Cl2/acetone 96:
4). Mp ) 148-150 °C (white crystals). [R]20 ) -71.5 (c )
D
0.770, CHCl3). 1H NMR (CDCl3) δ: 7.93 (2H, d, J ) 8.7 Hz,
Ph), 7.26 (2H, d, J ) 8.7 Hz, Ph), 6.46 (1H, s, 8-H), 6.16 (2H,
s, 2′,6′-H), 5.91 (2H, s, CH2O2), 5.72 (1H, s, 5-H), 4.70-4.59
(3H, m, 1,4-H, NH), 4.27 (2H, d, J ) 8.1 Hz, 11-H), 3.65 (6H,
s, 3′,5′-OCH3), 2.92-2.80 (2H, m, 2,3-H), 0.97 (9H, s, tBu), 0.10
(6H, s, 2Me). IR (CHCl3) ν: 3525 (NH), 2930 (aliphatic C-H),
2133 (N3), 1774 (CdO lactone), 1597, 1505, 1485 (aromatic Cd
C), 1336 (SO2). MS (CI) m/z: 712 [M + NH4]+. Anal.
(C33H38N4O9SSi) C, H, N.
4â-Octylsu lfon a m id o-4′-ter t-bu tyld im eth ylsilyloxy-4′-
O-d em eth yl-4-d esoxyp od op h yllotoxin
(7c). Reaction
time: 16 h. Yield: 94%. Rf ) 0.67 (CH2Cl2/acetone 96:4). Mp
) 110-112 °C (white crystals). [R]20 ) -57.0 (c ) 0.320,
D
CHCl3). 1H NMR (CDCl3) δ: 6.83 (1H, s, 5-H), 6.52 (1H, s, 8-H),
6.20 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 6.2 Hz, CH2O2), 4.82-
4.74 (1H, m, 11a-H), 4.56 (1H, br d, J ) 3.5 Hz, 1-H), 4.41-
4.21 (3H, m, 11b,4-H, NH), 3.67 (6H, s, 3′,5′-OCH3), 3.22-3.09
(2H, m, CH2SO2), 2.99-2.83 (2H, m, 2,3-H), 1.98-1.79 (2H,
m, CH2â), 1.61-1.41 (2H, m, CH2γ), 1.40-1.19 (8H, m, 4CH2),
0.98 (9H, s, tBu), 0.93-0.81 (3H, m, CH3), 0.10 (6H, s, 2Me).
IR (CHCl3) ν: 3395 (NH), 2930 (aliphatic C-H), 1776 (CdO
lactone), 1587, 1506, 1485 (aromatic CdC), 1335, 1132 (SO2).
MS (FAB) m/z: 712 [M + Na]+. Anal. (C35H51NO9SSi) C, H,
N.
4â-(Th iop h en -2-yl)su lfon a m id o-4′-ter t-bu tyld im eth yl-
silyloxy-4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7l). Re-
action time: 7 h. Yield: 96%. Rf ) 0.63 (CH2Cl2/acetone 96:
4). Mp ) 151-153 °C (white crystals). [R]20 ) -41.5 (c )
D
0.635, CHCl3). 1H NMR (CDCl3) δ: 7.80-7.71 (2H, m, Ph),
7.30-7.20 (1H, m, Ph), 6.47 (1H, s, 8-H), 6.18 (2H, s, 2′,6′-H),
5.90 (2H, s, CH2O2), 5.62 (1H, s, 5-H), 4.68-4.49 (3H, m, 1,4-
H, NH), 4.40-4.30 (2H, m, 11-H), 3.66 (6H, s, 3′,5′-OCH3),
3.00-2.82 (2H, m, 2,3-H), 0.98 (9H, s, tBu), 0.10 (6H, s, Me).
IR (CHCl3) ν: 3365 (NH), 2943 (aliphatic C-H), 1775 (CdO
lactone), 1559, 1507, 1485 (aromatic CdC), 1332, 1161 (SO2).
MS (CI) m/z: 677 [M + NH4]+. Anal. (C31H37NO9S2Si) C, H,
N.
4â-Hexadecylsu lfon am ido-4′-ter t-bu tyldim eth ylsilyloxy-
4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7d ). Reaction
time: 16 h. Yield: 92%. Rf ) 0.42 (CH2Cl2/acetone 96:4). mp
) 126-128 °C (white crystals). [R]20 ) -61.5 (c ) 0.275,
D
CHCl3). 1H NMR (CDCl3) δ: 6.84 (1H, s, 5-H), 6.52 (1H, s, 8-H),
6.22 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 6.8 Hz, CH2O2), 4.82-
4.76 (1H, m, 11a-H), 4.57 (1H, br d, J ) 3.3 Hz, 1-H), 4.42-
4.28 (3H, m, 11b,4-H, NH), 3.67 (6H, s, 3′,5′-OCH3), 3.21-3.11
(2H, m, CH2SO2), 2.94-2.88 (2H, m, 2,3-H), 1.95-1.80 (2H,
m, CH2â), 1.54-1.42 (2H, m, CH2γ), 1.38-1.20 (24H, m,
12CH2), 0.98 (9H, s, tBu), 0.88 (3H, t, J ) 6.6 Hz, CH3), 0.10
(6H, s, 2Me). IR (CHCl3) ν: 3388 (NH), 2929 (aliphatic C-H),
1776 (CdO lactone), 1587, 1506, 1485 (aromatic CdC), 1335,
1132 (SO2). MS m/z: (FAB) 802 [M + H]+, 824 [M + Na]+.
Anal. (C43H67NO9SSi) C, H, N.
4â-(2-Aceta m id o-4-m eth ylth ia zol-5-yl)su lfon a m id o-4′-
ter t-bu tyld im eth ylsilyloxy-4′-O-d em eth yl-4-d esoxyp od o-
p h yllotoxin (7m ). Reaction time: 20 h. Yield: 98%. Rf ) 0.50
(CH2Cl2/acetone 85:15). Mp ) 191-193 °C (white crystals).
[R]20 ) -100.0 (c ) 0.57, MeOH). 1H NMR (CDCl3) δ: 6.48
D
(1H, s, 8-H), 6.19 (2H, s, 2′,6′-H), 6.18 (1H, s, NH), 6.03 (1H,
s, 5-H), 5.94 (2H, d, J ) 3.8 Hz, CH2O2), 5.02 (1H, d, J ) 6.6
Hz, NH), 4.74-4.68 (1H, m, 4-H), 4.59-4.52 (1H, m, 1-H),
4.37-4.28 (2H, m, 11-H), 3.68 (6H, s, 3′,5′-OCH3), 2.97-2.89
(2H, m, 2,3-H), 2.54 (3H, s, CH3), 2.34 (3H, s, CH3), 0.98 (9H,
s, tBu), 0.10 (6H, s, Me). IR (CHCl3) ν: 3336 (NH), 2932
(aliphatic C-H), 1775 (CdO lactone), 1587, 1510, 1485 (aro-
matic CdC), 1337, 1150 (SO2). MS (CI) m/z: 732 [M +H]+.
Anal. (C33H41N3O10S2Si) C, H, N.
4â-(3-Ch lor op r op yl)su lfon a m id o-4′-ter t-b u t yld im et h -
ylsilyloxy-4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7e).
Reaction time: 22 h. Yield: 88%. Rf ) 0.53 (CH2Cl2/acetone
96:4). Mp ) 133-135 °C (white crystals). [R]20 ) -72.0 (c )
D
0.555, CHCl3). 1H NMR (CDCl3) δ: 6.87 (1H, s, 5-H), 6.53 (1H,
s, 8-H), 6.20 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 4.5 Hz, CH2O2),
4.85-4.76 (1H, m, 11a-H), 4.57 (1H, d, J ) 4.3 Hz, 1-H), 4.41-
4.25 (3H, m, 11b,4-H, NH), 3.81-3.70 (2H, m, CH2Cl), 3.67
(6H, s, 3′,5′-OCH3), 3.38 (2H, t, J ) 6.7 Hz, CH2SO2), 3.00-
2.88 (2H, m, 2,3-H), 2.48-2.25 (2H, m, CH2â), 0.98 (9H, s, tBu),
0.10 (6H, s, 2Me). IR (CHCl3) ν: 3385 (NH), 2930 (aliphatic
C-H), 1776 (CdO lactone), 1587, 1507, 1485 (aromatic Cd
C), 1336, 1132 (SO2). MS (CI) m/z: 671, 673 [M + NH4]+. Anal.
(C30H40ClNO9SSi) H, N; C, calcd 55.07; found 54.36.
4â-Dim eth yla m in osu lfon a m id o-4′-ter t-bu tyld im eth yl-
silyloxy-4′-O-dem eth yl-4-desoxypodoph yllotoxin (7n ). Re-
action time: 28 h. Yield: 96%. Rf ) 0.51 (CH2Cl2/acetone 96:
4). Mp ) 172-174 °C (white crystals). [R]20 ) -61.0 (c )
D
0.430, CHCl3). 1H NMR (CDCl3) δ: 6.97 (1H, s, 5-H), 6.51 (1H,
s, 8-H), 6.20 (2H, s, 2′,6′-H), 5.98 (2H, d, J ) 5.8 Hz, CH2O2),
4.80-4.69 (1H, m, 11a-H), 4.60-4.51 (1H, m, 1-H), 4.45-4.30
(2H, m, 4-H, NH), 4.28-4.19 (1H, m, 11b-H), 3.67 (6H, s, 3′,5′-
OCH3), 2.98-2.82 (8H, m, 2,3-H + 2CH3), 0.98 (9H, s, tBu),
0.10 (6H, s, 2Me). IR (CHCl3) ν: 3391 (NH), 2930 (aliphatic
C-H), 1775 (CdO lactone), 1587, 1506, 1485 (aromatic Cd
C), 1336, 1132 (SO2). MS (CI) m/z: 638 [M + NH4]+. Anal.
(C29H40N2O9SSi) C, H, N.
4â-(4-Met h ylsu lfon a m id op h en yl)su lfon a m id o-4′-ter t-
bu tyld im eth ylsilyloxy-4′-O-d em eth yl-4-d esoxyp od op h yl-
lotoxin (7h ). Reaction time: 5 h. Yield: 98%. Rf ) 0.22
(CH2Cl2/acetone 94:6). Mp ) 167-169 °C (white crystals).
[R]20D ) -40.0 (c ) 0.25, CHCl3). 1H NMR (CDCl3) δ: 8.21 (4H,
dd, J ) 8.6 Hz, Ph), 6.45 (1H, s, 8-H), 6.20 (2H, s, 2′6′-H), 5.89
(2H, s, CH2O2), 5.44 (1H, s, 5-H), 5.41 (1H, s, NH), 4.62-4.51
(2H, m, 1,4-H), 4.40-4.28 (1H, m, 11-H), 3.75 (6H, s, 3′,5′-
OCH3), 3.16 (3H, s, CH3), 2.95-2.83 (2H, m, 2,3-H), 0.86 (9H,
s, tBu), 0.01 (6H, s, 2Me). IR (CHCl3) ν: 3371 (NH), 2931
(aliphatic C-H), 1776 (CdO lactone), 1587, 1507, 1485 (aro-
matic CdC), 1333, 1132 (SO2). MS (FAB) m/z: 754 [M + Na]+.
Anal. (C34H41NO11S2Si) C, H, N.
4â-Dieth yla m in osu lfon a m id o-4′-ter t-bu tyld im eth ylsi-
lyloxy-4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7o). Re-
action time: 28 h. Yield: 89%. Rf ) 0.65 (CH2Cl2/acetone 92:
8). 1H NMR (CDCl3) δ: 7.04 (1H, s, 5-H), 6.52 (1H, s, 8-H),
6.23 (2H, s, 2′,6′-H), 6.00 (2H, d, J ) 4.9 Hz, CH2O2), 4.79-
4.70 (1H, m, 11a-H), 4.57 (1H, br d, J ) 2.5 Hz, 1-H), 4.47-
4.31 (2H, m, 4-H, NH), 4.01 (1H, d, J ) 6.2 Hz, 11b-H), 3.69
(6H, s, 3′,5′-OCH3), 3.36 (4H, q, J ) 7.2, 14.5 Hz, 2CH2), 2.98-
2.88 (2H, m, 2,3-H), 1.27 (6H, t, J ) 7.1 Hz, 2CH3), 0.98 (9H,
s, tBu), 0.10 (6H, s, 2Me). MS (CI) m/z: 666 [M + NH4]+.
4â-P en t a flu or op h en ylsu lfon a m id o-4′-ter t-b u t yld im e-
th ylsilyloxy-4′-O-dem eth yl-4-desoxypodoph yllotoxin (7i).
Reaction time: 24 h. Yield: 97%. Rf ) 0.60 (CH2Cl2/acetone
4â-Dibu tyla m in osu lfon a m id o-4′-ter t-bu tyld im eth ylsi-
lyloxy-4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7p ). Re-
action time: 48 h. Yield: 86%. Rf ) 0.55 (CH2Cl2/acetone 95:
1
96:4). H NMR (CDCl3) δ: 6.53 (1H, s, 8-H), 6.17 (2H, s, 2′,6′-
H), 6.03 (1H, s, 5-H), 5.96 (2H, d, J ) 5.1 Hz, CH2O2), 5.41
(1H, d, J ) 5.1 Hz, NH), 4.90-4.82 (1H, m, 4-H), 4.58 (1H, d,
J ) 4.2 Hz, 1-H), 4.40 (1H, t, J ) 6.9 Hz, 11a-H), 4.26 (1H, t,
5). Mp ) 112-114 °C (white crystals). [R]20 ) -45.0 (c )
D
0.235, CHCl3). 1H NMR (CDCl3) δ: 7.03 (1H, s, 5-H), 6.50 (1H,
s, 8-H), 6.27 (2H, s, 2′,6′-H), 5.98 (2H, d, J ) 6.0 Hz, CH2O2),

Sulfonamide Derivatives of Epipodophyllotoxin
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2371
4.80-4.70 (1H, m, 11a-H), 4.60-4.51 (1H, m, 1-H), 4.55-4.40
(2H, m, 4-H, NH), 3.91 (1H, d, J ) 6.0 Hz, 11b-H), 3.77 (6H,
s, 3′,5′-OCH3), 3.30-3.16 (4H, m, CH2R), 2.95-2.84 (1H, m,
2,3-H), 1.71-1.49 (4H, m, CH2â), 1.48-1.20 (4H, m, CH2γ),
1.02-0.90 (6H, m, CH3), 0.86 (9H, s, tBu), 0.10 (6H, s, Me).
IR (CHCl3) ν: 3338 (NH), 2937 (aliphatic C-H), 1775 (CdO
lactone), 1519, 1506, 1485 (aromatic CdC), 1333, 1118 (SO2).
MS (CI) m/z: 722 [M + NH4]+. Anal. (C35H52N2O9SSi) C, H,
N.
4â-P iper idin osu lfon am ido-4′-ter t-bu tyldim eth ylsilyloxy-
4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7q). Reaction
time: 28 h. Yield: 98%. Rf ) 0.62 (CH2Cl2/acetone 92:8). 1H
NMR (CDCl3) δ: 7.02 (1H, s, 5-H), 6.50 (1H, s, 8-H), 6.20 (2H,
s, 2′,6′-H), 5.98 (2H, d, J ) 3.4 Hz, CH2O2), 4.75-4.68 (1H, m,
11a-H), 4.55 (1H, br d, J ) 3.4 Hz, 1-H), 4.43-4.32 (2H, m,
4-H, NH), 4.17 (1H, d, J ) 6.7 Hz, 11b-H), 3.67 (6H, s, 3′,5′-
OCH3), 3.40-3.18 (4H, m, 2 CH2N), 2.97-2.88 (2H, m, 2,3-
H), 1.86-1.66 (4H, m, 2 CH2), 1.65-1.50 (2H, m, CH2), 0.98
(9H, s, tBu), 0.10 (6H, s, Me). MS (CI) m/z: 661 [M + H]+. 678
[M + NH4]+.
Filtration on a pad of Celite and rinsing with MeOH, followed
by evaporation of the filtrate, afforded a crude material flash-
chromatographed on silica gel (100 g, CH2Cl2/acetone 90:10)
to provide 7k in 96% yield. Rf ) 0.55 (CH2Cl2/acetone 90:10).
Mp ) 280-282 °C (white powder). [R]20 ) -73.0 (c ) 0.550,
D
MeOH). 1H NMR (CDCl3) δ: 7.72 (2H, d, J ) 8.7 Hz, Ph), 6.80
(2H, d, J ) 8.7 Hz, Ph), 6.44 (1H, s, 8-H), 6.17 (2H, s, 2′,6′-H),
5.90 (2H, s, CH2O2), 5.83 (1H, s, 5-H), 4.58-4.50 (1H, m, 1-H),
4.40-4.23 (4H, m, 11,4-H, NH), 3.65 (6H, s, 3′,5′-OCH3), 3.00-
2.80 (2H, m, 2,3-H), 1.58 (2H, br s, NH2), 0.97 (9H, s, tBu),
0.08 (6H, s, 2Me). IR (KBr) ν: 3482, 3378 (NH, NH2), 2930
(aliphatic C-H), 1774 (CdO lactone), 1597, 1505, 1485 (aro-
matic CdC), 1336, 1153 (SO2). MS (CI) m/z: 686 [M + NH4]+.
Anal. (C33H40N2O9SSi) H, N; C, calcd 59.26; found 59.70.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
8a -f, 8h -j, 8l, a n d 8n -r . Dowex 50 × 2-200 ion-exchange
resin (3 g), previously washed with water and then MeOH,
was added to a solution of 7 (0.6 mmol) in MeOH (65 mL).
The mixture was vigorously stirred for 16 h. The resin was
removed by filtration and thoroughly washed with MeOH.
Evaporation of the filtrate in vacuo gave the wanted compound
in the reported yields. The compounds were directly recrystal-
lized from acetone/heptane (for 8a -f and 8h -j) or benzene/
heptane (for 8l and 8n -r ).
4â-Mor p h olin osu lfon a m id o-4′-ter t-bu tyld im eth ylsily-
loxy-4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7r ). Reac-
tion time: 24 h. Yield: 95%. Rf ) 0.43 (CH2Cl2/acetone 96:4).
Mp ) 138-140 °C (white crystals). [R]20 ) -58.0 (c ) 0.595,
D
CHCl3). 1H NMR (CDCl3) δ: 6.99 (1H, s, 5-H), 6.51 (1H, s, 8-H),
6.20 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 5.4 Hz, CH2O2), 4.80-
4.70 (1H, m, 11a-H), 4.56 (1H, d, J ) 4.1 Hz, 1-H), 4.46-4.32
(2H, m, 4-H, NH), 4.31-4.22 (1H, m, 11b-H), 3.89-3.76 (4H,
m, CH2N), 3.67 (6H, s, 3′,5′-OCH3), 3.46-3.20 (4H, m, CH2O),
2.97-2.87 (2H, m, 2,3-H), 0.98 (9H, s, tBu), 0.09 (6H, s, 2Me).
IR (CHCl3) ν: 3392 (NH), 2930 (aliphatic C-H), 1776 (CdO
lactone), 1587, 1506, 1485 (aromatic CdC), 1337, 1132 (SO2).
MS (CI) m/z: 680 [M + NH4]+. Anal. (C31H42N2O10SSi) C, H,
N.
4â-Met h ylsu lfon a m id o-4′-O-d em et h yl-4-d esoxyp od o-
p h yllotoxin (8a ). Yield: 94%. Rf ) 0.52 (CH2Cl2/MeOH 90:
10). Mp ) 169-171 °C (white powder). [R]20 ) -76.5 (c )
D
0.225, CHCl3). 1H NMR (CDCl3) δ: 6.95 (1H, s, 5-H), 6.49 (1H,
s, 8-H), 6.24 (2H, s, 2′,6′-H), 5.98 (2H, d, J ) 9.7 Hz, CH2O2),
5.45 (1H, br s, 4′-OH), 4.88-4.72 (2H, m, 11a-H, NH), 4.60-
4.50 (1H, m, 1-H), 4.42-4.22 (2H, m, 11b,4-H), 3.75 (6H, s,
3′,5′-OCH3), 3.13 (3H, s, CH3SO2), 2.97-2.84 (2H, m, 2,3-H).
IR (CHCl3) ν: 3539 (OH), 3382 (NH), 2935 (aliphatic C-H),
1775 (CdO lactone), 1519, 1506, 1485 (aromatic CdC), 1330,
1155 (SO2). MS (CI) m/z: 495 [M + NH4]+. Anal. (C22H23NO9S)
C, H, N.
4â-(4-Meth ylp ip er a zin yl)su lfon a m id o-4′-ter t-bu tyld im -
eth ylsilyloxy-4′-O-dem eth yl-4-desoxypodoph yllotoxin (7s).
Reaction time: 30 h. Yield: 89%. Rf ) 0.46 (CH2Cl2/acetone
90:10). 1H NMR (CDCl3) δ: 7.01 (1H, s, 5-H), 6.51 (1H, s, 8-H),
6.20 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 5.4 Hz, CH2O2), 4.76-
4.72 (1H, m, 11a-H), 4.56 (1H, d, J ) 3.5 Hz, 1-H), 4.40-4.24
(3H, m, 11b,4-H, NH), 3.67 (6H, s, 3′,5′-OCH3), 3.40-3.25 (4H,
m, CH2N), 2.98-2.80 (2H, m, 2,3-H), 2.60-2.50 (4H, m, CH2N),
2.30 (3H, s, CH3), 0.98 (9H, s, tBu), 0.10 (6H, s, 2Me). MS (CI)
m/z: 676 [M + H]+.
4â-(3-Azid op r op yl)su lfon a m id o-4′-ter t-bu tyld im eth yl-
silyloxy-4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7f). To
a solution of 7e (496 mg, 0.758 mmol) in anhydrous DMF (27
mL), under argon atmosphere, was added dry sodium azide
(246 mg, 3.79 mmol). The mixture was heated at 60 °C for 60
h. After cooling, water (50 mL) was added and the reaction
medium was extracted with ethyl acetate (150 mL, then 5 ×
100 mL). Drying of the organic extract (MgSO4), followed by
filtration and then evaporation under reduced pressure, gave
a crude residue chromatographed on silica gel (90 g, CH2Cl2/
acetone 90:10). Because of the major formation of the depro-
tected product 8f, isolated in 61% yield (253 mg), the title
compound 7f is thus obtained in only 20% yield (100 mg). Rf
4â-Bu tylsu lfon am ido-4′-O-dem eth yl-4-desoxypodoph yl-
lotoxin (8b). Yield: 92%. Rf ) 0.56 (CH2Cl2/MeOH 90:10). Mp
) 222-224 °C (white powder). [R]20 ) -92.0 (c ) 0.485,
D
DMSO). 1H NMR (CDCl3) δ: 6.84 (1H, s, 5-H), 6.52 (1H, s,
8-H), 6.26 (2H, s, 2′,6′-H), 6.00 (2H, d, J ) 6.1 Hz, CH2O2),
5.41 (1H, br s, 4′-OH), 4.90-4.79 (1H, m, 11a-H), 4.64-4.52
(1H, m, 1-H), 4.46-4.30 (3H, m, 11b,4-H, NH), 3.77 (6H, s,
3′,5′-OCH3), 3.29-3.10 (2H, s, CH2SO2), 2.98-2.87 (2H, m, 2,3-
H), 1.98-1.80 (2H, m, CH2â), 1.61-1.52 (2H, m, CH2γ), 1.00
(3H, t, J ) 7.2 Hz, CH3). IR (CHCl3) ν: 3691 (OH), 3226 (NH),
2965 (aliphatic C-H), 1770 (CdO lactone), 1519, 1506, 1485
(aromatic CdC), 1331, 1116 (SO2). MS (CI) m/z: 537 [M +
NH4]+. Anal. (C25H29NO9S) C, H, N.
4â-Octylsu lfon a m id o-O-d em eth yl-4-d esoxyp od op h yl-
lotoxin (8c). Yield: 91%. Rf ) 0.64 (CH2Cl2/MeOH 90:10). Mp
) 138-140 °C (white powder). [R]20 ) -59.0 (c ) 0.505,
D
CHCl3). 1H NMR (CDCl3) δ: 6.84 (1H, s, 5-H), 6.51 (1H, s, 8-H),
6.25 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 8.6 Hz, CH2O2), 5.43 (1H,
br s, 4′-OH), 4.84-4.76 (1H, m, 11a-H), 4.72-4.61 (1H, m, 1-H),
4.52-4.37 (3H, m, 11b,4-H, NH), 3.77 (6H, s, 3′,5′-OCH3),
3.25-3.08 (2H, m, CH2SO2), 2.98-2.83 (2H, m, 2,3-H), 1.98-
1.80 (2H, m, CH2â), 1.65-1.42 (2H, m, CH2γ), 1.41-1.20 (8H,
m, 4 CH2), 0.98-0.80 (3H, m, CH3). IR (CHCl3) ν: 3545 (OH),
3282 (NH), 2929 (aliphatic C-H), 1775 (CdO lactone), 1519,
1507, 1485 (aromatic CdC), 1331, 1117 (SO2). MS (CI) m/z:
576 [M + H]+. Anal. (C29H37NO9S) C, H, N.
) 0.56 (CH2Cl2/acetone 90:10). Mp ) 110-112 °C (white
1
crystals). [R]20 ) -64.0 (c ) 0.52, CHCl3). H NMR (CDCl3)
D
δ: 6.86 (1H, s, 5-H), 6.53 (1H, s, 8-H), 6.20 (2H, s, 2′,6′-H),
6.00 (2H, d, J ) 5.5 Hz, CH2O2), 4.85-4.77 (1H, m, 11a-H),
4.57 (1H, d, J ) 4.4 Hz, 1-H), 4.44-4.25 (3H, m, 11b,4-H, NH),
3.67 (6H, s, 3′,5′-OCH3), 3.59 (2H, br t, J ) 6.0 Hz, CH2N3),
3.36-3.21 (2H, m, CH2SO2), 2.98-2.88 (2H, m, 2,3-H), 2.21-
2.08 (2H, m, CH2â), 0.98 (9H, s, tBu), 0.10 (6H, s, 2Me). IR
(CHCl3) ν: 3291 (NH), 2103 (N3), 2931 (aliphatic C-H), 1775
(CdO lactone), 1587, 1510, 1485 (aromatic CdC), 1335, 1132
(SO2). MS (CI) m/z: 678 [M + NH4]+. Anal. (C30H40N4O9SSi)
H, N; C, calcd 54.53; found 54.02.
4â-Hexadecylsu lfon am ido-4′-O-dem eth yl-4-desoxypodo-
p h yllotoxin (8d ). Yield: 96%. Rf ) 0.76 (CH2Cl2/MeOH 90:
10). Mp ) 132-133 °C (white powder). [R]20 ) -64.0 (c )
D
0.560, CHCl3). 1H NMR (CDCl3) δ: 6.84 (1H, s, 5-H), 6.51 (1H,
s, 8-H), 6.25 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 9.0 Hz, CH2O2),
5.43 (1H, br s, 4′-OH), 4.86-4.80 (1H, m, 11a-H), 4.60-4.55
(1H, m, 1-H), 4.45-4.29 (3H, m, 11b,4-H, NH), 3.77 (6H, s,
3′,5′-OCH3), 3.22-3.10 (2H, m, CH2SO2), 2.96-2.88 (2H, m,
2,3-H), 1.93-1.80 (2H, m, CH2â), 1.55-1.42 (2H, m, CH2γ),
1.39-1.20 (24H, m, 12 CH2), 0.88 (3H, t, J ) 6.6 Hz, CH3). IR
(CHCl3) ν: 3545 (OH), 3385 (NH), 2927 (aliphatic C-H), 1775
4â-(4-Am in op h en yl)su lfon a m id o-4′-ter t-bu tyld im eth yl-
silyloxy-4′-O-d em eth yl-4-d esoxyp od op h yllotoxin (7k ). A
solution of 7j (643 mg, 0.093 mmol) in dioxane (50 mL) was
vigorously stirred under hydrogen at atmospheric pressure in
the presence of 10% palladium on charcoal (100 mg) for 16 h.

2372 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Guianvarc’h et al.
(CdO lactone), 1519, 1507, 1485 (aromatic CdC), 1331, 1117
-54.5 (c ) 0.570, CHCl3). 1H NMR (CDCl3) δ: 7.78-7.76 (2H,
m, arom), 7.28-7.25 (1H, m, arom), 6.46 (1H, s, 8-H), 6.23 (2H,
s, 2′,6′-H), 5.93 (2H, d, J ) 2.2 Hz, CH2O2), 5.63 (1H, s, 5-H),
5.41 (1H, s, 4′-OH), 4.70-4.59 (2H, m, 4-H, NH), 4.54 (1H, d,
J ) 4.2 Hz, 1-H), 4.36 (2H, d, J ) 7.8 Hz, 11-H), 3.76 (6H, s,
3′,5′-OCH3), 3.00-2.85 (2H, m, 2,3-H). IR (CHCl3) ν: 3544
(OH), 3362 (NH), 2928 (aliphatic C-H), 1774 (CdO lactone),
1519, 1507, 1485 (aromatic CdC), 1332, 1160 (SO2). MS (CI)
m/z: 563 [M + NH4]+. Anal. (C25H23NO9S2) C, H, N.
(SO2). MS (CI) m/z: 688 [M + H]+. Anal. (C37H53NO9S) C, H,
N.
4â-(3-Ch lor op r op yl)su lfon a m id o-4′-O-d em eth yl-4-d es-
oxyp od op h yllotoxin (8e). Yield: 94%. Rf ) 1.57 (CH2Cl2/
20
MeOH 90:10). Mp ) 240-242 °C (white powder). [R]D
)
-87.5 (c ) 0.490, DMSO). 1H NMR (DMSO-d6) δ: 8.29 (1H,
br s, 4′-OH), 7.87 (1H, d, J ) 8.6 Hz, NH), 7.03 (1H, s, 5-H),
6.53 (1H, s, 8-H), 6.22 (2H, s, 2′,6′-H), 6.03 (2H, d, J ) 3.5 Hz,
CH2O2), 4.85-4.78 (1H, m, 4-H), 4.49 (1H, d, J ) 5.2 Hz, 1-H),
4.37 (1H, t, J ) 8.1 Hz, 11a-H), 4.12 (1H, t, J ) 9.5 Hz, 11b-
H), 3.81 (2H, t, J ) 6.2 Hz, CH2Cl), 3.64 (6H, s, 3′,5′-OCH3),
3.49-3.32 (2H, m, CH2SO2), 3.23 (1H, dd, J ) 5.3, 14.3 Hz,
2-H), 3.03-2.89 (1H, m, 3-H), 2.25-2.11 (2H, m, CH2â). IR
(KBr) ν: 3629 (OH), 3227 (NH), 2907 (aliphatic C-H), 1767
(CdO lactone), 1521, 1507, 1483 (aromatic CdC), 1363, 1116
4â-Dim et h yla m in osu lfon a m id o-4′-O-d em et h yl-4-d es-
oxyp od op h yllotoxin (8n ). Yield: 96%. Rf ) 0.64 (CH2Cl2/
MeOH 90:10). Mp ) 228-230 °C (white powder). [R]20
)
D
-77.5 (c ) 0.390, CHCl3). 1H NMR (CDCl3) δ: 6.98 (1H, s, 5-H),
6.50 (1H, s, 8-H), 6.26 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 7.1 Hz,
CH2O2), 5.41 (1H, s, 4′-OH), 4.80-4.70 (1H, m, 11a-H), 4.60-
4.55 (1H, m, 1-H), 4.45-4.31 (2H, m, 4-H, NH), 4.24 (1H, d, J
) 7.0 Hz, 11b-H), 3.77 (6H, s, 3′,5′-OCH3), 2.98-2.77 (8H, m,
2,3-H + 2CH3). IR (CHCl3) ν: 3539 (OH), 3391 (NH), 2941
(aliphatic C-H), 1775 (CdO lactone), 1519, 1506, 1482 (aro-
matic CdC), 1331, 1117 (SO2). MS (CI) m/z: 524 [M + NH4]+.
Anal. (C23H26N2O9S) C, H, N.
(SO2). MS (CI) m/z: 557, 559 [M + NH4]+. Anal. (C24H26
ClNO9S) H, N; C, calcd 53.38; found 52.96.
-
4â-(3-Azid op r op yl)su lfon a m id o-4′-O-d em et h yl-4-d es-
oxyp od op h yllotoxin (8f). Yield: 97%. Rf ) 0.61 (CH2Cl2/
acetone 90:10). Mp ) 207-209 °C (pale yellow powder). [R]20
D
1
) -57.0 (c ) 0.49, MeOH). H NMR (DMSO-d6) δ: 8.29 (1H,
4â-Dieth yla m in osu lfon a m id o-4′-O-d em eth yl-4-d esoxy-
p od op h yllotoxin (8o). Yield: 95%. Rf ) 0.66 (CH2Cl2/MeOH
br s, 4′-OH), 7.82 (1H, br s, NH), 7.04 (1H, s, 5-H), 6.52 (1H,
s, 8-H), 6.22 (2H, s, 2′,6′-H), 6.02 (2H, d, J ) 7.2 Hz, CH2O2),
4.81-4.75 (1H, m, 4-H), 4.49 (1H, d, J ) 5.3 Hz, 1-H), 4.37
(1H, t, J ) 7.8 Hz, 11a-H), 4.12 (1H, t, J ) 9.0 Hz, 11b-H),
3.64 (6H, s, 3′,5′-OCH3), 3.54 (2H, t, J ) 6.7 Hz, CH2N3), 3.41-
3.18 (3H, m, 2-H, CH2SO2), 3.02-2.88 (1H, m, 3-H), 2.01-1.90
(2H, m, CH2â). IR (KBr) ν: 3585 (OH), 3502 (NH), 3303
(aliphatic C-H), 2105 (N3), 1768 (CdO lactone), 1522, 1505,
1482 (aromatic CdC), 1330, 1154 (SO2). MS (CI) m/z: 564 [M
+ NH4]+. Anal. (C24H26N4O9S) C, H, N.
90:10). Mp ) 129-131 °C (white powder). [R]20 ) -73.0 (c )
D
0.480, CHCl3). 1H NMR (CDCl3) δ: 7.02 (1H, s, 5-H), 6.49 (1H,
s, 8-H), 6.26 (2H, s, 2′,6′-H), 5.98 (2H, d, J ) 6.1 Hz, CH2O2),
5.41 (1H, s, 4′-OH), 4.77-4.68 (1H, m,11a-H), 4.61-4.52 (1H,
m, 1-H), 4.47-4.30 (2H, m, 4-H, NH), 4.00 (1H, d, J ) 5.7 Hz,
11b-H), 3.77 (6H, s, 3′,5′-OCH3), 3.35 (4H, q, J ) 7.0 Hz, 2
CH2), 2.98-2.82 (2H, m, 2,3-H), 1.24 (6H, t, J ) 6.9 Hz, CH3).
IR (CHCl3) ν: 3541 (OH), 3392 (NH), 2940 (aliphatic C-H),
1775 (CdO lactone), 1519, 1506, 1485 (aromatic CdC), 1330,
1150 (SO2). MS (CI) m/z: 552 [M + NH4]+. Anal. (C25H30N2O9S)
C, H, N.
4â-(4-Meth ylsu lfon am idoph en yl)su lfon am ido-4′-O-dem -
eth yl-4-d esoxyp od op h yllotoxin (8h ). Yield: 87%. Rf ) 0.64
(CH2Cl2/MeOH 90:10). Mp ) 310-311 °C. [R]20 ) -111.0 (c
4â-Dibu tyla m in osu lfon a m id o-4′-O-d em eth yl-4-d esoxy-
p od op h yllotoxin (8p ). Yield: 93%. Rf ) 0.76 (CH2Cl2/MeOH
D
) 0.385, DMSO). 1H NMR (DMSO-d6) δ: 8.65 (1H, br s, 4′-
OH), 8.31 (1H, br s, NH), 8.24 (2H, d, J ) 8.4 Hz, Ph), 8.16
(2H, d, J = 8.4 Hz, Ph), 6.48 (1H, s, 8-H), 6.18 (2H, s, 2′,6′-H),
5.95 (2H, s, CH2O2), 5.88 (1H, s, 5-H), 4.77 (1H, d, J ) 4.4 Hz,
4-H), 4.47 (1H, d, J ) 5.2 Hz, 1-H), 4.09 (1H, t, J ) 8.0 Hz,
11a-H), 3.85 (1H, t, J ) 9.1 Hz, 11b-H), 3.62 (6H, s, 3′,5′-OCH3),
3.34 (3H, s, CH3), 3.16 (1H, dd, J ) 5.2, 14.3 Hz, 2-H), 3.00-
2.88 (1H, m, 3-H). IR (KBr) ν: 3474 (OH), 3261 (NH), 2922
(aliphatic C-H), 1775 (CdO lactone), 1519, 1508, 1485 (aro-
matic CdC), 1329 (SO2). MS (CI) m/z: 635 [M + NH4]+. Anal.
(C28H27NO11S2) C, H, N.
90:10). Mp ) 98-100 °C (white crystals). [R]20 ) -53.5 (c )
D
0.510, CHCl3). 1H NMR (CDCl3) δ: 7.03 (1H, s, 5-H), 6.50 (1H,
s, 8-H), 6.26 (2H, s, 2′,6′-H), 5.98 (2H, d, J ) 5.4 Hz, CH2O2),
5.42 (1H, s, 4′-OH), 4.79-4.70 (1H, m, 11a-H), 4.60-4.51 (1H,
m, 1-H), 4.46-4.30 (2H, m, 4-H, NH), 3.91 (1H, d, J ) 6.0 Hz,
11b-H), 3.77 (6H, s, 3′,5′-OCH3), 3.30-3.21 (4H, m, 2 CH2â),
2.94-2.83 (1H, m, 2,3-H), 1.70-1.50 (4H, m, CH2γ), 1.42-1.23
(9H, m, CH3), 1.02-0.88 (6H, m, 2CH3). IR (CHCl3) ν: 3538
(OH), 3339 (NH), 2964 (aliphatic C-H), 1775 (CdO lactone),
1519, 1506, 1485 (aromatic CdC), 1330, 1117 (SO2). MS (CI)
m/z: 608 [M + NH4]+. Anal. (C29H38N2O9S) C, H, N.
4â-P en t a flu or op h en ylsu lfon a m id o-4′-O-d em et h yl-4-
d esoxyp od op h yllotoxin (8i). Yield: 93%. Rf ) 0.59 (CH2-
4â-P iper idin osu lfon am ido-4′-O-dem eth yl-4-desoxypodo-
Cl2/MeOH 90:10). Mp ) 303-305 °C (white powder). [R]20
)
p h yllotoxin (8q). Yield: 87%. Rf ) 0.61 (CH2Cl2/MeOH 90:
D
-61.5 (c ) 0.615, MeOH). 1H NMR (CDCl3) δ: 6.48 (1H, s,
5-H), 6.30 (1H, s, 8-H), 6.23 (2H, s, 2′,6′-H), 5.94 (2H, d, J )
1.2 Hz, CH2O2), 5.54 (1H, br s, 4′-OH), 4.87 (1H, d, J ) 4.7
Hz, 4-H), 4.56 (1H, d, J ) 5.2 Hz, 1-H), 4.29 (1H, t, J ) 8.6
Hz, 11a-H), 4.14 (1H, t, J ) 9.0 Hz, 11b-H), 3.75 (6H, s, 3′,5′-
OCH3), 3.33 (1H, dd, J ) 5.2, 14.3 Hz, 2-H), 3.00-2.85 (1H,
m, 3-H). IR (KBr) ν: 3504 (OH), 3227 (NH), 2942 (aliphatic
C-H), 1774 (CdO lactone), 1519, 1505, 1434 (aromatic Cd
C), 1342, 1112 (SO2). MS (CI) m/z: 647 [M + NH4]+. Anal.
(C27H20F5NO9S) H, N; C, calcd 51.51; found 51.09.
10). Mp ) 249-250 °C (white powder). [R]20 ) -69.5 (c )
D
0.480, CHCl3). 1H NMR (CDCl3) δ: 7.03 (1H, s, 5-H), 6.49 (1H,
s, 8-H), 6.26 (2H, s, 2′,6′-H), 5.98 (2H, d, J ) 5.2 Hz, CH2O2),
5.41 (1H, s, 4′-OH), 4.76-4.70 (1H, m, 11a-H), 4.60-4.52 (1H,
m, 1-H), 4.42-4.33 (2H, m, 4-H, NH), 4.21 (1H, d, J ) 6.8 Hz,
11b-H), 3.77 (6H, s, 3′,5′-OCH3), 3.31-3.18 (4H, m, 2 CH2N),
2.93-2.84 (2H, m, 2,3-H), 1.80-1.69 (4H, m, 2 CH2), 1.60-
1.55 (2H, m, CH2). IR (CHCl3) ν: 3541 (OH), 3392 (NH), 2945
(aliphatic C-H), 1775 (CdO lactone), 1519, 1506, 1485 (aro-
matic CdC), 1331, 1159 (SO2). MS (CI) m/z: 564 [M + NH4]+.
Anal. (C26H30N2O9S) C, H, N.
4â-(4-Azid op h en yl)su lfon a m id o-4′-O-d em et h yl-4-d es-
oxyp od op h yllotoxin (8j). Yield: 92%. Rf ) 0.69 (CH2Cl2/
4â-Mor p h olin osu lfon a m id o-4′-O-d em et h yl-4-d esoxy-
p od op h yllotoxin (8r ). Yield: 98%. Rf ) 0.59 (CH2Cl2/MeOH
90:10). Mp ) 248-250 °C (white crystals). [R]20D ) -68.5 (c )
0.470, CHCl3). 1H NMR (CDCl3) δ: 7.00 (1H, s, 5-H), 6.50 (1H,
s, 8-H), 6.25 (2H, s, 2′,6′-H), 5.99 (2H, d, J ) 6.3 Hz, CH2O2),
5.42 (1H, s, 4′-OH), 4.80-4.70 (1H, m, 11a-H), 4.57 (1H, d, J
) 3.7 Hz, 1-H), 4.46-4.29 (3H, m, 11b,4-H, NH), 3.83 (4H, t,
J ) 4.6 Hz, 2CH2O), 3.77 (6H, s, 3′,5′-OCH3), 3.37-3.22 (4H,
m, 2CH2N), 2.98-2.90 (2H, m, 2,3-H). IR (CHCl3): 3526 (OH),
3386 (NH), 2920 (aliphatic C-H), 1776 (CdO lactone), 1519,
1506, 1485 (aromatic CdC), 1330, 1117 (SO2). MS (CI) m/z:
566 [M + NH4]+. Anal. (C25H28N2O10S) C, H, N.
MeOH 90:10). Mp ) 173-175 °C (pale yellow powder). [R]20
D
1
) -87.5 (c ) 0.360, CHCl3). H NMR (CDCl3) δ: 7.94 (2H, d,
J ) 8.7 Hz, Ph), 7.25 (2H, d, J ) 8.7 Hz, Ph), 6.45 (1H, s,
8-H), 6.21 (2H, s, 2′,6′-H), 5.91 (2H, s, CH2O2), 5.73 (1H, s,
5-H), 5.40 (1H, s, 4′-OH), 4.60-4.51 (3H, m, 1,4-H, NH), 4.30
(2H, d, J ) 8.1, 11-H), 3.75 (6H, s, 3′,5′-OCH3), 2.92-2.81 (2H,
m, 2,3-H). IR (CHCl3) ν: 3538 (OH), 3369 (NH), 2919 (aliphatic
C-H), 2133 (N3), 1774 (CdO lactone), 1519, 1506, 1486
(aromatic CdC), 1332, 1165 (SO2). MS (CI) m/z: 598 [M +
NH4]+. Anal. (C27H24N4O9S) C, H, N.
4â-(Th iop h en -2-yl)su lfon a m id o-4′-O-d em et h yl-4-d es-
oxyp od op h yllotoxin (8l). Yield: 95%. Rf ) 0.65 (CH2Cl2/
4â-(2-Aceta m id o-4-m eth ylth ia zol-5-yl)su lfon a m id o-4′-
O-d em eth yl-4-d esoxyp od op h yllotoxin (8m ). To a solution
MeOH 90:10). Mp ) 276-277 °C (white powder). [R]20
)
D

Sulfonamide Derivatives of Epipodophyllotoxin
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2373
of 7m (439 mg, 0.6 mmol) in anhydrous THF (10 mL) and
under inert atmosphere was added Bu4NF (1.6 mL of a 1 N
solution in THF, 1.6 mmol). The reaction mixture was stirred
overnight at room temperature. THF was removed in vacuo
and CH2Cl2 (25 mL) was added. The solution was then washed
with water (10 mL). After drying (MgSO4) and filtration, the
solvent was evaporated. The obtained crude material was
purified by flash column chromatography on silica gel (100 g,
eluent CH2Cl2/MeOH 90:10) to give pure 8m (303 mg, 82%).
Rf ) 0.58 (CH2Cl2/MeOH 90:10). Mp ) 202-204 °C (pale yellow
Chemicals. Compound were dissolved in dimethyl sulfoxide at
5 mM and then diluted to working concentrations in distilled
water immediately before use.
Top oisom er a se II-Med ia ted DNA Clea va ge Assa y. Su-
percoiled pBS DNA (0.1 µg) was incubated for 15 min at 30
°C, in a 50 mM Tris-HCl buffer, pH 7.5, containing 1 mM ATP,
120 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA,
and 30 µg BSA, in the presence of the drug at 50 µM (total
reaction volume 10 µL). Two units of human DNA topo-
isomerase II 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 (final concentration 0.5%) and of proteinase
K (Sigma) to 500 µg/mL, followed by incubation for 30 min at
55 °C. DNA samples were then added to the electrophoresis
dye mixture (5 µL) and electrophoresed (35 V/cm) in a 1%
agarose gel in TBE × 1, containing ethidium bromide (1 µg/
mL), at room temperature for 2 h. Gels were washed and
photographed under UV light.
crystals from acetone/heptane). [R]20 ) -133.5 (c ) 0.115,
D
MeOH). 1H NMR (DMSO-d6) δ: 8.55 (1H, br s, NH), 8.27 (1H,
s, 4′-OH), 6.50 (1H, s, 8-H), 6.22 (2H, s, 2′,6′-H), 5.98 (2H, d,
J ) 7.4 Hz, CH2O2), 4.70 (1H, d, J ) 4.1 Hz, 4-H), 4.47 (1H, d,
J ) 5.2 Hz, 1-H), 4.17 (1H, t, J ) 8.1 Hz, 11a-H), 3.95 (1H, t,
J ) 10.1 Hz, 11b-H), 3.63 (6H, s, 3′,5′-OCH3), 3.11 (1H, dd, J
) 5.3, 14.4 Hz, 2-H), 3.08-2.92 (1H, m, 3-H), 2.47 (3H, s, CH3),
2.21 (3H, s, CH3). IR (KBr) ν: 3626 (OH), 3271 (NH), 2990
(aliphatic C-H), 1772 (CdO lactone), 1520, 1505, 1485 (aro-
matic CdC), 1375, 1154 (SO2). MS (CI) m/z: 618 [M + H]+.
Anal. (C27H27 N3O10S2) C, H, N.
Cell Cu ltu r e a n d Cytotoxicity Assa ys. Cells were cul-
tivated in RPMI 1640 medium (Gibco) supplemented with 10%
fetal calf serum, 2 mM l-glutamine, 100 units/mL penicillin,
100 µg/mL streptomycin, and 10 mM HEPES buffer (pH 7.4).
Cytotoxicity was measured by the microculture tetrazolium
assay (MTA) as described.
4â-(4-Met h ylp ip er a zin yl)su lfon a m id o-4′-O-d em et h yl-
4-d esoxyp od op h yllotoxin (8s). Compound 8s was prepared
on a 0.5 mmol scale from 7s in an analogous manner to 8m
from 7m . Yield: 84%. Rf ) 0.37 (CH2Cl2/MeOH 90:10). Mp )
223-224 °C (white powder from benzene/heptane). [R]20
)
D
Briefly, adherent cells were seeded in 96 well-microplates
and incubated for 2 days. Tested compounds were then added
and plates incubated for four doubling times. The nonadherent
L1210 cells were directly incubated for 48 h with the com-
pounds. At the end of this period, 15 µL of 5 mg/mL 3-(4,5-
dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma)
was added to each well, and the plates were incubated for 4 h
at 37 °C. The medium was aspirated and the formazan
solubilized by 100 µL of DMSO. The IC50, the concentration
reducing by 50% the optical density at 540 nm, was calculated
by a linear regression performed on the linear zone of the
dose-response curve. All the measurements were performed
in triplicate.
Cell Cycle An a lysis.29,30 L1210 cells (2.5 × 105 cells/mL)
were incubated for 21 h with various concentrations of the
compounds. Cells were then fixed in 70% ethanol (v/v), washed,
and incubated in Dulbecco’s phosphate-buffered saline (D-PBS)
containing 100 µg/mL RNase A and 25 µg/mL propidium iodide
for 30 min at 20 °C. For each sample, 104 cells were analyzed
on an Epics XL/MCL flow cytometer (Beckman Coulter).
-75.5 (c ) 0.280, DMSO). 1H NMR (DMSO-d6) δ: 8.24 (1H, s,
4′-OH), 7.86 (1H, d, J ) 8.6 Hz, NH), 6.97 (1H, s, 5-H), 6.49
(1H, s, 8-H), 6.19 (2H, s, 2′,6′-H), 6.00 (2H, d, J ) 1.3 Hz,
CH2O2), 4.71-4.61 (1H, m, 4-H), 4.44 (1H, d, J ) 5.4 Hz, 1-H),
4.30 (1H, t, J ) 7.9 Hz, 11a-H), 4.12 (1H, t, J ) 10.4 Hz, 11b-
H), 3.61 (6H, s, 3′,5′-OCH3), 3.22 (2H, dd, J ) 5.3, 14.6 Hz,
2-H), 3.18-2.89 (9H, m, 3-H, 4 CH2), 2.16 (3H, s, CH3). IR
(KBr) ν: 3567 (OH), 3342 (NH), 2937 (aliphatic C-H), 1769
(CdO lactone), 1520, 1508, 1482 (aromatic CdC), 1337, 1151
(SO2). MS (CI) m/z: 579 [M + NH4]+. Anal. (C26H31N3O9S) C,
H, N.
4â-(3-Am in op r op yl)su lfon a m id o-4′-O-d em eth yl-4-d es-
oxyp od op h yllotoxin (8g). Compound 8g was prepared on a
0.4 mmol scale from 8f in an analogous manner to 7k from 7j:
yield 86%. Rf ) 0.73 (CH2Cl2/MeOH 90:10). Mp ) 204-206
°C (white powder from benzene/heptane). [R]20 ) -92.0 (c )
D
0.39, DMSO). 1H NMR (DMSO-d6) δ: 9.06 (1H, d, J ) 8.3 Hz,
NH), 6.82 (2H, s, NH2), 6.79 (1H, s, 5-H), 6.82 (2H, s, NH2),
6.77 (1H, s, 4′-OH), 6.55 (1H, s, 8-H), 6.26 (2H, s, 2′,6′-H), 6.02
(2H, d, J ) 5.7 Hz, CH2O2), 5.38-5.22 (1H, m, 4-H), 4.51 (1H,
d, J ) 4.7 Hz, 1-H), 4.30 (1H, t, J ) 7.8 Hz, 11a-H), 4.13 (1H,
t, J ) 10.5 Hz, 11b-H), 3.65 (6H, s, 3′,5′-OCH3), 3.16 (1H, dd,
J ) 5.1, 14.4 Hz, 2-H), 3.18-2.98 (1H, s, 3-H), (2H, m, CH2-
SO2) (The signals of CH2R, -â, -γ are masked by the presence
of a broad signal of H2O in the spectra). IR (KBr) ν: 3654 (OH),
3345, 3292 (NH, NH2), 2935 (aliphatic C-H), 1768 (CdO
lactone), 1523, 1505, 1482 (aromatic CdC), 1338, 1117 (SO2).
MS (CI) m/z: 521 [M + H]+, 538 [M + NH4]+. Anal.
(C24H28N2O9S) C, H, N.
An titu m or Activity in Vivo. Murine P388 leukemia tumor
model was used as previously described.31 B6D2F1 mice were
inoculated ip with 106 tumor cells, and drugs were adminis-
tered iv the first day of the experiment.
A549 human pulmonary tumor cells were cultured and
grafted into immunodeficient mice as previously described.32
Briefly, 106 cells in a volume of 100 L were implanted through
the chest wall into the left pleural space of anesthetized
BALB/C nude mice. Mice were treated iv when the tumors
were established, on days 14, 21, and 28 after the injection of
tumor cells.
4â-(4-Am in op h en yl)su lfon a m id o-4′-O-d em eth yl-4-d es-
oxyp od op h yllotoxin (8k ). Compound 8k was prepared from
8j in an analogous manner to 7k from 7j: Yield: 95%. Rf )
0.49 (CH2Cl2/MeOH 90:10). Mp ) 286-288 °C (pale yellow
All experiments were approved by an internal ethical
committee and in accordance with the guidelines approved by
the UKCCCR for the welfare of animals in experimental
neoplasia.33 The evaluation of the antitumor activity was
represented by the life span of mice. The median survival time
(MST) of the treated group (T) was compared with that of the
control group (C), and the results were expressed as T/C (%)
) (MST of treated group/MST of control group) × 100.
crystals from benzene/heptane). [R]20 ) -73.5 (c ) 0.385,
D
DMSO). 1H NMR (DMSO-d6) δ: 8.23 (1H, br s, 4′-OH), 7.74
(1H, d, J ) 7.6 Hz, NH), 7.50 (2H, d, J ) 7.8 Hz, Ph), 6.66
(2H, d, J ) 8.1 Hz, Ph), 6.41 (1H, s, 8-H), 6.16 (2H, s, 2′,6′-H),
6.04 (2H, br s, NH2), 5.98 (1H, s, 5-H), 5.91 (2H, d, J ) 4.2
Hz, CH2O2), 4.59-4.48 (1H, m, 4-H), 4.39 (1H, d, J ) 4.9 Hz,
1-H), 4.00 (1H, t, J ) 8.0 Hz, 11a-H), 3.91 (1H, t, J ) 9.1 Hz,
11b-H), 3.58 (6H, s, 3′,5′-OCH3), 3.09 (1H, dd, J ) 4.3, 14.3
Hz, 2-H), 2.96-2.73 (1H, m, 3-H). IR (KBr): 3649 (OH), 3392,
3355 (NH, NH2), 2940 (aliphatic C-H), 1770 (CdO lactone),
1521, 1507, 1486 (aromatic CdC), 1332, 1151 (SO2). MS (CI)
m/z: 572 [M + NH4]+. Anal. (C27H26N2O9S) C, H, N.
Tu bu lin e Test. Tubulin polymerization inhibition was
determined as previously reported.34
Ack n ow led gm en t. This work was supported by
grants from Fondation de France (to D.G.) and from
Ligue National contre le Cancer and Ministe`re de la
Recherche et de l’Industrie (ACI “Mole´cules et Cibles
The´rapeutiques”) (to P.B.A.).
DNA a n d Bioch em ica ls. The plasmid pBS was obtained
from Stratagene. Purified human topoisomerase IIR was
purchased from TopoGEN Inc. and etoposide from Sigma

2374 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Guianvarc’h et al.
(20) Arimondo, P.; Boukarim, C.; Bailly, C.; Dauzonne, D.; Monneret,
C. Design of two etoposide-amsacrine conjugates: Topoisomerase
II and tubuline polymerization inhibition and relation to cyto-
toxicity. Anti-Cancer Drug Design 2000, 15, 413-421.
(21) Imbert, T.; Guminski, Y. Method for preparing 4′-demethylepi-
podophyllotoxin from podophyllotoxin. PCT Int. Appl. WO 97
21, 713 (Cl. CO7D493/04), 19 J un 97; Fr. appl. 95/14, 875, 14
Dec 1995; pp 20 (Fr). Chem. Abstr. 1997, 127, 121598a.
(22) Zhou, X.-M.; Wang, Z.-Q.; Chang, J .-Y.; Chen, H.-X.; Cheng, Y.-
C, Lee, K.-H. Antitumor agents. 120. New 4-substituted benzy-
lamine and benzyl ether derivatives of 4′-O-demethylepipodo-
phyllotoxin as potent inhibitors of human DNA topoisomerase
II. J . Med. Chem. 1991, 34, 3346-3350.
(23) Chen, M.; Beck, W. T. Differences in inhibition of chromosome
reparation and G2 arrest by DNA topoisomerase II inhibitors
merbarone and VM-26. Cancer Res. 1995, 55, 1509-1516.
(24) Guilbaud, N.; Kraus-Berthier, L.; Meyer-Losic, F.; Malivet, V.;
Chacun, C.; J an, M.; Tillequin, F.; Michel, S.; Koch, M.; Pfeiffer,
B.; Atassi, G.; Hickman, J .; Pierre´, A. Marked antitumor activity
of a new potent acronycine derivative in orthotopic models of
human solid tumors. Clin. Cancer Res. 2001, 7, 2573-2580.
(25) Binkley, W. W.; Degering, E. F. Organic syntheses with sulfuryl
chloride. J . Am. Chem. Soc. 1939, 61, 3250-3251.
(26) Chatterjee, S.; Ator, M. A.; Bozyczko-Coyne, D.; J osef, K.; Wells,
G.; Tripathy, R.; Iqbal, M.; Bihovsky, R.; Senadhi, S. E.; Mallya,
S.; O’Kane, T. M.; McKenna, B. A.; Siman, R.; J ohn, P.; Mallamo,
J . P. Synthesis and biological activity of a series of potent
fluoromethyl ketone inhibitors of recombinant human calpain
I. J . Med. Chem. 1997, 40, 3820-3828.
(27) Denivelle, L. Action de l′ammoniac et des amines sur les
chlorosulfates d′aryle et les N-chlorosulfonyl-sulfonamides. Bull.
Soc. Chim. Fr. 1936, 3, 2143-2152.
(28) Matier, W. L.; Comer, W. T. Sulfamoyl azides. Hydrolysis rates
and hypotensive activity. J . Med. Chem. 1972, 15, 538-541.
(29) Leonce, S.; Anstett, M.; Combe-Perez, V.; Pierre, A. Flow
cytometric evaluation of the cell cycle perturbations induced by
S12363, a new vinca alkaloid derivative. Anti-Cancer Drugs
1990, 1, 179-183.
(30) Pierre´, A.; Perez, V.; Leonce, S.; Boutin, J . A.; Saint-Dizier, D.;
Hautefaye, P.; Lavielle, G.; Atassi, G. Relationship between the
cellular accumulation and the cytotoxicity of S12363, a new
Vinca alkaloid derivative. Cancer Chemother. Pharmacol. 1992,
29, 367-374.
Refer en ces
(1) Slevin, M. L. The clinical pharmacology of etoposide. Cancer
1991, 67, 319-329.
(2) Hande, K. R. Etoposide: Four decades of development of a
topoisomerase II inhibitor. Eur. J . Cancer 1998, 34, 1514-1521.
(3) Chabner, B. A.; Longo, D. L. Cancer chemotherapy and bio-
therapy. Principles and practice, 2nd ed.; Lippincott-Raven
Publishers: New York, 1996.
(4) Monneret, C.; Daley, L. Nouveaux analogues hydrosolubles de
l′e´toposide. Actualite´s Chim. The´r. 1997, 23, 267-279.
(5) Wang, J . C. DNA topoisomerases. Annu. Rev. Biochem. 1996,
65, 635-692.
(6) Burden, D. A.; Osheroff, N. Mechanism of action of eukaryotic
topoisomerase II and drugs targeted to the enzyme. Biochim.
Biophys. Acta 1998, 1400, 139-154.
(7) Leroy, D.; Kajava, A. V.; Frei, C.; Gasser, S. M. Analysis of
etoposide binding to subdomains of human DNA topoisomerase
II alpha in the absence of DNA. Biochemistry 2001, 40, 1624-
1634.
(8) Pedersen-Bjergaard, J . Radiotherapy- and chemotherapy-in-
duced myelodysplasia and acute myeloid leukemia. A review.
Leuk. Res. 1992, 16, 61-65.
(9) Felix, C. A. Secondary leukemias induced by topoisomerase-
targeted drugs. Biochim. Biophys. Acta 1998, 1400, 233-255.
(10) Lazo, J . S.; Li, T.; Woo, E. S.; Settineri, C. E.; Allan, W. P.;
Yalovich, J . C. Chemical synthesis and biological activity of a
novel fluorescent etoposide derivative. Biochem. Pharmacol.
1997, 53, 715-722.
(11) Zhu, X.-K.; Guan, J .; Tachibana, Y.; Bastow, K. F.; Cho, S. J .
Cheng, H.-H.; Cheng, Y.-C.; Gurwith, M.; Lee, K.-H. Antitumor
agents. 194. Synthesis and biological evaluations of 4â-mono-,
-di-, and -trisubstituted aniline-4′-O-demethyl-podophyllotoxin
and related compounds with improved pharmacological profiles.
J . Med. Chem. 1999, 42, 2441-2446.
(12) Xiao, Z.; Xiao, Y.-D.; Feng, J .; Golbraikh, A.; Tropsha, A.; Lee,
K.-H. Antitumor agents. 213. Modeling of epipodophyllotoxin
derivatives using variable selection k nearest neighbor QSAR
method. J . Med. Chem. 2002, 45, 2294-2309.
(13) Saito, H.; Yoshikawa, H.; Nishimura, Y.; Kondo, S.; Takeuchi,
T.; Umezawa, H. Studies on lignan lactone antitumor agents.
II. Synthesis of N-alkylamino- and 2,6-dideoxy-2-aminoglycosidic
lignan variants related to podophyllotoxin. Chem. Pharm. Bull.
1986, 34, 3741-3746.
(14) Daley, L.; Guminski, Y.; Demerseman, P.; Kruczynski, A.;
Etievant, C.; Imbert, T.; Hill, B. T.; Monneret, C. Synthesis and
antitumor activity of new glycosides of epipodophyllotoxin,
analogues of etoposide, and NK 611. J . Med. Chem. 1998, 41,
4475-4485.
(15) Zheng, J .; Wang, H.-K.; Bastow, K. F.; Zhu, X.-K.; Cho, S. J .;
Cheng, Y.-C.; Lee, K.-H. Antitumor agents. 177. Design, syn-
theses, and biological evaluation of novel etoposide analogues
bearing pyrrolecarboxamidino group as DNA topoisomerase II
inhibitors. Bioorg. Med. Chem. Lett. 1997, 7, 607-612.
(16) Lee, K.-H.; Wang, H.-K. Antitumor agents. 165. Current status
of bioanalysis of etoposide and related compounds. J . Food Drug
Anal. 1995, 3, 209-232.
(17) Macdonald, T. L.; Lehnert, E. K.; Loper, J . T.; Chow, K.-C.; Ross,
W. E. DNA topoisomerases in Cancer; Oxford University Press:
New York, 1991; pp 199-214.
(18) Fosse´, P.; Rene´, B.; Saucier, J . M.; Henichart, J . P.; Waring, M.
J .; Colson, P.; Houssier, C.; Bailly, C. Stimulation of site-specific
topoisomerase II-mediated DNA cleavage by an N-methylpyr-
rolecarboxamide-anilinoacridine conjugate: Relation to DNA
binding. Biochemistry 1994, 33, 9865-9874.
(31) Guilbaud, N.; Kraus-Berthier, L.; Saint-Dizier, D.; Rouillon, M.
H.; J an, M.; Burbridge, M.; Visalli, M.; Bisagni, E.; Pierre´, A.;
Atassi, G. In vivo antitumor activity of S 16020-2, a new olivacine
derivative. Cancer Chemother. Pharmacol. 1996, 38, 513-521.
(32) Kraus-Berthier, L.; J an, M.; Guilbaud, N.; Naze, M.; Pierre´, A.;
Atassi, G. Histology and sensitivity to anticancer drugs of two
human nonsmall cell lung carcinomas implanted in the pleural
cavity of nude mice. Clin. Cancer Res. 2000, 6, 297-304.
(33) Workman, P.; Balmain, A.; Hickman, J . A.; McNally, N. J .;
Rohas, A. M.; Mitchinson, N. A.; Pierrepoint, C. G.; Raymond,
R.; Rowlatt, C.; Stephens, T. C.; Wallace, J .; Straughan, D. W.
UKCCCR guidelines for the welfare of animals in experimental
neoplasia. Lab. Anim. 1988, 22, 195-201.
(34) Zavala; F.; Guenard, D.; Robin, J .-P.; Brown, E. Structure-
Antitubulin Activity Relationships in Steganacin Congeners and
Analogues. Inhibition of Tubulin Polymerization in Vitro by (()-
Isodeoxypodophyllotoxin. J . Med. Chem. 1980, 23, 546-549.
(19) Rene´, B.; Fosse´, P.; Khe´lifa, T.; J acquemin-Sablon, A.; Bailly,
C. The 1′-substituent on the anilino ring of the antitumour drug
amsacrine is a critical element for topoisomerase II inhibition
and cytotoxicity. Mol. Pharmacol. 1996, 49, 343-350.
J M031117B