Synthesis and antitumor activity of new glycosides of epipodophyllotoxin, analogues of etoposide, and NK 611

Article abstract of DOI:10.1021/jm9800752

A series of 3-amino- and 3-alkylamino-2-deoxy-β-D-ribo- and β-D- arabino-glycosides of 4'-demethylepipodophyllotoxin have been synthesized by means of an improved trimethylsilyl-iodide procedure for the podophyllotoxin- 4'-demethylepipodophyllotoxin conversion, an efficient and high yielding synthesis of silyl glycoside donors of 3-azido-2,3-dideoxy-β-D-ribo-and β- D-arabino-hexopyranosides and stereoselective glycosylations. In vitro evaluation of cytotoxic effects against murine L1210 leukemia critically demonstrates the essential role played by a 4,6-acetal for biological activity. Among the most cytotoxic compounds, 3-amino-2,3-dideoxy-and 3-N,N- (dimethylamino)-2,3-dideoxy etoposide analogues, 17 and 27-29 are at least as potent as etoposide on the in vivo P388 (iv/ip) murine leukemia models. However, surprisingly enough, none of these compounds inhibits the human DNA topoisomerases I or II or binds to tubulin to prevent its polymerization and microtubule assembly. Therefore, their mechanism of action remains to be cleared up.

Full text of DOI:10.1021/jm9800752

J . Med. Chem. 1998, 41, 4475-4485
4475
Syn th esis a n d An titu m or Activity of New Glycosid es of Ep ip od op h yllotoxin ,
An a logu es of Etop osid e, a n d NK 611
Laurent Daley,† Yves Guminski,‡ Pierre Demerseman,† Anna Kruczynski,§ Chantal Etie´vant,‡ Thierry Imbert,‡
Bridget T. Hill,§ and Claude Monneret*,†
UMR 176 CNRS/ Institut Curie, Section Recherche, 26 rue d’Ulm, F-75248 Paris Cedex 05, France, and Pierre Fabre
Me´dicament, C.R.P.F., 17 Avenue J ean-Moulin, F-81106 Castres, France
Received February 2, 1998
A series of 3-amino- and 3-alkylamino-2-deoxy-â-D-ribo- and â-D-arabino-glycosides of 4′-
demethylepipodophyllotoxin have been synthesized by means of an improved trimethylsilyl-
iodide procedure for the podophyllotoxin-4′-demethylepipodophyllotoxin conversion, an efficient
and high yielding synthesis of silyl glycoside donors of 3-azido-2,3-dideoxy-â-D-ribo- and â-D-
arabino-hexopyranosides and stereoselective glycosylations. In vitro evaluation of cytotoxic
effects against murine L1210 leukemia critically demonstrates the essential role played by a
4,6-acetal for biological activity. Among the most cytotoxic compounds, 3-amino-2,3-dideoxy-
and 3-N,N-(dimethylamino)-2,3-dideoxy etoposide analogues, 17 and 27-29 are at least as
potent as etoposide on the in vivo P388 (iv/ip) murine leukemia models. However, surprisingly
enough, none of these compounds inhibits the human DNA topoisomerases I or II or binds to
tubulin to prevent its polymerization and microtubule assembly. Therefore, their mechanism
of action remains to be cleared up.
In tr od u ction
further development of this drug in phase II trials is
soon expected.
Following the discovery of the antitumor activity of
the natural lignan, podophyllotoxin,1 clinical trials were
undertaken in the 1950s.2 However, these trials were
stopped because of excessive toxicity and lack of clinical
response. Nevertheless, as a result of a general pro-
gram in attempts to find a less toxic podophyllotoxin
analogue and/or derivative, 4′-demethylpodophyllotoxin
4-(4,6-O-ethylidene)-â-D-glucopyranoside (or etoposide
or VP-16) and the corresponding 4,6-O-thenylidene (or
teniposide or VM-26) emerged as the most interesting
glucosides in the 1970s.3
Etoposide is now widely used in clinical cancer
chemotherapy, alone or in association, for the treatment
of testicular carcinomas and small cell lung cancer4 but
its current therapeutic use is somewhat limited by
myelosuppression, particularly neutropenia.5 Unlike
podophyllotoxin, etoposide and teniposide do not inhibit
tubuline polymerization but induce dose-dependent
DNA-strand breakage associated with inhibition of
DNA-topoisomerase II.6 Therefore, the researchers’
efforts in this area were directed toward modification
of epipodophyllotoxin glucosides as potent topoisomerase
II inhibitors by synthesizing amino glycoside,7 thio
glycoside,8 and N- or C-glycoside analogues.9,10
In this report, we describe the synthesis of new
glycosides of epipodophyllotoxin and the in vivo struc-
ture-activity relationships of this new series.
Although less active than NK 611, 3-amino-â-D-
glucoside analogues were nevertheless found7a to have
higher activity than etoposide against murine L1210
leukemia cell lines. In view of these results, we were
interested in further modifications of the sugar moiety.
Therefore, we turned our attention toward 3-amino-2,3-
dideoxy sugars, closely related to the corresponding
3-amino-2,3,6-trideoxy-L-hexoses12 occurring in natural
antibiotics belonging to the anthracycline family13 and
to the vancomycin group.14 Lacking the OH group at
C-2, such 3-amino-2,3-dideoxy D-sugars were revealed
to possess an interesting balance between hydrophilicity
and lipophilicity with respect to intracellular penetra-
tion while keeping the faculty to afford water-soluble
salts such as hydrochlorides. This feature has already
been used to prepare potent antitumor nitrosourea-
sugar derivatives such as ecomustine,15 a compound
endowed of potent antitumor activity and, above all,
highly active against colon-38 carcinoma.16
Ch em istr y
Among all these new glycosides, NK 611,7b which
differs from etoposide in the replacement of the glucose
moiety with the 2-N,N-dimethylglucosamine residue,
has raised clinical trials for the past few years. Like
etoposide, NK 611 inhibits topoisomerase II activity but
is, advantageously, 120-fold more soluble in water than
etoposide. Following preliminar clinical information,11
Ten years ago, some of us reported17 a highly efficient
synthesis of 3-amino-2,3,6-trideoxy-L-hexoses starting
from 3,4-di-O-acetyl-L-rhamnal (or 3,4-di-O-acetyl-6-
deoxy-L-glucal) and, very recently,18 this synthesis was
extended to the 3,4,6-tri-O-acetyl-D-glucal 1. Based
upon Michael addition of NaN3 to the corresponding
hex-2-enopyranose, this methodology straightly afforded
3-azido-2,3-dideoxy precursors 2 in a one-pot reaction
(Scheme 1). The problem was the control of the stere-
ochemistry at the anomeric position in order to separate
the arabino and the ribo isomers. This could be
† UMR 176 CNRS/Institut Curie, Section Recherche, Paris.
‡ Pierre Fabre Me´dicament C.R.P.F., Division de Chimie Me´dicinale.
§ Pierre Fabre Me´dicament C.R.P.F., Division Cance´rologie Expe´ri-
mentale.
10.1021/jm9800752 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/17/1998

4476 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Daley et al.
Sch em e 1a
Sch em e 2a
a
Reagents: (a) TBDMSCl, imidazol; (b) MeONa/MeOH; (c)
ClCH2COCl, pyridine; (d) CH3CO(OEt)2, APTS.
achieved by using pivaloyl chloride and pyridine, since
only â-anomers were observed under these conditions
resulting from a kinetically preferred â-alkylation.19 In
a next investigation, we decided to look toward the
formation of 1-O-tert-butyldimethylsilyl glycosides which
are known as excellent glycosyl donors for the synthesis
of 2-deoxy glycosides.20,21 Moreover usually silylation
of the free anomeric OH group in DMF/imidazole leads
predominantly, or stereoselectively, to the â-anomers in
the range of 80-95%.
Indeed treatment of the crude azido sugar mixture 2
with tert-butyldimethylsilyl chloride in the presence of
imidazole afforded the azido derivatives of â-D-ribo and
â-D-arabino configuration, 3 and 4, respectively, in 75%
overall yield and 1:2 ratio. Subsequent transesterifi-
cation (NaOMe-MeOH) led to the corresponding 4,6-
diols 5 and 6 which were immediatly protected as their
chloroacetyl esters 7 and 8.22 Alternatively, these azido
sugars 5 and 6 were converted into their 4,6-O-eth-
ylidene acetals 9 and 10, with the acetal group present
in etoposide itself, by treatment with 1,1-diethoxy-
ethane/TsOH.
a
Reagents: (a) HBr then BaCO3; (b) TMSI then BaCO3; (c)
PhCH2OCOCl, Et3N; (d) BF3-Et2O; (e) TMSOTf; (f) Amberlite
OH-; (g) CH3CH(OEt)2, TsOH; (h) H2, Pd/C.
quently protected at the 4′-position as its benzyl carbon-
ate, leading to 13 (≈70% overall yield)31 (Scheme 2).
The first coupling reaction of 13 was undertaken with
the glycosyl donor of D-ribo configuration 7, in the
presence of trimethylsilyltriflate (TMSOTf). The â-gly-
coside 14 was selectively isolated in 50% yield and then
deprotected (Amberlite OH-) on the sugar moiety to give
15 in 97% yield. This was treated with 1,1-diethoxy-
ethane in the presence of p-TsOH to afford (66%) the
corresponding 4,6-O-ethylidene derivative 16. Alterna-
tively, 16 was also prepared in 36% yield by glycosyla-
tion of 13 with the glycosyl donor having the 4,6-acetal
group already present, i.e., 9, in the presence of TM-
SOTf. However, in this case, better yield was obtained
when boron trifluoride etherate was used (47%) instead
of TMSOTf. The â-ribo-glycoside 16 was subsequently
converted into 17 (H2, 10% Pd/C, 88% yield).
On the other hand, classical conversion of podophyl-
lotoxin 11 into 4′-demethyl-epipodophyllotoxin simul-
taneously and selectively deprotected at the 4′-position,
such as 12, involved treatment of 11 with HBr in 1,2-
dichloroethane-ether (10:1) at 0 °C followed by hy-
drolysis (BaCO3 in H2O).23 A slightly modified version
was next proposed by Lee et al.24 with HBr gas in CH2-
Cl2 but in an overall yield not exceeding 52%. Consid-
ering these literature data (low yield and lack of
reproductive yield on higher scale), we decided to
explore the possibility of using trimethylsilyl iodide
(TMSI) instead of HBr. Indeed, it has been shown that
TMSI is able not only to selectively cleave25 the 2-OMe
ether in the 2,3-dimethoxytoluene but also to convert a
hydroxyl group into the corresponding iodo derivative
with inversion of configuration.26-29 Moreover, it should
be noticed that the potential usefulness of TMSI for the
two reactions aforementioned has been exemplified in
one report30 on the synthesis of structural analogues of
epipodophyllotoxin, although not in a sequential ap-
plication.
The highly stereoselective formation of the â-glycoside
may be explained in terms of kinetic product resulting
from the attack of the intermediate enoxonium ion on
the opposite side of the azido group leading to antipar-
rallel dipoles at C1-C3. This hypothesis seems to be in
agreement with the next result obtained with the azido
sugar of D-arabino configuration 8. In this case, con-
densation with epipodophyllotoxin 13, under the same
conditions as above, resulted in the exclusive formation
of the R-glycoside 18 in 65% yield (Scheme 3). In this
latter case, the higher yield and stereoselectivity can
be seen as the result of both kinetic and thermodynamic
controls. This R-arabino-glycoside 18 was then depro-
tected (19) (Amberlite OH-) and successively converted
Therefore, podophyllotoxin 11 was treated with TMSI
and barium carbonate to afford 12 which was subse-

New Glycosides of Epipodophyllotoxin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4477
Sch em e 3a
Sch em e 4a
a
Reagents: (a) BF3Et2O; (b) TMSOTf; (c) Amberlite OH-; (d)
CH3CH(OEt)2, TsOH; (e) H2, Pd/C.
into 20 (1,1-diethoxyethane, p-TsOH, 90%) and 21 (H2,
10% Pd/C, 75%).
The remaining problem was to prepare the â-glyco-
sides of arabino configuration, since it has been reported
that only the â-glycosides of epipodophyllotoxin were
endowed with antitumor properties.32 One way to
overcome this difficulty could be found in a glycosidation
reaction type Kuhn,33 which involved a reverse addition
mechanism by formation of a benzylic cation on the
aglycon moiety and attack from a 1-â-OH sugar. At first
compound 10 was treated with Bu4NF in THF to deliver
the reduced analogue by cleavage of the O-Si bond.
This led to the E-unsaturated compound 22 proceeding
from a retro-Michae¨l reaction with departure of the
azido group. To avoid this problem, the azido group was
reduced prior to the desilylation step and the amino
derivative 23 protected as its benzyloxycarbonyl deriva-
tive 24 (70% overall yield). In this case, desilylation of
24 gave the expected reduced analogue 25 but as a
mixture of R- and â-anomers in a 1:1 ratio after
a
Reagents: (a) Bu4NF/THF; (b) H2, Pd/C; (c) PhCH2OCOCl,
Et3N; (d) BF3/Et2O; (e) HCHO, NaBH3CN.
Sch em e 5a
1
extraction, as shown by H NMR spectrum. One way
of avoiding this epimerization, which probably occurs
during the workup, could be through the formation of
free sugar in situ, followed by immediate glycosidation
without any purification. Consequently, we decided to
undertake a “one-pot” reaction involving successive
additions to 24 of Bu4NF (1.2 equiv), epipodophyllotoxin
derivative 13 (1.1 equiv), and lastly, BF3‚Et2O catalyst
(15 equiv). Such a hypothesis proved to be right, since
this exclusively afforded the â-glycoside 26 in 54% yield.
The next transformation involved reductive hydro-
genolysis to afford 27 (90%).
a
Reagents: (a) HCHO, NaBH3CN; (b) ICH2CN, Et3N; (c)
ICH2CH2NHZ, Et3N; (d) H2, Pd/C; (e) (ICH2CH2)2O, Et3N.
mediate 31 was deprotected by hydrogenolysis in the
presence of 10% Pd/C.
To compare the activity of new glucosides with NK
611, our next objective was to prepare the 3-dimethyl-
amino derivatives, namely 28 and 29, by reductive
alkylation (HCHO, NaBH3CN) of 17 and 27, respec-
tively (Schemes 4 and 5). For their part, N-alkyl
derivatives such as N-cyanoethyl, N-aminoethyl, and
N-morpholino residues, 30, 32, and 33, were obtained
(Scheme 5) by condensation of the amino-glycoside of
ribo configuration 17 with the appropriate alkyl iodide
in the presence of Et3N. In the case of 32, the inter-
To ascertain whether the presence of an acetal group
at C-4-C-6 was a requisite condition for biological
activity as observed in the glycoside series, the azido
sugar 5 was converted (Scheme 6) into the key inter-
mediate 6-iodo-6-deoxy monosaccharide 36 via selective
tosylation at C-6, replacement of the tosyloxy group by
iodine, and then chloroacetylation (5 f 34 f 35 f 36)
in 50% overall yield.
This iodo sugar 36 was condensed with 13 to cleanly
afford (48%) the â-glycoside 37. Introduction of an azido

4478 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Daley et al.
Sch em e 6a
a
Reagents: (a) TsCl, pyridine; (b) NaI, acetone; (c) ClCH2COCl, pyridine; (d) BF3/Et2O; (e) NaN3; (f) Amberlite OH-; (g) H2, Pd/C.
Ta ble 1. In Vitro Cytotoxic Activity
Ta ble 2. In Vitro Activity on Topoisomerase and Tubulin
L1210
topoisomerase II
TPI
compd
IC50 (µM)a
compd
EC50 (µM)a
IC50 (µM)b
17
21
27
28
29
30
32
33
40
42
NK 611
etoposide
0.23
1.3
0.07
0.11
0.09
2.0
17
21
27
28
29
30
32
33
40
42
>100
>100
>100
>100
>100
>1
>10
>50
>50
>50
>50
>100
>10
NT
11
>10
NT
NT
3
13
1.7
>100
>1
>1.0
0.21
12
0.33
0.20
>100
5.6
56
etoposide
NK 611
podophyllotoxin
4′-DMEP
56
NTc
NT
a
IC50: concentration of drug required to reduce cell growth to
50% of that obtained with control cell.
a
EC50: concentration at which any inhibitory effect could be
detected. The EC50 values reported here represent the mean of
two separate experiments. IC50: concentration leading to 50%
group by reacting 37 with NaN3 led to the triazido
glycoside 38 (90%) resulting from the simultaneous Cl
substitution as present in the chloroacetyl group. 3,6-
Diamino glycoside 40 was obtained by treatment of 38
with Amberlite (IRA 410 OH-) and hydrogenolysis (89%
overall yield) of intermediate 39. Alternatively, 37 was
subsequently transformed by Amberlite treatment into
41, and hydrogenolysis of 41 (36% overall yield) led to
the corresponding 3-amino-6-deoxy glycoside 42.
b
tubulin polymerization. The IC50 values reported here were
determined from five independent experiments. c Not tested.
trideoxy glycoside 40 was endowed with potent cytotoxic
activity. Indeed this latter result is in contrast with
the accepted results within the etoposide or NK 611
series, where an ethylidene acetal was proved to be an
important structural requirement to enhance cytotoxic
properties.7a,b
To investigate the mechanism of action of these new
glycosides, inhibition of topoisomerases I and II and
tubulin polymerization inhibition experiments were
carried out, and results are shown in Table 2. None of
the synthesized compounds interfered with topo-
isomerase II, considered as a classical target for epi-
podophyllotoxin derivatives,34 up to a 10-4 M concentra-
tion. Only one compound (42), without the ethylidene
acetal moiety, consistently inhibited the capacity of
topoisomerase II (EC50 ) 5.6 µM) to cleave double-
stranded kDNA with a 10-fold higher activity than NK
611 and etoposide (IC50 ) 56 µM). As compound 42 is
not cytotoxic and constitutes a potent topoisomerase II
inhibitor, correlatively with 27, 28, 17, 29, and 40 which
are cytotoxic and not topoisomerase II inhibitors, the
Resu lts a n d Discu ssion
In vitro cytotoxic activity results assessed with the
L1210 murine leukemia model are shown in Table 1.
â-glycosides of 4′-demethylepipodophyllotoxin having a
3-amino-2,3-dideoxy or a 3-(dimethylamino)-2,3-dideoxy
sugar moiety along with a 4,6-O-ethylidene acetal, i.e.,
17, 27, 28, and 29 showed the same order of magnitude
in terms of cytotoxic potency, compared to etoposide or
NK 611 reference compounds. The R-glycosyl derivative
21 as well as the aminoalkyl derivatives 30-33 were
less cytotoxic. Among the two compounds which did not
feature an ethylidene group, the 3′-amino-2′,3′,6′-
trideoxy glycoside 42 was nearly inactive whereas, for
a priori unexplainable reason, the 3′,6′-diamino-2′,3′,6′-

New Glycosides of Epipodophyllotoxin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4479
Ta ble 3. In Vivo P388 Murine Leukemia (iv/ip)
reference compounds (i.e., 27, 28, 17, 29, 40). No in
vitro activity was shown against topoisomerase I, II, or
tubulin for compounds which displayed in vivo activity.
Potent in vivo activity was displayed for two compounds,
27 and 28, on the P388 (iv/ip) murine leukemia model,
relative to the reference compounds, while two others,
17 and 29, proved as potent as etoposide. Though 10
times more potent than the reference compounds against
topoisomerase II enzyme, compound 42 did not show
any in vivo antitumor activity in the P388 test.
water
solubility
(mg/mL)
ED50
(mg/kg)
T/C max (%)
[dose (mg/kg)]
compd
17 (R-NH2)
11
10
9
5
300 [160]
21 (â-NH2)
27 (â-NH2)
28 (â-NMe2)
29 (R-NMe2)
30 (R-NHCH2CN)
32 (R-NH(CH2)2NH2)
33 (R-N-morpholino)
40
inact. (40)
1.7
1.7
193 [40]
243 [40]
257 [40]
186 [160]
24
26
ins.
40
ins.
42
13
15
5
20
inact. (40)
inact. (40)
inact. (40)
inact. (40)
0.9
Further studies are currently underway to assess the
potential activity of these analogues against DNA as
minor grove binders or against putative target enzymes
and to enlighten their mechanism of action.
42
NK 611
228 [20]
100 [40]
242 [160]
etoposide
0.01
5
Exp er im en ta l Section
Ch em ica l Meth od s. Gen er a l P r oced u r e. Melting points
were determined using an Electrothermal apparatus and are
uncorrected. UV spectra were determined on a Varian-Cary/
3E spectrophotometer. IR spectra were obtained with a
Perkin-Elmer 1710 spectrophotometer. 1H NMR spectra were
recorded in the given solvent with a Bruker AC-250 spectrom-
eter. Chemical shifts are reported as δ values in parts per
million. The splitting pattern abbreviations are as follows: s
) singlet, d ) doublet, dd ) double doublet, dt ) double triplet,
t ) triplet, br ) broad, m ) multiplet. Chemical ionization
(CI) mass spectra were recorded on a Nermag R 10,10C
spectrometer. Elemental analyses, performed by the Service
de Microanalyse du CNRS (Vernaison-Lyon, France), were
within 0.4% of the theoretical values calculated for C, H, and
N. The thin-layer chromatographic analyses were performed
using precoated silica gel (60F254) plates, and the spots were
examined with UV light and phosphomolybdic acid spray.
Column chromatography was carried out on Merck silica gel
(230-240 mesh). Extraction in usual manner refers to wash-
ing the organic layer with water, drying it over MgSO4, and
evaporating the solvent under reduced pressure.
ter t-Bu tyld im eth ylsilyl 3-Azid o-4,6-d i-O-a cetyl-2,3-d i-
d eoxy-â-D-r ibo (3) a n d â-D-a r a bin o (4) Hexop yr a n osid es.
To a cooled solution (0 °C) of the crude mixture 218 (16.1 g, 58
mmol) in anhydrous dichloromethane (200 mL) were added
imidazole (6 g, 87.8 mmol) and tert-butyldimethylsilyl chloride
(13.24 g, 87.8 mmol). The reaction mixture was stirred at the
same temperature for 0.5 h and then allowed to reach room
temperature with further stirring for 18 h. It was poured into
H2O (500 mL) and extracted with CH2Cl2 (300 mL). The
organic layer was separated and dried over anhydrous MgSO4.
Flash chromatography (9:1 cyclohexane/EtOAc) of the residue
(18.7 g) successively led to 3 (4.5 g, 20%), to a mixture of 3
and 4 (2.7 g, 12%), and then to 4 (9.5 g, 43%).
problem of direct correlation between inhibition of this
enzyme and cytotoxic activity is posed in this new series.
Though tubulin polymerization inhibition (TPI) was
lost in the epipodophyllotoxin series relative to podo-
phyllotoxin, as already known, this inhibition test was
also performed. Interestingly, compound 40 showed a
modest but consistent inhibition of TPI (IC50 ) 11 µM)
closely related to that of 4′-demethyl-epipodophyllotoxin
(4′-DMEP) with an IC50 ) 13 µM. The 4′-DMEP proved
to be slightly less cytotoxic than podophyllotoxin (in-
ternal results). Hence, the cytotoxic activity of 40 (IC50
) 0.21 µM) could hardly be attributed to its TPI
mechanism.
In vivo antitumor activity displayed by the studied
compounds against the P388 murine leukemia model
(iv/ip) is recorded in Table 3. The compounds of the
arabino series 27 and 28, with a 3′′-â-amino group in
equatorial position relative to the pyran ring, appeared
nearly as potent as NK 611, with an ED50 equal to 1.7
mg/kg versus 0.9 mg/kg for NK 611. They exhibited a
relatively greater potency than the ribo analogues 17
and 29, with a 3′′-R amino group in an axial position
relative to the pyran ring. Compounds 17 and 29, with
an ED50 ) 5 mg/kg, were shown to be as potent as
etoposide. No difference was shown between primary
and tertiary amine substitution, 27 versus 28 and 17
versus 29. Moreover, bulky axial amino groups, as in
30, 32, and 33, reduced (30) or totally abolished activity
(32, 33). These results indicated the unfavorable oc-
cupancy of the R-side of the glucoside moiety. It is
noteworthy that compounds 27, 28, 17, and 29 induced
increased the life span of P388 leukemia-bearing mice
with a T/C ratios ranging from 193 to 300%; these
values are identical or higher than that for NK 611 or
etoposide. Consequently, compounds 27, 28, and 29
could be administrated up to 40 mg/kg and 17 up to 160
mg/kg to obtain the greatest increase of lifespan, in
comparison with NK 611. So, these compounds ap-
peared less toxic than NK 611 and they would afford a
better putative flexibility in the dose schedule than NK
611, with which activity was lost at 40 mg/kg. Water
solubility made these compounds attractive, the values
recorded in Table 3 proved to be sufficiently adequate
to overcome administration problems.
20
Compound 3, syrup: Rf 0.52 (4:1 cyclohexane/EtOAc); [R]D
-10° (c 1.0, CHCl3); IR (CDCl3) 2104 (N3), 1745 (CdO) cm-1
;
1H NMR (300 MHz, CDCl3) δ 5.02 (dd, 1H, J ) 8.5, 2 Hz, H1),
4.89 (dd, 1H, J ) 9.5, 3.5 Hz, H4), 4.22-4.17 (m, 3H, H3, H6,
H6′), 4.12-4.05 (m, 1H, H5), 2.13 (s, 3H, CH3CO), 2.05 (s, 3H,
CH3CO), 2.03 (ddd, 1H, J ) 14, 4, 2 Hz, H2e), 1.64 (m, 1H, J
) 14, 8.5, 3.5 Hz, H2a), 0.88 (s, 9H, t-Bu), 0.10 (s, 6H, CH3-
Si); MS (CI) m/z 405 (M + NH4)+. Anal. (C16H19N3O6S) C, H,
N.
20
Compound 4, syrup: Rf 0.45 (4:1 cyclohexane/EtOAc); [R]D
+10° (c 1.4, CHCl3); IR (CDCl3) 2103 (N3), 1746 (CdO) cm-1
;
1H NMR (300 MHz, CDCl3) δ 4.86 (dd, 1H, J ) 9.5, 1.5 Hz,
H1), 4.86 (t, 1H, J ) 9.5, 9.5 Hz, H4), 4.20 (dd, 1H, J ) 12, 6
Hz, H6), 4.10 (dd, 1H, J ) 12, 2.5 Hz, H6′), 3.62-3.55 (m, 2H,
H3, H5), 2.25 (ddd, 1H, J ) 12.5, 4.5, 1.5 Hz, H2e), 2.15 (s, 3H,
CH3CO), 2.05 (s, 3H, CH3CO), 1.72 (m, 1H, J ) 12.5, 12.5, 9.5
Hz, H2a), 0.92 (s, 9H, t-Bu), 0.14 (s, 3H, CH3-Si), 0.13 (s, 3H,
CH3-Si); MS (CI): m/z 405 (M + NH4)+. Anal. (C16H19N3O6S)
C, H, N.
In conclusion, new water-soluble epipodophyllotoxin
derivatives were synthesized and evaluated in vitro and
in vivo. In vitro L1210 cytotoxic assay demonstrated
an equipotent activity for five compounds compared with
ter t-Bu tyld im eth ylsilyl 3-Azid o-2,3-d id eoxy-â-D-r ibo-
h exop yr a n osid e (5). To a stirred solution of 3 (1.1 g, 2.8
mmol) in anhydrous MeOH (15 mL) kept under argon was

4480 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Daley et al.
added a 1 M solution of sodium methoxide (0.7 mL). After
stirring for 1.5 h at room temperature and subsequent
neutralization by addition of ion-exchange resin IRC 50 S,
filtration, followed by concentration under reduced pressure,
afforded 5 (0.87 g, 99%) as crystals: Rf 0.32 (2:1 cyclohexane/
ter t-Bu tyld im eth ylsilyl 3-Azid o-2,3-d id eoxy-4,6-O-eth -
ylid en e-â-D-a r a bin o-h exop yr a n osid e (10). A solution of 6
(0.2 g, 0.6 mmol) in acetonitrile (5 mL) in the presence of 1,1-
diethoxyethane (0.94 mL, 6.6 mmol) and p-TsOH (0.015 g, 0.08
mmol) was treated as described above for the preparation of
9. This led to 10 (0.19 g, 85%) after flash chromatography
(95:5 cyclohexane/EtOAc), syrup: Rf 0.89 (7:3 cyclohexane/
20
EtOAc); mp 70-72 °C; [R]D +15° (c 1.0, CHCl3); IR (CDCl3)
3599, 3441 (OH), 2119 (N3) cm-1; 1H NMR (300 MHz, CDCl3)
δ 5.04 (dd, 1H, J ) 9, 2 Hz, H1), 4.07 (q, 1H, J ) 3.5, 3.5, 3.5
Hz, H3), 3.82-3.78 (m, 3H, H4, H6, H6′), 3.67 (m, J ) 9.5, 4, 4
Hz, H5), 2.98 (m, OH), 2.03 (ddd, 1H, J ) 14, 3.5, 2 Hz, H2e),
1.75 (ddd, J ) 14, 9, 3.5 Hz, H2a), 0.89 (s, 9H, t-Bu), 0.10 (s,
6H, CH3-Si); MS (CI) m/z 304 (M + H)+, 321 (M + NH4)+.
Anal. (C12H25N3O4Si) C, H, N.
EtOAc); [R]D20 -19° (c 1.1, CHCl3); IR (CDCl3) 2114 (N3) cm-1
;
1H NMR (300 MHz, CDCl3) δ 4.80 (dd, 1H, J ) 9.5, 2 Hz, H1),
4.68 (q, 1H, J ) 5 Hz, CH-CH3), 4.03 (dd, 1H, J ) 10, 4.5 Hz,
H6), 3.60-3.54 (m, 1H, H3), 3.50 (t, 1H, J ) 10, 10 Hz, H6′),
3.29-3.16 (m, 2H, H4, H5), 2.10 (ddd, 1H, J ) 13, 4.5, 2 Hz,
H2e), 1.55 (ddd, 1H, J ) 13, 13, 9.5 Hz, H2a), 1.30 (d, 3H, J )
5 Hz, CH3-CH), 0.82 (s, 9H, t-Bu), 0.04 (s, 6H, CH3-Si). Anal.
(C14H27N3O4Si), C, H, N.
ter t-Bu tyldim eth ylsilyl 3-Azido-2,3-dideoxy-â-D-a r a bin o-
h exop yr a n osid e (6). Obtained in 97% yield from 4 (3 g, 7,7
mmol) by similar treatment as above for 5: Rf 0.36 (2:1
4-Dem eth yl-ep ip od op h yllotoxin (12). To a solution of
podophyllotoxin 11 (3.22 g, 7.77 mmol) in anhydrous CH2Cl2
(100 mL) kept under argon, a solution of trimethylsilyl iodide
(3.30 mL, 23.31 mmol) in CH2Cl2 (10 mL) was added dropwise
at 0 °C over a period of 0.5 h. The mixture was stirred for an
additional 4.5 h at 0 °C, and the reaction was quenched with
acetone/H2O (1/1, 100 mL) and then BaCO3 (1.5 g). Half an
hour later, the mixture was extracted with CH2Cl2 (100 mL).
The organic layer was separated and washed with 10%
aqueous solution of sodium thiosulfate (500 mL). After the
mixture was dried over anhydrous MgSO4 and concentrated
in vacuo, flash chromatography (92:8 CH2Cl2/acetone) afforded
4′-demethyl-epipodophyllotoxin 12 (2.23 g, 72%) pure enough
for the next step. A sample was recrystallized from acetone-
20
cyclohexane/EtOAc); mp 70-72 °C (EtOAc); [R]D -26° (c 1,
CHCl3); IR (CDCl3) 3598, 3445 (OH), 2103 (N3) cm-1; 1H NMR
(300 MHz, CDCl3) δ 4.85 (dd, 1H, J ) 9, 2 Hz, H1), 3.82-3.78
(m, 2H, H6, H6′), 3.52 (t, 1H, J ) 9, 9 Hz, H4), 3.55-3.42 (m,
1H, H3), 3.30 (m, 1H, J ) 9, 3.5, 3.5 Hz, H5), 2,15 (ddd, 1H, J
) 12.5, 4.5, 2 Hz, H2e), 1.62 (m, 1H, J ) 12.5, 12.5, 9 Hz, H2a),
0.89 (s, 9H, t-Bu), 0.11 (s, 3H, CH3-Si), 0.10 (s, 3H, CH3-Si);
MS (CI) m/z 304 (M + H)+, 321 (M + NH4)+. Anal.
(C12H25N3O4Si) C, H, N.
ter t-Bu tyld im eth ylsilyl 3-Azid o-4,6-d i-O-ch lor oa cetyl-
2,3-dideoxy-â-D-r ibo-h exopyr an oside (7). Chloroacetyl chlo-
ride (920 µL, 7.9 mmol) was added under argon atmosphere
to a cooled solution (-10 °C) of 5 (0.87 g, 2.9 mmol) in a
mixture of anhydrous CH2Cl2 (10 mL) and pyridine (930 µL).
After stirring for 4 h at -10 °C, the crude mixture was poured
into H2O (20 mL). The separated aqueous layer was extracted
twice with CH2Cl2 (2 × 10 mL), and the combined organic
phases were dried over anhydrous MgSO4 and concentrated
under reduced pressure. This afforded a residue (1.3 g) which
was purified by flash chromatography (9:1 cyclohexane/
EtOAc), giving pure 7 (1.2 g, 92%) as a syrup: Rf 0.53 (4:1
20
isopropyl ether: mp 248-250 °C; [R]D -69° (c 0.4, CHCl3);
lit.23 mp 244-249 °C (acetone); [R]D -69° (c 0.63, CHCl3).
20
4′-Ben zyloxyca r bon yl-ep ip od op h yllotoxin (13). A so-
lution of the crude material 12 (2 g, 4.8 mmol) was dissolved
into anhydrous CH2Cl2 (120 mL) and cooled to 0 °C. To this
solution were added Et3N (1.2 mL, 8.8 mmol) and benzylchlo-
roformate (1.1 mL, 7.2 mmol). After the crude mixture was
stirred for 2 h at 0 °C, it was washed twice with water and
dried over MgSO4. Concentration under reduced pressure
followed by flash chromatography (96:4 then 92:8 CH2Cl2/
acetone) led to 13 (2.6 g, 97%) which recrystallized from
cyclohexane/EtOAc); [R]D +14° (c 1.6, CHCl3); 1H NMR (300
20
MHz, CDCl3) δ 5.09 (dd, 1H, J ) 9, 2 Hz, H1), 4.98 (dd, 1H, J
) 9.5, 3.5 Hz, H4), 4.31 (d, 2H, H6, H6′), 4.20-4.10 (m, 1H, J
) 9.5, 4, 4 Hz, H5), 4.10 (d, 4H, ClCH2CO), 4.07 (q, 1H, J )
3.5, 3.5, 3.5 Hz, H3), 2.07 (ddd, 1H, J ) 14, 3.5, 2 Hz, H2e),
1.85 (ddd, 1H, J ) 14, 9, 3.5 Hz, H2a), 0.89 (s, 9H, t-BuSi),
0.11 (s, 6H, CH3-Si); MS (CI) m/z 474 (M + NH4)+.
20
acetone-isopropyl ether: mp 205-206 °C; [R]D -43.5° (c )
1, CHCl3); lit.23b mp 205-207 °C; [R]D -44.5° (c ) 0.506,
20
CHCl3); Rf 0.37 (92:8 CH2Cl2/acetone).
4-O-(3′′-Azid o-2′′,3′′-d id eoxy-4′′,6′′-d i-O-ch lor oa cetyl-â-
D-r ibo-h exop yr a n osyl)-4′-b en zyloxyca r b on yl-ep ip od o-
p h yllotoxin (14). To a cooled (-35 °C) mixture of 7 (0.235
g, 0.51 mmol) and 13 (0.25 g, 0.51 mmol) in anhydrous CH2-
Cl2 (10 mL) in the presence of 4 Å powdered molecular sieves
(1.4 g) was added trimethylsilyl triflate (270 µL, 1.4 mmol).
After it was stirred for 22 h at -35 °C, the reaction mixture
was diluted with CH2Cl2 (50 mL) and washed successively with
a citrate buffer (pH 5, 20 mL) and with H2O (20 mL). Usual
workup followed by flash chromatography (7:3 cyclohexane/
EtOAc) led to 14 (0.2 g, 50%) as a crystalline residue: mp 95
ter t-Bu tyld im eth ylsilyl 3-Azid o-4,6-d i-O-ch lor oa cetyl-
2,3-d id eoxy-â-D-a r a bin o-h exop yr a n osid e (8). It was ob-
tained from 6 (0.6 g, 1.9 mmol) in 92% yield, as 7 from 5, syrup:
20
Rf 0.50 (4:1 cyclohexane/EtOAc); [R]D -12° (c 1.5, CHCl3);
1
IR (CDCl3) 2104 (N3), 1763 (CdO) cm-1; H NMR (300 MHz,
CDCl3) δ 4.90 (t, 1H, J ) 10, 10 Hz, H4), 4.87 (dd, 1H, J ) 9.5,
2 Hz, H1), 4.28 (m, 2H, H6, H6′), 4.11 (d, 4H, ClCH2CO), 3.70-
3.65 (m, 1H, H5), 3.65-3.57 (m, 1H, J ) 13, 10, 5 Hz, H3),
2.07 (ddd, 1H, J ) 13, 5, 2 Hz, H2e), 1.85 (ddd, 1H, J ) 13, 13,
9.5 Hz, H2a), 0.90 (s, 9H, t-Bu), 0.12 (s, 3H, CH3-Si), 0.11 (s,
3H, CH3-Si); MS (CI) m/z 474 (M + NH4)+.
20
°C; Rf 0.69 (1:1 cyclohexane/EtOAc); [R]D -69° (c 1, CHCl3);
ter t-Bu tyld im eth ylsilyl 3-Azid o-2,3-d id eoxy-4,6-O-eth -
ylid en e-â-D-r ibo-h exop yr a n osid e (9). To a solution of 5 (5.8
g, 19.1 mmol) in acetonitrile (100 mL) were added 1,1-
diethoxyethane (27.2 mL, 191 mmol) and p-TsOH (1.1 g, 5.7
mmol). The mixture was stirred at room temperature for 1 h
and then diluted with EtOAc (200 mL). The organic layer was
separated and washed successively with an aqueous saturated
solution of NaHCO3 (200 mL) and with H2O (200 mL). After
the mixture was dried over anhydrous MgSO4, the concentra-
tion was reduced under pressure followed by flash chroma-
tography (95:5 cyclohexane/EtOAc), giving 3 g (48%) of 9 as a
IR (CDCl3) 2105 (N3), 1768 (CdO) cm-1; MS (CI) m/z 875 (M
+ NH4)+.
4-O-(3′′-Azid o-2′′,3′′-d id eoxy-â-D-r ibo-h exop yr a n osyl)-
4′-b en zyloxyca r bon yl)-ep ip od op h yllot oxin (15). To a
solution of the azido glycoside 14 (0.2 g, 0.02 mmol) in a
mixture of CH2Cl2/MeOH (2/1, 6 mL) kept under argon
atmosphere was added Amberlite IRA 410 ion-exchange (OH-)
resin. After it was stirred for 1.5 h at room temperature, the
reaction mixture was filtered, and the filtrate was washed with
a solution of phosphate buffer (pH 7, 5 mL). The organic layer
was separated, dried over anhydrous MgSO4, and concentrated
under reduced pressure, giving 15 (0.16 g, 97%) as a crystalline
20
syrup: Rf 0.77 (4:1 cyclohexane/EtOAc); [R]D -35° (c 1.1,
CHCl3); IR (CDCl3) 2115 (N3) cm-1; 1H NMR (300 MHz, CDCl3)
δ 5.02 (dd, 1H, J ) 9, 2 Hz, H1), 4.71 (q, 1H, J ) 5 Hz, CH-
CH3), 4.13-4.07 (m, 2H, H3, H6), 3.81 (m, 1H, H5), 3.56-3.48
(m, 2H, H4, H6′), 1.98 (m, 1H, H2e), 1.75 (ddd, 1H, H2a), 1.35
(d, 3H, J ) 5 Hz, CH3-CH), 0.87 (s, 9H, t-Bu), 0.09 (s, 6H,
CH3-Si); MS (CI) m/z 330 (M + H)+, 347 (M + NH4)+. Anal.
(C14H27N3O4Si) C, H, N.
20
residue: mp 140 °C; Rf 0.20 (97:3 CH2Cl2/MeOH); [R]D -67°
(c ) 1.2, CHCl3); IR (CDCl3) 3614 (OH), 2115 (N3), 1768 (Cd
O) cm-1; MS (CI) m/z 723 (M + NH4)+. Anal. (C35H35N3O13
C, H, N.
)
4-O-(3′′-Azido-2′′,3′′-dideoxy-4′′,6′′-O-eth yliden e-â-D-r ibo-
h exop yr a n osyl)-4′-b en zyloxyca r b on yl-ep ip od op h yllo-

New Glycosides of Epipodophyllotoxin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4481
toxin (16). (1) F r om 15. Acetalation of 15 (0.16 g, 0.23
mmol) (cf. preparation of 9 and 10) afforded 16 (0.11 g, 66%).
4-O-(3′′-Am in o-2′′,3′′-d id eoxy-4′′,6′′-O-et h ylid en e-r-D-
a r a bin o-h exop yr a n osyl)-ep ip od op h yllotoxin (21). Azido
glycoside 20 (0.13 g, 0.18 mmol) was treated under conditions
similar to those described for the preparation of 19, giving 21
(0.08 g, 75%) as a crystalline residue: mp 255 °C; Rf 0.28 (95:5
CH2Cl2/MeOH); [R]D20 +7° (c 1, CHCl3); IR (CDCl3) 3540 (NH2,
(2) F r om 9 a n d 13 in th e P r esen ce of Tr im eth ylsilyl-
tr ifla te. A mixture of 9 (0.27 g, 0.82 mmol) and 13 (0.44 g,
0.82 mmol) was treated under conditions similar to those
described for the preparation of 14, giving 16 in 36% yield (0.22
g) after flash chromatography using cyclohexane/EtOAc (65:
35) as eluent.
1
OH), 1779 (CdO) cm-1; H NMR (300 MHz, CDCl3) δ 6.86 (s,
1H, H5), 6.49 (s, 1H, H8), 6.25 (s, 2H, H2′, H6′), 5.97 (d, 2H,
O-CH2-O), 4.96 (d, 1H, J ) 3.5 Hz, H1′′), 4.74-4.69 (m, 2H,
H4, H7′′), 4.60 (d, 1H, J ) 5.3 Hz, H1), 4.36 (t, 1H, J ) 8, 8 Hz,
H9a), 4.09-4.04 (m, 2H, H9b, H6′′), 3.52 (s, 3H, CH3O), 3.53-
3.49 (m, 2H, H5′′, H6′′′), 3.32 (dd, 1H, J ) 14, 5.3 Hz, H2), 3.21-
3.17 (m, 1H, H3′′), 3.10 (t, 1H, J ) 9, 9 H4′′), 2.86 (m, 1H, H3),
2.18 (dd, 1H, J ) 13.5, 4.5 Hz, H2′′e), 1.75 (m, 1H, J ) 13.5,
12, 3.5 Hz, H2′′a), 1.31 (d, 3H, J ) 5 Hz, CH3); MS (CI) m/z 572
(M + H)+, 589 (M + NH4)+. Anal. (C29H33NO11) C, H, N.
2,3-Did eoxy-4,6-O-et h ylid en e-D-th r eo-h ex-2-en op yr a -
n ose (22). To a solution of 10 (0.50 g, 1.5 mmol) in THF (10
mL) was added Bu4NF (1.8 mL, 1.8 mmol) (1 M solution in
THF). After it was stirred for 10 min at room temperature,
the mixture was poured into H2O (20 mL) and extracted with
ether (6 × 20 mL). The organic layer was washed over
anhydrous MgSO4, concentrated under reduced pressure, and
flash-chromatographed (7:3 cyclohexane/EtOAc) to give 22 as
a syrup (0.15 g, 57%): Rf 0.39 (1:1 cyclohexane/EtOAc); IR
(3) F r om 9 a n d 13 in th e P r esen ce of BF 3‚Et2O. To a
cooled (-15 °C) solution of 13 (1.85 g, 3.46 mmol) and 9 (1.20
g, 3.64 mmol) in anhydrous CH2Cl2 (100 mL) was added BF3‚
Et2O (425 µL, 3.46 mmol). After the solution was stirred for
2 h, it was diluted with CH2Cl2 (100 mL) and poured into a
satured aqueous solution of NaHCO3 (200 mL). The organic
layer was separated and dried over anhydrous MgSO4. Flash
chromatography (65:35 cyclohexane/EtOAc) afforded 16 (1.19
g, 47%) which was recrystallized from Et2O/hexane: mp 139
°C; Rf 0.41 (6:4 cyclohexane/EtOAc); [R]D20 -101° (c 1.1, CHCl3);
IR (CDCl3) 2114 (N3), 1768 (CdO) cm-1; MS (CI) m/z 749 (M
+ NH4)+. Anal. (C37H37N3O13) C, H, N.
4-O-(3′′-Am in o-2′′,3′′-d id eoxy-4′′,6′′-O-et h ylid en e-â-D-
r ibo-h exop yr a n osyl)-ep ip od op h yllotoxin (17). To a solu-
tion of 16 (0.26 g, 0.35 mmol) were added EtOAc (15 mL),
triethylamine (20 µL), and palladium-on-charcoal 10% (0.15
g). After stirring for 2 h at room temperature under hydrogen
atmosphere, the catalyst was eliminated by filtration. The
filtrate, concentrated under reduced pressure, was purified by
flash chromatography (98.5:1.5 then 96:4 CH2Cl2/MeOH),
affording 17 (0.18 g, 88%) as a crystalline product: mp 188
(CDCl3) 1692 (CdO), 1644 (CdC) cm-1 1H NMR (90 MHz,
;
CDCl3) δ 9.60 (d, 1H, H aldehyde), 7.10 (dd, 1H, H3), 6.40 (ddd,
1H, H2), 4.20-4.00 (m, 2H, H4, H6), 3.80-3.50 (m, 2H, H5, H6′),
1.40 (d, 3H, CH3).
ter t-Bu tyld im eth ylsilyl 3-Am in o-2,3-d id eoxy-4,6-O-eth -
ylid en e-â-D-a r a bin o-h exop yr a n osid e (23). A solution of 10
(2 g, 6 mmol) in EtOAc (30 mL) was stirred overnight under
H2 atmosphere (1 atm) in the presence of Et3N (50 µL) and
10% of palladium on activated carbon (0.5 g). After removal
of the catalyst by filtration, the filtrate was concentrated in
vacuo, furnishing the corresponding amino sugar 23 (1.82 g,
20
°C; Rf 0.39 (95:5 CH2Cl2/MeOH); [R]D -100° (c 1.05, CHCl3);
1
IR (CDCl3) 3541 (NH2, OH), 1774 (CdO) cm-1; H NMR (300
MHz, CDCl3) δ 6.86 (s, 1H, H5), 6.51 (s, 1H, H8), 6.25 (s, 2H,
H2′, H6′), 5.97 (d, 1H, O-CH-O), 5.93 (d, 1H, O-CH-O), 5.38
(dd, 1H, J ) 9, 2 Hz, H1′′), 4.91 (d, 1H, J ) 3.4 Hz, H4), 4.78
(q, 1H, J ) 5 Hz, H7′′), 4.57 (d, 1H, J ) 5.2 Hz, H1), 4.42 (dd,
1H, J ) 10.5, 9 Hz, H9a), 4.19 (t, 1H, J ) 9, 8 Hz, H9b), 4.16
(dd, 1H, J ) 10, 5 Hz, H6′′), 4.02-3.94 (m, 1H, H5′′), 3.74 (s,
3H, CH3O), 3.59 (m, 1H, H3′′), 3.53 (t, 1H, J ) 10, 10 Hz, H6′′′),
3.42 (dd, 1H, J ) 9.5, 3.5 Hz, H4′′), 3.22 (dd, 1H, J ) 14, 5.2
Hz, H2), 2.83 (m, 1H, H3), 1.90 (m, 1H, H2′′e), 1.73 (m, 1H, J )
13.5, 9, 3.5 Hz, H2′′a), 1.35 (d, 3H, J ) 5 Hz, CH3); MS (CI) m/z
98%) as a colorless syrup: Rf 0.23 (1:1 cyclohexane/EtOAc);
[R]D -28° (c 1.3, CHCl3); IR (CDCl3) 3428, 3370 (NH2) cm-1
;
20
1H NMR (300 MHz, CDCl3) δ 4.89 (dd, 1H, J ) 9, 2 Hz, H1),
4.70 (q, 1H, J ) 5 Hz, CH-CH3), 4.07 (dd, 1H, J ) 10, 5 Hz,
H6), 3.55 (t, 1H, J ) 10, 10 Hz, H6′), 3.22 (m, 1H, J ) 10, 10,
5 Hz, H5), 3.05-2.91 (m, 2H, H3, H4), 1.98 (m, 1H, J ) 13, 4.5,
2 Hz, H2e), 1.49 (m, 1H, H2a), 1.33 (d, 3H, J ) 5 Hz, CH3-
CH), 0.88 (s, 9H, t-Bu), 0.10 (s, 6H, CH3-Si); MS (CI) m/z 304
(M + H)+. Anal. (C14H29NO4Si) C, H, N.
572 (M + H)+, 589 (M + NH4)+. Anal. (+ H2O) (C29H33NO11
C, H, N.
)
4-O-(3′′-Azid o-2′′,3′′-d id eoxy-4′′,6′′-d i-O-ch lor oa cetyl-r-
D-a r a bin o-h exopyr an osyl)-4′-ben zyloxycar bon yl-epipodo-
p h yllotoxin (18). Trimethylsilyl triflate (32.6 mL, 0.18 mmol)
was added to a cooled (-35 °C) solution of 13 (0.1 g, 0.18 mmol)
and 8 (0.85 g, 0.18 mmol) in anhydrous CH2Cl2 (10 mL)
containing 4 Å powdered molecular sieves (0.6 g). After the
mixture was stirred for 48 h at -35 °C, the reaction was
quenched by addition of citrate buffer (pH 5, 10 mL). The
separated organic layer was dried over anhydrous MgSO4 and
concentrated in vacuo. Flash chromatography of the crude
residue (2:1 cyclohexane/EtOAc) afforded 18 as a crystalline
compound (0.1 g, 65%): Rf 0.58 (1:1 cyclohexane/EtOAc); IR
(CDCl3) 2107 (N3), 1767 (CdO) cm-1; MS (CI) m/z 875 (M +
NH4)+.
ter t-Bu t yld im et h ylsilyl 3-Am in ob en zyloxyca r b on yl-
2,3-d id eoxy-4,6-O-et h ylid en e-â-D-a r a bin o-h exop yr a n o-
sid e (24). Benzyloxycarbonyl chloride (1.10 mL, 7.88 mmol)
was added to a cooled (0-5 °C) solution of 23 (1.82 g, 6 mmol)
in dichloromethane (30 mL) and in the presence of Et3N (1.25
mL, 9 mmol). After the crude mixture was stirred for 3 h, it
was poured into water (100 mL) and dichloromethane was
subsequently added (100 mL). The organic layer was sepa-
rated, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by flash chromatography
(6:1 then 4:1 cyclohexane/EtOAc), giving 24 (1.9 g, 72%) as
crystals: Rf 0.84 (1:1 cyclohexane/EtOAc); mp 102 °C (CH2-
20
Cl2); [R]D -27° (c 1.14, CHCl3); IR (CDCl3) 3439 (NH), 1718
(CdO) cm-1; 1H NMR (300 MHz, CDCl3) δ 7.37-7.30 (m, 4H,
Har), 5.10 (s, 2H, CH2Ph), 4.89 (dd, 1H, J ) 9, 2 Hz, H1), 4.71
(q, 1H, J ) 5 Hz, CH-CH3), 4.08 (dd, 1H, J ) 10, 5 Hz, H6),
3.82 (m, 1H, H3), 3.55 (t, 1H, J ) 10, 10 Hz, H6′), 3.32 (m, 1H,
J ) 10, 10, 5 Hz, H5), 3.18 (t, J ) 10, 10 Hz, H4), 2.45 (m, 1H,
4-O-(3′′-Azid o-2′′,3′′-d id eoxy-r-D-a r a bin o-h exop yr a n o-
syl)4′-ben zyloxyca r bon yl-ep ip od op h yllotoxin (19). Azido
glycoside 18 (0.074 g, 0.08 mmol) was treated under conditions
similar to those described for the preparation of 15 from 14,
giving 19 (0.06 g, 98%) as a crystalline residue: mp 120 °C
20
(from pentane); Rf 0.28 (93:7 CH2Cl2/MeOH); [R]D +26° (c 1,
H
2e), 1.52 (m, 2H, J ) 12, 12, 9 Hz, H2a), 1.35 (d, 3H, J ) 5
CHCl3); IR (CDCl3) 3603 (OH), 2106 (N3), 1769 (CdO) cm-1
;
Hz, CH3), 0.88 (s, 9H, t.Bu), 0.10 (s, 6H, CH3-Si). Anal.
(C22H35NO6Si) C, H, N.
MS (CI) m/z 706 (M + H)+, 723 (M + NH4)+. Anal.
(C35H35N3O13) C, H, N.
3-Am in ob en zyloxyca r b on yl-2,3-d id eoxy-4,6-O-et h yli-
d en e-D-a r a bin o-h exop yr a n ose (25). To a solution of 24
(0.59 g, 1.35 mmol) in THF (30 mL) was added Bu4NF (1.35
mL, 1.35 mmol; 1 M solution in THF). After it was stirred for
30 min at room temperature, the reaction mixture was poured
into H2O (60 mL) and extracted with CH2Cl2 (2 × 60 mL). The
organic layer was dried over anhydrous MgSO4 and concen-
trated under reduced pressure to quantitatively give 25 (0.43
4-O-(3′′-Azido-2′′,3′′-dideoxy-4′′,6′′-O-eth yliden e-â-D-r ibo-
h exop yr a n osyl)-4′-b en zyloxyca r b on yl-ep ip od op h yllo-
toxin (20). Acetalation of 19 (0.12 g, 0.17 mmol) (cf. prepa-
ration of 9 and 10) afforded 20 (0.112 g, 90%) as a crystalline
20
product: mp 137 °C; Rf 0.57 (1:1 cyclohexane/EtOAc); [R]D
+30° (c 1, CHCl3); IR (CDCl3) 2108 (N3), 1768 (CdO) cm-1
;
MS (CI) m/z 749 (M + NH4)+. Anal. (C37H37N3O13) C, H, N.

4482 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Daley et al.
g) as a mixture of R- and â-isomers (in 1 to 1 ratio): Rf 0.24
(1:1 cyclohexane/EtOAc); 1H NMR (300 MHz, CDCl3) δ 5.05
(d, J ) 3 Hz, H1eq), 4.71 (dd, J ) 9, 1.5 Hz, H1ax); MS (CI) m/z
306 (M + H-H2O)+.
To a solution of 17 (0.029 g, 0.05 mmol) in acetonitrile (1 mL)
were added successively formaldehyde (10.5 µL) and sodium
cyanoborohydride (0.012 g). After it was stirred for 2 h at room
temperature, the reaction mixture was diluted in CH2Cl2 (20
mL) and washed with H2O (20 mL).The organic layer was dried
over anhydrous MgSO4 and concentrated under reduced pres-
sure. The residue, submitted again to the above treatment,
was purified by flash chromatography (97:3 CH2Cl2/MeOH)
affording 29 (0.029 g, 95%) as crystals: mp 140 °C; Rf 0.5 (95:5
4-O-(3′′-Am in oben zyloxyca r bon yl-2′′,3′′-d id eoxy-4′′,6′′-
O-eth ylid en e-â-D-a r a bin o-h exop yr a n osyl)-4′-ben zyloxy-
ca r bon yl-ep ip od op h yllot oxin (26). To the solution of
amino sugar 24 (2 g, 4.57 mmol) in 100 mL of anhydrous CH2-
Cl2 at -20 °C was added Bu4NF (5.05 mL, 1.1 M solution in
THF, 5.56 mmol). After reacting for 5 min at -20 °C, the
temperature was slowly heightened until disappearance of the
starting compound. The mixture was then cooled to -20 °C
before successive addition of epipodophyllotoxin derivative 13
(2.57 g, 4.80 mmol) and BF3‚Et2O (8.45 mL, 68.6 mmol). After
it was stirred for 1 h at the same temperature, the reaction
mixture was diluted with CH2Cl2 (100 mL) and poured into a
saturated solution of NaHCO3 (600 mL). The organic layer
was dried over anhydrous MgSO4, concentrated under reduced
pressure, and the crude residue (6.2 g) was purified by flash
chromatography (98:2 then 97:3 CH2Cl2/acetone), affording 26
(2.08 g, 54%) as crystals: mp 175 °C; Rf 0,60 (92:8 CH2Cl2/
20
CH2Cl2/MeOH); [R]D -85° (c 1.06, CHCl3); IR (CDCl3) 3539
(OH), 1773 (CdO) cm-1; 1H NMR (300 MHz, CDCl3) δ 6.80 (s,
1H, H5), 6.52 (s, 1H, H8), 6.25 (s, 2H, H2′, H6′), 5.98 (d, 1H,
O-CH-O), 5.96 (d, 1H, O-CH-O), 5.21 (dd, 1H, J ) 9.5, 1.5
Hz, H1′′), 4.88 (d, 1H, J ) 3.4 Hz, H4), 4.62-4.57 (m, 2H, H1,
H
7′′), 4.43 (dd, 1H, J ) 10.5, 9 Hz, H9a), 4.22-4.02 (m, 3H,
H9b, H5′′, H6′′), 3.75 (s, 3H, CH3O), 3.61 (t, 1H, J ) 10, 10 Hz,
6′′′), 3.52 (dd, 1H, J ) 9, 3 Hz, H4′′), 3.23 (dd, 1H, J ) 14, 5.3
H
Hz, H2), 2.85 (m, 1H, H3), 2.62 (m, 1H, H3′′), 2.36 (s, 6H, CH3N),
2.20 (m, 1H, H2′′e), 1.55 (m, 1H, H2′′a), 1.35 (d, 3H, J ) 5 Hz,
CH3); MS (CI) m/z 600 (M + H)+.
4-O-(3′′-Cya n om eth yla m in o-2′′,3′′-d id eoxy-4′′,6′′-O-eth -
yliden e-â-D-r ibo-h exopyr an osyl)-epipodoph yllotoxin (30).
To a solution of 17 (0.1 g, 0.18 mmol) in DMF (5 mL) were
added successively Et3N (180 µL, 1.29 mmol) and iodoaceto-
nitrile (94 µL, 1.29 mmol). The reaction mixture was stirred
for 27 h at room temperature, poured into H2O (20 mL), and
extracted with EtOAc (30 mL). The organic layer was washed
with H2O (4 × 20 mL), dried over anhydrous MgSO4, and
concentrated under reduced pressure. Flash chromatography
(92:8 CH2Cl2/acetone) gave 30 (0.1 g, 85%) and recovery
material 17 (0.04 g, 15%). Compound 30: Rf 0.25 (95:5 CH2-
Cl2/MeOH); [R]D20 -86° (c 0.80, CHCl3); IR (CDCl3) 3543 (NH,
20
acetone); [R]D -74° (c 1.09, CHCl3); IR (CDCl3) 3441 (NH),
1768 and 1723 (CdO) cm-1; MS (CI) m/z 857 (M + NH4)+. Anal.
(C45H45NO15) C, H, N.
4-O-(3′′-Am in o-2′′,3′′-d id eoxy-4′′,6′′-O-et h ylid en e-â-D-
a r a bin o-h exop yr a n osyl)-ep ip od op h yllotoxin (27). To a
solution of 26 (0.28 g, 0.33 mmol) in EtOAc (20 mL) were
successively added triethylamine (30 µL) and 10% palladium
on activated carbon (0.15 g). The reaction was left under
hydrogen atmosphere and under atmospheric pressure and
was stirred for 1.5 h at room temperature. The catalyst was
then eliminated by filtration, and the filtrate, concentrated
under reduced pressure, was purified by flash chromatography
(97:3 CH2Cl2/MeOH), giving 27 (0.17 g, 90%) as a crystalline
1
OH), 1774 (CdO) cm-1; H NMR (300 MHz, CDCl3) δ 6.78 (s,
1H, H5), 6.53 (s, 1H, H8), 6.25 (s, 2H, H2′, H6′), 6.00 (d, 1H,
O-CH-O), 5.99 (d, 1H, O-CH-O), 5.17 (dd, 1H, J ) 9.5, 2
Hz, H1′′), 4.90 (d, 1H, J ) 3.3 Hz, H4), 4.75 (q, 1H, J ) 5 Hz,
H7′′), 4.59 (d, 1H, J ) 5.3 Hz, H1), 4.41 (dd, 1H, J ) 10.5, 9 Hz,
H9a), 4.19 (t, 1H, J ) 9, 8 Hz, H9b), 4.14 (dd, 1H, J ) 10, 5 Hz,
H6′′), 3.88 (m, 1H, H5′′), 3.76 (s, 3H, CH3O), 3.56-3.51 (m, 4H,
20
compound: mp 219 °C; Rf 0.31 (95:5 CH2Cl2/MeOH); [R]D
1
-120° (c 1.05, CHCl3); H NMR (300 MHz, CDCl3) δ 6.75 (s,
1H, H5), 6.55 (s, 1H, H8), 6.24 (s, 2H, H2′, H6′), 6.00 (d, 1H,
O-CH-O), 5.98 (d, 1H, O-CH-O), 4.94 (d, 1H, J ) 3.3 Hz,
H4), 4.85 (dd, 1H, J ) 9, 2 Hz, H1′′), 4.75 (q, 1H, J ) 5 Hz,
H
4′′, H6′′′, CH2CN), 3.46 (m, 1H, H3′′), 3.25 (dd, 1H, J ) 14, 5.3
H
7′′), 4.59 (d, 1H, J ) 5.2 Hz, H1), 4.41 (dd, 1H, J ) 10.5, 9 Hz,
Hz, H2), 2.86 (m, 1H, H3), 1.93 (m, 1H, H2′′e), 1.69 (m, 1H, J )
13, 9.5, 3 Hz, H2′′a), 1.35 (d, 3H, J ) 5 Hz, CH3); MS (CI) m/z
611 (M + H)+, 628 (M + NH4)+.
H9a), 4.21 (t, 1H, J ) 9, 8 Hz, H9b), 4.15 (dd, 1H, J ) 10, 5 Hz,
H6′′), 3.75 (s, 3H, CH3O), 3.57 (t, 1H, J ) 10, 10 Hz, H6′′′), 3.30
(m, 1H, H5′′), 3.28 (dd, 1H, J ) 14, 5.2 Hz, H2), 3.05-2.99 (m,
2H, H3′′, H4′′), 2.88 (m, 1H, H3), 2.05 (m, 1H, H2′′e), 1.51 (m,
1H, H2′′a), 1.36 (d, 3H, J ) 5 Hz, CH3); MS (CI) m/z 594 (M +
Na)+, 610 (M + K)+. Anal. (C29H33NO11) C, H, N.
4-O-(3′′-Am in o-N-eth yla m in oben zyloxyca r bon yl-2′′,3′′-
d id eoxy-4′′,6′′-O-eth ylid en e-â-D-r ibo-h exop yr a n osyl)-ep i-
p od op h yllotoxin (31). To a solution of 17 (0.173 g, 0.30
mmol) in DMF (10 mL) were added successively Et3N (127 µL,
0.91 mmol) and N-benzyloxycarbonyl-2-iodoethylamine (0.28
g, 0.91 mmol). The reaction mixture was stirred for 5 days at
room temperature, poured into H2O (30 mL), and extracted
with EtOAc (30 mL). The organic layer was washed with H2O
(5 × 20 mL), dried over anhydrous MgSO4, and concentrated
under reduced pressure. Flash chromatography (97:3 CH2-
Cl2/MeOH) gave 31 (0.155 g, 68%) and recovery material 17
(0.055 g, 32%). Compound 31: mp 110 °C; Rf 0.70 (95:5 CH2-
Cl2/MeOH); [R]D20 -74° (c 1.17, CHCl3); MS (CI) m/z 749 (M +
H)+.
4-O-(3′′-Dim et h yla m in o-2′′,3′′-d id eoxy-4′′,6′′-O-et h yli-
d en e-â-D-a r a bin o-h exop yr a n osyl)-ep ip od op h yllot oxin
(28). To a solution of 27 (0.19 g, 0.33 mmol) in CH2Cl2 (15
mL) were added successively formaldehyde (135 µL, 1.66
mmol) and sodium cyanoborohydride (0.085 g, 1.33 mmol).
After the mixture was stirred for 0.75 h at room temperature,
an additional amount of formaldehyde (135 µL) and sodium
cyanoborohydride (0.085 g) was poured into the mixture with
additional stirring for 0.75 h at room temperature. The
reaction mixture was diluted in CH2Cl2 (30 mL) and washed
with H2O (40 mL).The organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure. Flash
chromatography (97:3 CH2Cl2/MeOH) gave 28 (0.1 g, 51%) as
crystals: mp 270 °C; Rf 0.40 (95:5 CH2Cl2/MeOH); [R]D20 -121°
4-O-(3′′-Am in o-N-(et h yla m in o)-2′′,3′′-d id eoxy-4′′,6′′-O-
et h ylid en e-â-D-r ibo-h exop yr a n osyl)-ep ip od op h yllot ox-
in (32). To a solution of 31 (0.15 g, 0.20 mmol) in a mixture
of EtOAc and ethanol (1:1, 10 mL) were successively added
triethylamine (30 µL) and 10% palladium on activated carbon
(0.1 g). The reaction, kept under hydrogen atmosphere, was
stirred for 1.5 h at room temperature in the presence of
hydrogen at atmospheric pressure, and the catalyst was
eliminated by filtration. The filtrate, concentrated under
reduced pressure, was purified by flash chromatography (97:3
CH2Cl2/MeOH(NH3)) to give 32 (0.1 g, 84%): mp 130 °C; Rf
(c 1.0, CHCl3); IR (CDCl3) 3541 (OH), 1774 (CdO) cm-1 1H
;
NMR (300 MHz, CDCl3) δ 6.76 (s, 1H, H5), 6.55 (s, 1H, H8),
6.25 (s, 2H, H2′, H6′), 6.00 (d, 1H, O-CH-O), 5.97 (d, 1H,
O-CH-O), 4.95 (d, 1H, J ) 3.2 Hz, H4), 4.82 (dd, 1H, J ) 9.5,
2 Hz, H1′′), 4.74 (q, 1H, J ) 5 Hz, H7′′), 4.59 (d, 1H, J ) 5.2 Hz,
H1), 4.42 (dd, 1H, J ) 10.5, 9 Hz, H9a), 4.21 (t, 1H, J ) 9, 8
Hz, H9b), 4.16 (dd, 1H, J ) 10, 5 Hz, H6′′), 3.75 (s, 3H, CH3O),
3.58 (t, 1H, J ) 10, 10 Hz, H6′′′), 3.38 (t, 1H, J ) 9, 9 Hz, H4′′),
3.35-3.25 (m, 2H, H2, H5′′), 2.91-2.82 (m, 2H, H3, H3′′), 2.33
(s, 6H, CH3-N), 1.96 (m, 1H, H2′′e), 1.55 (m, 1H, J ) 12.5, 12.5,
9.5 Hz, H2′′a), 1.38 (d, 3H, J ) 5 Hz, CH3); MS (CI) m/z 600 (M
+ H)+.
20
0.22 (95:5 CH2Cl2/MeOH(NH3)); [R]D -77° (c 1, CHCl3); IR
1
(CDCl3); H NMR (300 MHz, CDCl3) δ 6.80 (s, 1H, H5), 6.52
(s, 1H, H8), 6.25 (s, 2H, H2′, H6′), 5.99 (d, 1H, O-CH-O), 5.96
(d, 1H, O-CH-O), 5.27 (dd, 1H, J ) 9.5, 2 Hz, H1′′), 4.89 (d,
1H, J ) 3.4 Hz, H4), 4.77 (q, 1H, J ) 5 Hz, H7′′), 4.58 (d, 1H,
J ) 5.2 Hz, H1), 4.43 (dd, 1H, J ) 10.5, 9 Hz, H9a), 4.16 (t, 1H,
4-O-(3′′-N,N-Dim eth yla m in o-2′′,3′′-d id eoxy-4′′,6′′-O-eth -
yliden e-â-D-r ibo-h exopyr an osyl)-epipodoph yllotoxin (29).

New Glycosides of Epipodophyllotoxin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4483
EtOAc), compound 36 was isolated (1.1 g, 90%) as a syrup: Rf
J ) 9, 8 Hz, H9b), 4.12 (dd, 1H, J ) 10, 5 Hz, H6′′′), 3.98 (m,
1H, H5′′), 3.75 (s, 3H, CH3O), 3.55-3.45 (m, 2H, H4′′, H6′′′), 3.25
(dd, 1H, J ) 14, 5.2 Hz, H2), 3.20 (m, 1H, H3′′), 2.90-2.74 (m,
3H, H3, CH2NH2), 2.67 (t, 2H, CH2NH), 2.02 (m, 1H, H2′′e), 1.55
(m, 1H, H2′′a), 1.35 (d, 3H, J ) 5 Hz, CH3); MS (CI) m/z 615 (M
+ H)+.
4-O-(3′′-Mor p h olin o-2′′,3′′-d id eoxy-4′′,6′′-O-eth ylid en e-
â-D-r ibo-h exop yr a n osyl)-ep ip od op h yllotoxin (33). To a
solution of 17 (0.06 g, 0.10 mmol) in DMF (2 mL) were added
successively triethylamine (58 µL, 0.42 mmol) and diiodoethyl
ether (0.5 g, 1.57 mmol). The reaction mixture was stirred
for 68 h at room temperature in the dark and diluted with
H2O (30 mL) and EtOAc (30 mL). The organic layer was
washed with H2O (4 × 30 mL), dried over anhydrous MgSO4,
and concentrated under reduced pressure. Flash chromatog-
raphy (92:8 CH2Cl2/acetone) gave 33 (0.046 g, 68%) as a
20
0.46 (6:1 cyclohexane/EtOAc); [R]D -14° (c 1.03, CHCl3); IR
(CDCl3) 2110 (N3), 1766 (CdO) cm-1 1H NMR (300 MHz,
;
CDCl3) δ 5.08 (dd, 1H, J ) 9, 2 Hz, H1), 4.83 (dd, 1H, J ) 9.5,
3.5 Hz, H4), 4.23 (m, 1H, J ) 3.5, 3.5, 3.5 Hz, H3), 4.13 (s, 2H,
ClCH2CO), 3.90 (m, 1H, J ) 9.5, 8, 3 Hz, H5), 3.35 (dd, 1H, J
) 11, 3 Hz, H6), 3.15 (dd, 1H, J ) 11, 8 Hz, H6′), 2.07 (m, 1H,
J ) 14, 3.5, 2 Hz, H2e), 1.86 (m, 1H, J ) 14, 9, 3.5 Hz, H2a),
0.92 (s, 9H, t-Bu), 0.19 (s, 3H, CH3-Si), 0.18 (s, 3H, CH3-Si);
MS (CI) m/z 507 (M + NH4)+. Anal. (C14H25N3O4ClISi) C, H,
N.
4-O-(3′′-Azid o-6′′-iod o-4′′-O-ch lor oa cetyl-2′′,3′′,6′′-tr id e-
oxy-â-D-r ibo-h exop yr a n osyl)-4′-b en zyloxyca r b on yl-ep i-
p od op h yllotoxin (37). To a mixture of 13 (1 g, 1.85 mmol)
and 36 (1 g, 2.04 mmol) in anhydrous CH2Cl2 (100 mL) cooled
to -15 °C was added BF3‚Et2O (455 µL, 3.7 mmol). After
reacting for 5 h (-15 °C f 0 °C), the reaction mixture was
diluted with CH2Cl2 (100 mL) and poured into a solution of
saturated NaHCO3 (200 mL). The organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure.
The crude residue was purified by flash chromatography (7:3
cyclohexane/EtOAc), giving 37 (0.8 g, 48%) as crystals: mp
20
syrup: Rf 0.31 (92:8 CH2Cl2/acetone); [R]D -98° (c 1.04,
CHCl3); 1H NMR (300 MHz, CDCl3) δ 6.73 (s, 1H, H5), 6.54 (s,
1H, H8), 6.25 (s, 2H, H2′, H6′), 6.00 (d, 1H, O-CH-O), 5.97 (d,
1H, O-CH-O), 5.20 (dd, 1H, J ) 9.5, 2 Hz, H1′′), 4.89 (d, 1H,
J ) 3.4 Hz, H4), 4.62-4.57 (m, 2H, H1, H7′′), 4.43 (dd, 1H, J )
10.5, 9 Hz, H9a), 4.20 (t, 1H, J ) 9, 8 Hz, H9b), 4.16-4.08 (m,
2H, H5′′, H6′′), 3.76-3.66 (m, 7H, CH2O, CH3O), 3.57 (dd, 1H,
J ) 9, 3 Hz, H4′′), 3.48 (t, 1H, J ) 12, 12 Hz, H6′′′), 3.23 (dd,
1H, J ) 14, 5.2 Hz, H2), 2.90-2.84 (m, 3H, H3, CH-N), 2.80
(q, 1H, J ) 3, 3, 3 Hz, H3′′), 2.42 (m, 2H, CH-N), 2.15 (m, 1H,
20
143 °C; Rf 0.58 (1:1 cyclohexane/EtOAc); [R]D -85° (c 1.25,
CHCl3); IR (CDCl3) 2103 (N3), 1773 (CdO) cm-1; MS (CI) m/z
909 (M + NH4)+. Anal. (C37H35N3O13ClI) C, H, N.
4-O-(3′′,6′′-Dia zid o-2′′,3′′,6′′-tr id eoxy-4′′-O-a zid oa cetyl-
â-D-r ibo-h exop yr a n osyl)-4′-ben zyloxyca r bon yl-ep ip od o-
p h yllotoxin (38). To a solution of 37 (0.45 g, 0.51 mmol) in
DMF (10 mL) was added NaN3 (0.1 g, 1.5 mmol). The reaction
mixture was stirred for 64 h at room temperature and diluted
with H2O (30 mL) and EtOAc (30 mL). The organic layer was
washed with H2O (4 × 20 mL), dried over MgSO4, and
concentrated under reduced pressure. Flash chromatography
(7:3 cyclohexane/EtOAc) afforded 38 (0.36 g, 90%): mp 120
H
2′′e), 1.55 (ddd, 1H, J ) 13, 9.5, 1 Hz, H2′′a), 1.33 (d, 3H, J )
5 Hz, CH3); MS (CI) m/z 642 (M + H)+.
ter t-Bu tyld im eth ylsilyl 3-Azid o-2,3-d id eoxy-6-O-tosyl-
â-D-r ibo-h exop yr a n osid e (34). A solution of tosyl chloride
(1.55 g, 8.15 mmol) in pyridine (10 mL) was added dropwise
to a cooled solution (0 °C) of diol 5 (2.06 g, 6.79 mmol). After
the solution was stirred for 1 h at 0 °C, it was allowed to reach
room temperature and stirred for an additional 18 h. The
reaction mixture was then extracted with CH2Cl2 (2 × 100
mL), and the organic layer was washed with H2O (2 × 50 mL),
dried over anhydrous MgSO4, and concentrated under reduced
pressure. The residue was purified by flash chromatography
(8:2 cyclohexane/EtOAc), giving 2.02 g (65%) of 34 and starting
material 5 (0.4 g, 26%). Compound 34, syrup: Rf 0.46 (2:1
cyclohexane/EtOAc); 1H NMR (300 MHz, CDCl3) δ 7.82-7.79,
7.37-7.34, (m, 4H, Har), 4.99 (dd, 1H, J ) 9, 2 Hz, H1), 4.21
(m, 2H, H6, H6′), 4.10 (m, 1H, H3), 3.80-3.73 (m, 2H, H4, H5),
2.46 (s, 3H, CH3), 2.07 (m, 1H, J ) 14, 3.5, 2 Hz, H2e), 1.78
(m, 1H, J ) 14, 9, 3.5 Hz, H2a), 0.90 (s, 9H, t-Bu), 0.09 (s, 6H,
CH3-Si); MS (CI) m/z 475 (M + NH4)+. Anal. (C19H31N3O6-
SSi) C, H, N.
20
°C; Rf 0.44 (6:4 cyclohexane/EtOAc); [R]D -64° (c 1, CHCl3);
IR (CDCl3) 2109 (N3), 1767 (CdO) cm-1; MS (CI) m/z 831 (M
+ NH4)+. Anal. (C37H35N9O13) C, H, N.
4-O-(3′′,6′′-Dia zid o-2′′,3′′,6′′-tr id eoxy-â-D-r ibo-h exop yr a -
n osyl)-4′-ben zyloxyca r bon yl-ep ip od op h yllotoxin (39). To
a solution of azido glycoside 38 (0.07 g, 0.08 mmol) in a CH2-
Cl2/MeOH mixture (2:1, 3 mL) was added Amberlite resin IRA
410 (OH-). After reaction for 3 h at 20 °C, the reaction mixture
was filtered and concentrated under reduced pressure to give
39 (0.06 g, 94%) as crystals: mp 125 °C; Rf 0.25 (6:4 cyclo-
20
hexane/EtOAc); [R]D -53° (c 1.04, CHCl3); IR (CDCl3) 2103
(N3), 1768 (CdO) cm-1; MS (CI) m/z 748 (M + NH4)+.
4-O-(3′′,6′′-Dia m in o-2′′,3′′,6′′-tr id eoxy-â-D-r ibo-h exop y-
r a n osyl)-ep ip od op h yllotoxin (40). To a solution of 39 (0.11
g, 0.15 mmol) in EtOAc/ethanol (1:1, 10 mL) were successively
added triethylamine (20 µL) and 10% palladium on activated
carbon (0.07 g). After the mixture was stirred for 30 h at room
temperature in the presence of hydrogen under atmospheric
pressure, the catalyst was eliminated by filtration and the
filtrate was concentrated under reduced pressure to afford 40
(0.078 g, 95%): mp 110 °C; Rf 0.06 (90:10 CH2Cl2/MeOH(NH3));
[R]D20 -86° (c 1.04, MeOH); IR (CDCl3) 3544, 3406 (NH2, OH),
ter t-Bu tyld im eth ylsilyl 3-Azid o-2,3,6-tr id eoxy-6-iod o-
â-D-r ibo-h exop yr a n osid e (35). A solution of 34 (2.02 g, 4.42
mmol) in 120 mL acetone was heated under reflux for 72 h in
the presence of sodium iodide (2.65 g, 17.6 mmol). After
cooling, the crude mixture was concentrated under reduced
pressure to ca. 30 mL and then diluted with CH2Cl2 (100 mL).
The solution was then washed with 10% aqueous solution of
sodium thiosulfate and dried over anhydrous MgSO4. Evapo-
ration under reduced pressure followed by flash chromatog-
raphy (8:2 cyclohexane/EtOAc) gave 35 (1.5 g, 82%) as a syrupy
1
1772 (CdO) cm-1; H NMR (300 MHz, CDCl3) δ 6.83 (s, 1H,
20
residue: Rf 0.50 (4:1 cyclohexane/EtOAc); [R]D -30° (c 1.0,
H5), 6.53 (s, 1H, H8), 6.26 (s, 2H, H2′, H6′), 5.99 (s, 1H, O-CH-
O), 5.96 (s, 1H, O-CH-O), 5.22 (dd, 1H, J ) 2, 9 Hz, H1′′),
4.91 (d, 1H, J ) 3.4 Hz, H4), 4.58 (d, 1H, J ) 5.3 Hz, H1), 4.47
(t, 1H, J ) 9, 10.5 Hz, H9a), 4.20 (t, 1H, J ) 8, 9 Hz, H9b), 3.76
(s, 3H, CH3O), 3.70 (m, 1H, H5′′), 3.61 (dd, 1H, J ) 4, 9 Hz,
H4′′), 3.46 (m, 1H, H3′′), 3.26 (dd, 1H, J ) 5.3, 14 Hz, H2), 3.18
(dd, 1H, J ) 5, 12 Hz, H6′′), 2.96 (t, 1H, J ) 8, 12 Hz, H6′′′),
2.86 (m, 1H, H3), 1.82 (m, 1H, H2′′e), 1.73 (m, 1H, H2′′a); MS
(CI) m/z 545 (M + H)+. Anal. (C27H32N2O10) C, H, N.
CHCl3); IR (CDCl3) 2123 (N3) cm-1; 1H NMR (300 MHz, CDCl3)
δ 5.07 (dd, 1H, J ) 9, 2 Hz, H1), 4.08 (m, 1H, H3), 3.58-3.48
(m, 3H, H4, H5, H6), 3.25 (dd, 1H, J ) 10, 7 Hz, H6′), 2.15 (m,
1H, J ) 14, 3.5, 2 Hz, H2e), 1.85 (m, 1H, J ) 14, 9, 3.5 Hz,
H
2a), 0.92 (s, 9H, t-Bu), 0.19 (s, 3H, CH3-Si), 0.18 (s, 3H, CH3-
Si); MS (CI) m/z 431 (M + NH4)+. Anal. (C12H24N3O3ISi) C,
H, N.
ter t-Bu tyld im eth ylsilyl 3-Azid o-4-O-ch lor oa cetyl-2,3,6-
t r id eoxy-6-iod o-â-D-r ibo-h exop yr a n osid e (36). Chloro-
acetyl chloride (395 µL, 5 mmol) was added to a cooled (-10
°C) solution of 35 (1.03 g, 2.5 mmol) in CH2Cl2 (20 mL) and
pyridine (405 µL, 5 mmol). After stirring for 1 h at the same
temperature, CH2Cl2 was added (30 mL). The organic solution
was washed with water (3 × 20 mL) and dried over MgSO4.
After evaporation and flash chromatography (10:1 cyclohexane/
4-O-(3′′-Azid o-6′′-iod o-2′′,3′′,6′′-tr id eoxy-â-D-r ibo-h exo-
pyr an osyl)-4′-ben zyloxycar bon yl-epipodoph yllotoxin (41).
To a solution of the azido glycoside 37 (0.26 g, 0.29 mmol) in
a CH2Cl2/MeOH mixture (2:1, 15 mL) was added Amberlite
resin IRA 410 (OH-). After reacting for 3 h at 20 °C, the
reaction mixture was filtered and concentrated under reduced
pressure to give 41 (0.22 g, 94%) as crystals: mp 115 °C; Rf

4484 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Daley et al.
20
0.46 (1:1 cyclohexane/EtOAc); [R]D -57° (c 1.02, CHCl3); IR
and 2 mM GTP. Reaction mixtures containing 2 mg/mL of
purified tubulin in 0.7 M monosodium glutamate, pH 6.6, 1
mM MgCl2, and 0.4% DMSO were preincubated at 37 °C for
15 min without GTP, chilled on ice for 10 min, and then 0.4
mM GTP was added at the same time as the test compound.36
Adsorbance of the solution of tubulin was determined at 350
nm. The IC50 values for inhibition of assembly or disassembly
were those concentrations of test compound that reduced the
assembly or disassembly by 50% as compared to solvent
controls.37
Assa y for DNA Rela xa tion Activity of Top oisom er a se
I. The method described by Larsen et al.38 was followed.
Briefly, 18 µL of buffer A (50 mM Tris, pH 7.5, 60 mM KCl,
0.5 mM DTT, 0.5 mM EDTA), containing 200 ng of pBR322,
and the amount of purified topoisomerase I prepared from calf
thymus which resulted in 100% relaxation were added to an
Eppendorf tube containing 2 µL of either vehicle alone or the
test compound in vehicle. After 30 min incubation, the
reaction mixture was analyzed on a 1% agarose gel and run
at 35 mA overnight in Tris-borate-EDTA (90 mM Tris, 90 mM
borate, 2 mM EDTA, pH 8.3) buffer. These so-called neutral
gels permitted the detection of compounds which inhibit
relaxation. Gels were stained with ethidium bromide, photo-
graphed under UV illumination, and the EC50 value, defined
as the 50% efficacy concentration (i.e., the concentration at
which 50% of the assays were positive for inhibition, which
was generally the calculated intermediate value between the
last active and the first active concentration calculated as the
log mean value), were determined.
Assa y for k DNA Deca t en a t ion Act ivit y of Top o-
isom er a se II. Briefly, 18 µL of buffer A (50 mM Tris, pH
7.5, 60 mM KCl, 0.5 mM DTT, 0.5 mM EDTA) containing 200
ng of kDNA and one unit of Drosophila topoisomerase II,
identified as the amount of enzyme which resulted in the
complete decatenation of 200 ng of kDNA, were added to an
Eppendorf tube containing 2 µL of either vehicle (DMSO) alone
or the test compound in DMSO. After 30 min incubation, the
reaction mixture was analyzed on a 1% agarose gel and run
at 35 mA overnight in Tris-borate-EDTA (89 mM Tris, 89 mM
borate, 2 mM EDTA, pH 8.3) buffer. Gels were stained with
ethidium bromide, photographed under UV illumination, and
the EC50 value, defined as the 50% efficacy concentration, were
calculated as described above for topoisomerase I inhibition.
(CDCl3) 2105 (N3), 1768 (CdO) cm-1; MS (CI) m/z 833 (M +
NH4)+.
4-O-(3′′-Am in o-2′′,3′′,6′′-tr id eoxy-â-D-r ibo-h exop yr a n o-
syl)-ep ip od op h yllotoxin (42). To a solution of 41 (0.20 g,
0.25 mmol) in EtOAc (10 mL) were successively added tri-
ethylamine (100 µL) and 10% palladium on activated carbon
(0.2 g). After stirring for 30 h at room temperature in the
presence of hydrogen under atmospheric pressure, the catalyst
was eliminated by filtration, and the filtrate was concentrated
under reduced pressure. Flash chromatography (92:8 CH2-
Cl2/MeOH) afforded 42 (0.05 g, 38%) as crystals: mp 140 °C;
20
Rf 0.33 (90:10 CH2Cl2/MeOH); [R]D -90° (c 0.5, CHCl3); IR
(CDCl3) 3543 (NH2, OH), 1768 (CdO) cm-1; 1H NMR (300 MHz,
CDCl3) δ 6.82 (s, 1H, H5), 6.54 (s, 1H, H8), 6.26 (s, 2H, H2′,
H6′), 6.00 (d, 1H, O-CH-O), 5.97 (d, 1H, O-CH-O), 5.01 (dd,
1H, J ) 2, 9 Hz, H1′′), 4.96 (d, 1H, J ) 3.3 Hz, H4), 4.59 (d, 1H,
J ) 5.3 Hz, H1), 4.48 (dd, 1H, J ) 9, 10.5 Hz, H9a), 4.24 (t, 1H,
J ) 8, 9 Hz, H9b), 3.76 (s, 3H, CH3O), 3.65 (m, 1H, H5′′), 3.35
(m, 1H, J ) 4, 4, 4 Hz, H3′′), 3.31-3.25 (m, 2H, H2, H4′′), 2.85
(m, 1H, H3), 1.90 (m, 1H, J ) 2, 4, 14 Hz, H2′′e), 1.78 (m, 1H,
J ) 4, 9, 14 Hz, H2′′a), 1.34 (d, 3H, J ) 6 Hz, CH3); MS (CI)
m/z 530 (M + H)+.
In Vivo Stu d ies. Female DBA/2 (Iffa Credo) and hybrid
CDF1 (Balb/c × DBA/2) mice were used for implanting with
the murine P388 leukemia. The P388 leukemia was passaged
as ip implants. For chemotherapy testing, tumors were
transplanted in the same strain or in the appropriate F1
hybrid. All mice were over 18 g at the start of each study.
One million P388 cells/mouse were implanted iv in CDF1 mice
on day 0. After randomization into treatment cages, test
compounds were administered ip on day 1 as single doses,
taken from the geometric series 2.5, 10, 40, 160 mg/kg. Mice
were checked daily for any adverse clinical reactions, which
were then noted and the day of death was recorded. Mice were
weighed on days 1, 4, and 8. Evaluation of antitumor activity
was measured in terms of life span. An increase of life span
was defined as the median survival of treated mice/median
survival of control mice × 100 (T/C, %). According to NCI
standard criteria for the P388 tumor model, 125% e T/C e
175% is the minimum level for activity.
In Vitr o Stu d ies. Gr ow th In h ibition Assa ys Usin g
L1210 Cells. The murine lymphocytic L1210 leukemia cell
line was obtained from the American Type Culture Collection
(ATCC). Cells were cultured in RPMI 1640 medium, supple-
mented with 10% heat-inactivated horse serum, 4 mM L-
glutamine, 100 U/L penicillin, 100 µg/mL streptomycin, and
1.25 µg/mL fungizone at 37 °C in an humidified atmosphere
of 5% CO2 in air. Cells were split twice a week and used for
studies over 40 in vitro passages. Under these conditions the
population doubling time of these cells was 8-10 h. Effects
of test compounds on L1210 cell proliferation were determined
using a standard growth inhibition assay. Exponentially
growing L1210 cells (1.5 × 105 cells/well) in a 24-well plate
were exposed to a range of concentrations of test compounds
for 48 h, after which the control and treated cells were counted
using an electronic particle counter. Computerized data was
processed using an in-house custom-designed program. The
IC50 values, i.e., the concentration of test compound to reduce
the cell number to 50% of that obtained with control cells, were
then determined from replicates of 6.
Ack n ow led gm en t. We thank Dr. S. Cros (Toulouse,
France) for a generous gift of murine P388 leukemia
cell culture. Thanks are due also to Laboratoires Pierre
Fabre for a fellowship (L. Daley). The authors are
indebted to G. Flad (Inst. Curie) for performing the
NMR spectra and to E. Chazottes, J . Astruc, J .-M.
Barret, and A. Limouzy (P. Fabre) who realized the
pharmacological experimentation.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR data for
all compounds which do not appear in the Experimental
Section (3 pages). Ordering information is given on any
current masthead page.
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