Practical demethylation of podophyllotoxin and efficient preparation of 4-amino-4-deoxy-4′-demethylepipodophyllotoxin

Article abstract of DOI:10.1080/00397911.2011.567436

(Chemical Equation Presented) 4′-Demethylepipodophyllotoxin (4′-DMEP) was readily available through demethylation of podophyllotoxin using methionine in methanesulfonic acid in the presence of TFA (or acetone/water). Thus, 4-amino-4-deoxy-4′-demethylepipodophyllotoxin was obtained in three steps in excellent yield by a Ritter reaction on 4′-DMEP, followed by treatment with thiourea in AcOH. Copyright Institut de Recherche Pierre Fabre.

Full text of DOI:10.1080/00397911.2011.567436

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Practical Demethylation of
Podophyllotoxin and Efficient
Preparation of 4-Amino-4-deoxy-4′-
demethylepipodophyllotoxin
Yves Guminski a , Martial Grousseaud a , Sandrine Cugnasse a &
Thierry Imbert a
a Centre de Recherche Pierre Fabre, Division de Chimie Médicinale ,
Castres , France
Accepted author version posted online: 02 Jan 2012.Published
online: 29 May 2012.
To cite this article: Yves Guminski , Martial Grousseaud , Sandrine Cugnasse & Thierry
Imbert (2012) Practical Demethylation of Podophyllotoxin and Efficient Preparation of 4-
Amino-4-deoxy-4′-demethylepipodophyllotoxin, Synthetic Communications: An International
Journal for Rapid Communication of Synthetic Organic Chemistry, 42:18, 2780-2789, DOI:
10.1080/00397911.2011.567436
To link to this article: http://dx.doi.org/10.1080/00397911.2011.567436
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Synthetic Communications1, 42: 2780–2789, 2012
Copyright # Institut de Recherche Pierre Fabre
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2011.567436
PRACTICAL DEMETHYLATION OF PODOPHYLLOTOXIN
AND EFFICIENT PREPARATION OF 4-AMINO-4-
DEOXY-4’-DEMETHYLEPIPODOPHYLLOTOXIN
Yves Guminski, Martial Grousseaud, Sandrine Cugnasse, and
Thierry Imbert
´
Centre de Recherche Pierre Fabre, Division de Chimie Medicinale, Castres,
France
GRAPHICAL ABSTRACT
Abstract 40-Demethylepipodophyllotoxin (40-DMEP) was readily available through
demethylation of podophyllotoxin using methionine in methanesulfonic acid in the presence
of TFA (or acetone/water). Thus, 4-amino-4-deoxy-40-demethylepipodophyllotoxin was
obtained in three steps in excellent yield by a Ritter reaction on 40-DMEP, followed by
treatment with thiourea in AcOH.
Keywords 4-Amino-4-deoxy-40-demethylepipodophyllotoxin; demethylation; 40-DMEP;
methionine; thiourea
INTRODUCTION
As part of our ongoing program to identify new antitumor compounds within
the podophyllotoxin series, we needed easy and cheap access to 4-amino-4-deoxy-
40-demethylepipodophyllotoxin 1 as a building block, which is presented here. In
addition, we report our investigations about the demethylation of podophyllotoxin 2.
Demethylation of aromatic methoxy groups is a usual and challenging problem
in synthetic organic chemistry, and this reaction is particularly useful in the field of
natural products, as reviewed by Bhatt and Kulkarni.[1] We needed a reliable and
efficient method of synthetic value to yield a large amount of the key intermediate
40-demethylepipodophyllotoxin (40-DMEP) 3, as starting material, without any tedi-
ous chromatographic purification step. This compound 3 was formerly obtained by
Kuhn et al.,[2] according to a method involving the treatment with hydrogen bromide
Received February 20, 2011.
Address correspondence to Yves Guminski, Centre de Recherche Pierre Fabre, Division de Chimie
´
Medicinale, 17 av. Jean Moulin, 81106 Castres Cedex, France. E-mail: yves.guminski@pierre-fabre.com
2780

SYNTHESIS OF 4-AMINO-4-DEOXY-40-DMEP
2781
Table 1. Demethylation of podophyllotoxin 2 or -4-substituted podophyllotoxin 4a, 4b to 40-DMEP 3,
using MsOH (30 eq.), D,L-methionine (5 eq.), and various carbocation trapping reagents
Entry
Starting material
Reagent
Temperature (ꢀC)
Time
Yield (%)
1
2
3
4
5
6
7
2
4a
4b
2
No
No
RT
ꢁ10
ꢁ10
40
5 min
1 h
2 h
Degradation
52
54
93
88
48
No
TFA
45 min
6 h
25 min
2
H3PO4
0 ->RT
40
2
HCOOH
Acetone=water (5=1)
Gram scale
Large scale
2
RT
40 ->RT
2 h
2 h
90
80
as a gas in methylene chloride=Et2O at 0 ꢀC, followed by a basic treatment (BaCO3).
This procedure was then routinely used by different groups,[3–6] giving an average
yield of 40% in our hands and requiring long reaction and purification times. Daley
et al.[7] improved this reaction using ISiMe3. However, this procedure was not
convenient for scale-up purposes. Kamal et al.[8] used methanesulfonic acid=sodium
iodide as a reagent system to elegantly overcome this problem.
Classical reagents[1] were attempted for such a transformation (AlCl3=EtSH;
pyridine=HCl; BBr3; BCl3; AlCl3; LiI=2,4,6-collidine) but proved unsuccessful
because degradation of the starting material was observed.
A method for demethylation involving methanesulfonic acid and methionine as
[13]
reagent was previously mentioned by Yajima et al.[9–12] Particularly, Andre et al.
´
pointed out its use for demethylation in the naloxone synthesis, leading with trans-
position[14–18] to the apo series. This method has been used only rarely up to
now.[19,20]
This reagent was used without success when applied to podophyllotoxin 2. The
reaction medium rapidly turned dark red and gave only degradation products, aro-
matization of the C ring, or tars (Table 1, entry 1). It was rationalized that the
known lability of the benzylic hydroxy group of podophyllotoxin in a strongly acidic
medium might undergo this degradation during demethylation. In contrast, encour-
aging results were obtained when demethylation was carried out with derivatives
with a chloro, bromo substituent at this 4-position. These starting materials (4a,
4b) were conveniently prepared by treatment with HCl and HBr as already
described.[21] In these cases, 40-demethylation with MsOH=methionine gave fair
yields of 3 (Table 1, entries 2 and 3). It was necessary to work with an excess of meth-
ionine for a nearly complete reaction. However this methodology needed a two-step
process.
Interestingly, the HBr-AcOH method proved to be efficient in the case of
4-deoxypodophyllotoxin.[22] In the case of 2, the first intermediate formed was the
benzylic carbocation generated at the 4-position under acidic medium (Lewis acid),
in agreement to Kuhn and Von Wartburg.[23] This species is not stable enough dur-
ing the demethylation process involving methanesulfonic acid and methionine. How-
ever, the benzylic cation might be protected against its degradation by trapping this
species with an appropriate group, yielding the 4-hydroxy in the b-position after

2782
Y. GUMINSKI ET AL.
Scheme 1.
hydrolysis. Thus, trifluoroacetic acid was used both as a cosolvent and as an entity
protecting the carbocation immediately after its formation. This putative mech-
anism, related to that published by Berenyi et al.,[17] is shown in Scheme 1. The best
results were obtained by addition of trifluoroacetic acid (93% yield) (Table 1, entry
4). Phosphoric acid gave good yields (Table 1, entry 5) but a less convenient reaction
for scaling up because of the difficulty of stirring the reaction medium. Acetic or
formic acid (Table 1, entry 6) worked as well, but gave lesser yields.
In a second series of experiments, to reduce this difficulty on a large scale, we
used acetone as a cosolvent. However, an excess of acetone lowered the yield. The
optimum ratio for the mixture of methanesulfonic acid=acetone was 10=1. Next, it
was thought that H2O might trap the transient carbocation. It was observed that
the reaction proceeded without degradation. So, the presence of water was not det-
rimental for such a reaction. Thus, trifluroacetic acid (TFA) could be replaced by a
small amount of water in acetone (mixture 5=1), giving good yields (90% or 80%
according to the scale of experiment) (Table 1, entry 7).
Badet and Julia[24] prepared trimethylsulfonium salt with dimethylsulfide
(DMS) and anisole in MsOH. Based on this demethylation procedure, our objective
was to use DMS as a demethylating reagent. Experiments with several sulfides were
undertaken. Use of DMS gave results similar to methionine, featuring well-stirred
slurry (Table 2, entries 1–4). A longer reaction time or an excess of demethylating
reagent led to a small amount of di- and tri-demethylation, yielding 3 and a mixture
of 3’,40-dihydroxy 5a[25] and 3’,4’,50-trihydroxy 5b that was easy to separate (Table 2,
entry 3 and 4). Conversely, a shorter reaction time and an excess of DMS led to 3
and a little amount (2%) of the 4-epipodophyllotoxin 4c (Table 2, entry 2). The best
conditions were determined as 7 molar excess of DMS in 30 molar excess of MsOH

SYNTHESIS OF 4-AMINO-4-DEOXY-40-DMEP
2783
Table 2. Demethylation of podophyllotoxin 2 to 40-DMEP 3, using MsOH and various sulfides as
demethylating reagent in acetone=water (5=1) at room temperature
Entry
Demethylating reagent
Time
Yield (%)
1
2
3
4
5
6
7
8
DMS (5 eq.)
DMS (7 eq.)
1 h
4 h
60
90a
DMS (10 eq.)
DMS (40 eq.)
48 h
15 min
2 h
95b
95c
50
Dithiane (5 eq.)
Thiophene, thioethanol, thioanisole
Thiodiacetic acid
Methylthioacetic acid (5 eq.)d
—
—
Degradation
Degradation
60
3 h
aPresence of 2% of 4c (4-epimer of starting material).
bPresence of di- and tri-demethylated compounds 5a (1%) and 5b (0.5%).
cPresence of di-demethylated compound 5a (0.5%).
dIn the absence of acetone=water.
in the presence of a mixture of acetone=water (5=1) (Table 2, entry 2). Under the best
conditions, the crude crystalline compound 40-DMEP 3 was obtained in 90% yield,
with a purity of 83% (by analytical high-performance liquid chromatography
HPLC), the by-products essentially consisted of an equilibrated proportion of 4-epi-
mer of starting material 4c, and di- and tridemethylated compounds, 5a and 5b. It
was considered adequate for most synthetic purposes. Nevertheless, a purity
enhanced up to 95% could be obtained by a further recrystallization in hot AcOEt,
with an overall yield of 69% from podophyllotoxin 2.
Some other sulfides such as dithiane gave 50% of 3 (Table 2, entry 5). Thio-
phene, thioethanol, and thioanisole did not react as expected (Table 2, entry 6).
Methylthioacetic acid and thiodiacetic acid were tried for their ability to both trap
the intermediate and act as demethylating reagent. Only methylthioacetic acid gave
60% of 3 (Table 2, entry 7).
The conventional approach to give 4-amino-40-O-demethylepipodophyllotoxin
1 from 40-DMEP 3 was the reduction of the 4-azido intermediate, prepared from 3
with the couple NaN3=TFA. This methodology actually led to the mixture of both
a and b amino epimers. Though this problem was partially resolved by Zhou and
coworkers,[26] it needed improvement for scale-up purposes. Another method has
been reported with HN3=Et2O-BF3 at ꢁ15 ꢀC and reduction with SmI2 in t-BuOK
or FeSO4=NH3.[27,28] Besides the necessary chromatographic purification of both
azido epimers,[29] the drawbacks of these methods were (1) the risk of using an azido
derivative, potentially explosive, during large-scale synthesis and (2) the cost of a
catalytic reduction step. We present here our results to overcome these problems.
Within the podophyllotoxin series, Ritter reaction with chloroacetonitrile was
reported,[3] and this reaction was applied to 40-DMEP 3. Chloroacetonitrile was used
as a solvent (10 eq) and H2SO4 at room temperature, this reaction led directly to the
already known[30] 4-chloroacetamido-4-deoxy-40-demethylepipodophyllotoxin 6,
with an excellent yield (Scheme 1). The configuration at C4 was confirmed by large
coupling value in the NMR spectrum (trans relationship JH3-H4 ¼ 7 Hz). The
cleavage of chloroacetamido group to amino with thiourea in ethanol in the presence

2784
Y. GUMINSKI ET AL.
Table 3. Experimental conditions for the preparation of 4-amino-4-deoxy-40-demethylepipo-
dophyllotoxin 1 from 4-acetamido-4-deoxy-40-demethylepipodophyllotoxin 6
Reaction time
(h)
Entry
1
Conditions
EtOH=AcOH (5=1)
Yield (%)
Purity (%)
10
<10
Presence of starting material
and degradation products
2
3
4
5
Glacial AcOH at 80 ꢀC
EtOH=HCl1 N
2
9
93
90
60
70
>95
95
EtOH=H2O=AcOH (5=1=1)
Dioxan or DMA=AcOH=H2O (5=1=1)
10
5-6
95
95
of AcOH is a method already reported by Jirgensons et al.[31] We used this procedure
with some modifications to adapt it to our purpose. In our hands, the reported pro-
cedure afforded the target compound 1 with a poor yield (<10%). A longer reaction
time did not improve the transformation and promoted degradation. However, when
40-DMEP 3 was heated at 80 ꢀC in glacial AcOH, followed by addition of thiourea,
the reaction was completed in less than 2 h. The initial insoluble slurry became quite
limpid and then a precipitate was observed, leading to the 4-amino-4-deoxy-
40-demethylepipodophyllotoxin 1 in 93% yield.
Different conditions were tested (Table 3), demonstrating that (1) the presence
of water (i.e., HCl 1 N, entry 3) with ethanol appeared to be more efficient than
anhydrous medium and (2) in contrast to the previously described procedure, the
presence of ethanol (entry 4) or other solvents such as dioxan or DMA (entry 5) gave
less yield than glacial AcOH alone (entry 2).
In conclusion, 4-amino-4-deoxy-40-demethylepipodophyllotoxin
1
was
obtained in three steps without purification from natural podophyllotoxin 2 with a
70% overall yield. Demethylation of podophyllotoxin with methionine or DMS in
methanesulfonic acid in the presence of acetone and water (80% yield), followed
by a Ritter reaction with chloroacetonitrile (93% and yield), and then, finally, an
acidic cleavage with AcOH gave the requested 4-amino-4-deoxy-40-demethylepipo-
dophyllotoxin 1 (93% yield). This three-step method appeared to be the best way
for a large-scale reaction.[32,33]
EXPERIMENTAL
All solvents and reagents used were obtained commercially as ‘‘pure for syn-
thesis’’ grade (min. 98%) and were used without further purification unless indicated
otherwise in the experimental section. Melting points were determined using an Elec-
trothermal 9300 capillary apparatus and were uncorrected. Elemental analyses were
performed on a Fisons 1108 microanalyzer and were within 0.4% of the theoretical
values. Karl-Fisher analyses were performed on a Mettler DL 37 coulometer. The
reaction progress was monitored by thin-layer chromatography (TLC) on silica-gel
plates (60 F-254, Merck art. 1.05554), examined under ultraviolet (UV) light at
254 nm and sprayed with Salkowski or Draggendoff reagent. Purification was
performed by flash-column chromatography using Merck silica gel (35–70 m) with
Intelliflash 280 (Analogix), gradient, and flow of 40 or 80 mL=min for 30- or

SYNTHESIS OF 4-AMINO-4-DEOXY-40-DMEP
2785
1
45-cm columns respectively. H and 13C NMR spectra were recorded in dimethyl-
sulfoxide (DMSO-d6) solution (unless otherwise specified) on Brucker AC 200 and
Advance DPX 400 spectrometers; chemical shifts are given in parts per million
(ppm) relative to tetramethylsilane (TMS) as internal standard. Liquid chromato-
graphy coupled with mass spectra was performed on ThermoElectron MSQ (simple
quadripole) spectrometer, and mass spectra for structural analysis were recorded on
TSQ ThermoElectron (triple quadripole).
4’-Demethylepipodophyllotoxin (4’-DMEP) 3
Procedure using trifluoroacetic acid (Table 1, Entry 4). Methanesulfonic
acid (84 mL, 140 mol) and D,L-methionine (21.6 g, 140 mmol) were introduced to a
stirred solution of podophyllotoxin 2 (12 g, 29 mmol) in 20 mL of trifluoroacetic acid.
The temperature rose to 40 ꢀC, and stirring was maintained for 45 min. Reaction
mixture was poured into 500 mL of acetone and cold water (1=1). The stirred mixture
was cautiously neutralized up to pH 5 with K2CO3 and extracted (twice) with
AcOEt. Organic layers were separated, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was crystallized with iPr2O and gave 10.7 g
1
(93%) of 40-DMEP 3; mp 252 ꢀC. H NMR (200 MHz, d6-DMSO): d 8.30(s, 1H),
6.94 (s, 1H), 6.50 (s, 1H), 6.21 (s, 2H), 5.99 (d, 2H, J ¼ 3.9 Hz), 4.71 (d, 1H,
J ¼ 3.4 Hz), 4.49 (d, 1H, J ¼ 5.2 Hz), 4.33 (dd, 1H, J ¼ J0 ¼ 8 Hz), 4.17 (dd, 1H,
J ¼ 8.4 Hz, J0¼10.5 Hz), 3.61 (s, 6H), 3.24 (dd, 1H, J ¼ 5.25 Hz, J0 ¼ 14.2 Hz), 2.77
(m, 1H).
Procedure using 4-chloroepipodophyllotoxin 4a (Table 1, Entry
2). Podophyllotoxin 2 (1 g, 2.4 mmol) was dissolved in 15 mL CH2Cl2 and 5 mL
Et2O. Through this solution, cooled at ꢁ10 ꢀC, a light stream of hydrochloric acid
was introduced for 15 min. The solution was evaporated at rt and then were added
to the residue methane sulfonic acid (5 mL, 8.3 mmol) and D,L-methionine (0.54 g,
3.5 mmol). After solubilization and stirring for 1 h at rt, the slurry was poured into
a mixture of acetone=water (1=1) and BaCO3 was added up to pH 7. Extraction with
AcOEt yielded after evaporation a residue, which was crystallized with Et2O to
afford 0.5 g (52%) of 40-DMEP 3, as white crystals.
Procedure using phosphoric acid (Table 1, Entry 5). Podophyllotoxin 2
(5 g, 12 mmol) was added to the viscous mixture of orthophosphoric acid (2.5 mL),
methanesulfonic acid (40 mL, 600 mmol) and methionine (11 g, 72 mmol) at 0 ꢀC.
The stirring continued for 6 h at rt. The reaction mixture was poured into ice water
(500 mL) to yield a white precipitate. This solid was extracted (three times) with
AcOEt, and the organic layers were washed with NaHCO3 solution. The organic
layers were dried over Na2SO4 and filtered. After evaporation, 4.2 g (88%) of
40-DMEP 3 were obtained (HPLC purity 79%).
Procedure using formic acid (Table 1, Entry 6). A drop of MsOH,
D,L-methionine (10.5 g, 68 mmol), and 49 mL of MsOH were introduced to a limpid
solution of podophyllotoxin (7 g, 17 mmol) in 3.5 mL of formic acid and under stir-
ring. The temperature rose to 40 ꢀC, and again D,L-methionine (10.5 g, 68 mmol)
was added. Stirring was maintained during 15 min, and the reaction mixture was

2786
Y. GUMINSKI ET AL.
poured into ice water. The obtained precipitate was extracted with AcOEt and
washed with a solution of NaHCO3. The organic layer was decanted, dried over
Na2SO4, filtered, and evaporated. The residue was crystallized in a mixture of
iPr2O=CH2Cl2, to yield 3.2 g (48%) of 40-DMEP 3.
Procedure using D,l-Methionine/acetone/water (Table 1, Entry 7). A
mixture of D,L-methionine (4 g, 27 mmol) and methanesulfonic acid (20 mL,
310 mmol) with H2O (0.4 mL) was added to a solution of podophyllotoxin 2 (2 g,
4.8 mmol) in 2 mL of acetone under stirring at rt. After 2 h, the reaction medium
was poured into ice water (200 mL) and extracted with AcOEt. The organic layers
were washed with a solution of NaHCO3, dried over Na2SO4, and evaporated,
affording 1.75 g of 40-DMEP 3 (yield 90%).
Large Scale Procedure
A solution of podophyllotoxin 2 (50 g, 0.12 mol) in acetone (50 mL) was added
under stirring at room temperature to a mixture of D,L-methionine (100 g, 0.67 mol),
methane sulfonic acid (500 mL, 7.7 mol), and water (10 mL). The temperature rose to
40 ꢀC, and stirring was maintained for 2 h. Then, the mixture was poured onto ice to
obtain a precipitate, which was extracted with ethyl acetate (3 ꢂ 100 mL). The
organic layers were washed with a NaHCO3 solution, separated, dried over anhy-
drous Na2SO4, filtered, and concentrated under vacuum to afford the crude
demethylated compound (45 g, yield 94%) as a white solid. The residue was taken
up in isopropyl ether, and the 40-DMEP 3 (38 g, 80%) was crystallized as a white
powder. A sample was recrystallized from acetone–water mixture. Mp 248–250 ꢀC.
Procedure using DMS in acetone/water (Table 2, Entry 2). DMS
(5.2 mL) and MsOH (23 mL, 0.36 mol) were successively added to a stirred suspen-
sion of podophyllotoxin 2 (5 g, 12 mmol) in 2.5 mL of a mixture of acetone=H2O
(5=1). The temperature rose up to 40 ꢀC, and stirring was maintained for 1.5 h at
rt. The reaction mixture was poured into ice water (500 mL) to yield a white precipi-
tate. This solid was filtered and washed with water and Et2O to give 3 (4 g crude yield
90%). The purity was determined as 83% with HPLC. These obtained crystals could
be recrystallized if necessary in AcOEt, affording 3.3 g of pure 40-DMEP 3 (overall
yield 69%). Another experiment with 500 g of podophyllotoxin gave essentially the
same yield. A lengthened reaction time up to 5 h gave a purity of 75% (analytical
HPLC).
Procedure using dithiane (Table 2, Entry 5). Dithiane (3 mL, 25 mmol) and
MsOH (15 mL, 0.23 mol) were added to a suspension of podophyllotoxin (2 g,
5 mmol) in 2 mL of a mixture of acetone=H2O (5=1). Stirring was maintained for
2 h at rt. The reaction mixture was poured into ice water, and the white precipitate
was filter and washed with water and Et2O to afford 1.2 g (yield 50%) of 40-DMEP 3.
Procedure using methylthioacetic acid. (Table 2, Entry 7). Methylthioa-
cetique acid (1.28 g, 12 mmol) was added to a stirred solution of podophyllotoxin
(1 g, 2.4 mmol) in MsOH (10 mL). The stirred slurry was kept 3 h at rt and poured
into water. The formed precipitate was filtered, washed with water, and dried with
Et2O, affording 580 mg (yield 60%) of 40-DMEP 3.

SYNTHESIS OF 4-AMINO-4-DEOXY-40-DMEP
2787
Preparation of 3’,4’-Didemethylated Epipodophyllotoxin 5a and
3’,4’,5’-Tridemethylated Epipodophyllotoxin 5b
Podophyllotoxin 2 (10 g, 24 mmol) was dissolved in trifluoroacetic acid
(60 mL). DMS (5.4 mL, 72 mmol), and MsOH (47 mL, 72 mmol) were successively
added. Stirring was maintained for 9 h. Then the medium was poured onto ice
(600 mL) and extracted with ethyl acetate (3 ꢂ 300 mL). The organic layers were
washed with water and then with a NaHCO3 solution to neutral pH. After drying
over anhydrous Na2SO4, the solution was filtered and concentrated under vacuum.
Crude demethylation products were obtained. Flash chromatography on silica gel
(elution : CH2Cl2=acetone 9=1) successively afforded 550 mg of 40-DMEP 3, 1.1 g
of 30,40-didemethylepipodophyllotoxin 5a, (C20H18O8, 0.15 H2O; calc. C%: 61.74;
H%: 4.74; found: C%: 61.67, H%: 4.68) and 1.9 g of 30,40,50-tridemethy-
lepipodophyllotoxin 5b (1H NMR 400 MHz d6-DMSO) d 8.65 (m, 2H), 7.95 (m,
1H), 6.71 (s, 1H), 6.47 (s, 1H), 5.98 (d, 2H, J ¼ 2 Hz), 5.93 (s, 2H), 4.68 (d, 1H,
J ¼ 3.2 Hz), 4.34 (t, 1H, J ¼ 8 Hz), 4.29 (d, 1H, J ¼ 5.2 Hz), 4.16 (dd, 1H, J ¼ 8 Hz,
J0 ¼ 10 Hz), 3.17 (dd, 1H, J ¼ 5.2 Hz and J’ ¼ 14 Hz), 2.76 (m, 1H).
Preparation of 4-Chloroacetamido-4-deoxy-4’-
demethylepipodophyllotoxin 6
To a suspension of 30 g of 40-DMEP 3 (0.075 mol) in 47.5 mL (0.75 mol) of
chloroacetonitrile was added 0.5 mL of concentrated H2SO4 at rt under stirring.
Solubilization was observed and followed by reprecipitation, and 300 mL of iPrOH
were added under vigorous stirring. The precipitate was filtered washed with 200 mL
of iPrOH and water to pH 7. The obtained white powder was dried under vacuum at
40 ꢀC to provide 32.9 g (yield 93%) of 4-chloroacetamido-4-deoxy-40-deme-
thylepipodophyllotoxin 6.[29] TLC Rf 0.44 (CH2Cl2=MeOH=NH4OH 95=4.5=0.5)
1
mp 240 ꢀC. H NMR (400 MHz, d6-DMSO) d 8.65 (d, 1H, J ¼ 7 Hz), 8.26 (s, 1H),
6.78 (s, 1H), 6.54 (s, 1H), 6.24 (s, 2H), 5.99 (d, 2H, J ¼ 11.3 Hz), 5.17 (dd, 1H,
J ¼ 4.56 and 7 Hz), 4.51 (d, 1H, J ¼ 5,2 Hz), 4.29 (t, 1H, J ¼ 8 Hz), 4.10 (s, 2H),
3.97 (m, 1H), 3.78 (dd, 1H, J ¼ 8 Hz and 10 Hz), 3.63 (s, 6H), 3.15 (dd, 1H,
J ¼ 5.2 and 14 Hz).
Preparation of 4-Amino-4-deoxy4’-demethylepipodophyllotoxin 1
A suspension of 4-chloracetamido-4-deoxy-40-demethylepipodophyllotoxin 6
(17 g, 0.0358 mol) in glacial acetic acid was heated at 80 ꢀC under stirring. Thiourea
(4.2 g, 0.0537 mol) was added and the slurry was stirred at this temperature for 1
h 30 min. Dissolution and precipitation were observed. The reaction mixture was fil-
trated, washed with glacial acetic acid (75 mL) and diisopropyl ether. The obtained
white solid was dried under vacuum at 40 ꢀC to give 1 as its hydrochloride (14.6 g,
93%).[28] Mp > 260 ꢀC.
1H NMR (400 MHz, d6-DMSO) 8.63 (m, 2H), 8.32 (m, 1H), 7.23 (s, 1H), 6.60
(s, 1H), 6.8 (s, 2H), 6.05 (d, 2H, J ¼ 2.1 Hz), 4.73 (d, 1H, J ¼ 4.5 Hz), 4.56 (d, 1H,
J ¼ 5.2 Hz), 4.34 (m, 2H), 3.65 (dd, 1H, J ¼ 5.2 Hz), 3.62 (s, 6H), 3.06 (m, 1H).

2788
Y. GUMINSKI ET AL.
ACKNOWLEDGMENTS
We thank Daniel Dauzonne and Claude Monneret from Institut Curie (Paris)
for their interest and exploitation of the demethylation procedure.
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