Synthesis and cytotoxicity of podophyllotoxin analogues modified in the A ring

Article abstract of DOI:10.1016/S0223-5234(02)00007-7

Several podophyllotoxin derivatives lacking the methylenedioxy group or with different functionalization of the A-ring of the cyclolignan skeleton have been prepared and evaluated for their cytotoxic activities on four neoplastic cell lines (P-388, A-549,

Full text of DOI:10.1016/S0223-5234(02)00007-7

European Journal of Medicinal Chemistry 38 (2003) 65ꢀ74
/
www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxicity of podophyllotoxin analogues modified in
the A ring
Angeles Castro a,*, Jose´ M. Miguel del Corral a, Marina Gordaliza a,
Concepcio´n Grande a, Antonia Go´mez-Zurita a, Dolores Garc´ıa-Gra´valos b,
Arturo San Feliciano a
a Departamento de Qu´ımica Farmace´utica, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spain
b Biomar S.A. Calera 3, Tres Cantos, 28760 Madrid, Spain
Received 21 August 2002; received in revised form 6 November 2002; accepted 13 November 2002
In memory of Dr. Benedicto del Rey
Abstract
Several podophyllotoxin derivatives lacking the methylenedioxy group or with different functionalization of the A-ring of the
cyclolignan skeleton have been prepared and evaluated for their cytotoxic activities on four neoplastic cell lines (P-388, A-549, HT-
29 and MEL-28). Most of them maintained their cytotoxicity at the mM level.
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Podophyllotoxin; Cyclolignans; A-ring modifications; Cytotoxicity
1. Introduction
QSAR methods by Lee and coworkers [3] leaving the
cyclolignan skeleton nearly untouched.
Podophyllotoxin belongs to the natural product
family of lignans, well known for their antineoplastic
and antiviral properties [1]. These properties prompted
the scientific community to perform numerous modifi-
cations on the podophyllotoxin skeleton, in order to
avoid several side effects, which have resulted in the
clinical introduction of etoposide, teniposide and eto-
pophos [1,2] (Fig. 1).
The chemical modifications that led to etoposide and
derivatives, led also to a change in the mechanism of
action: from antimicrotubule in the parent compound
podophyllotoxin to antitopoisomerase II in etoposide
and congeners. This change is related to three main
chemical modifications: demethylation at C-4? of the E
ring, a C-7 epimerization and the presence of a glicosidic
moiety or similar at the C-7 position on the C ring.
Those facts were the origin of a great number of
derivatives that have been synthesized and analyzed by
Over the years, we have been involved in the chemical
transformation of podophyllotoxin and we have pre-
pared a large number of cyclolignans, some of which
displayed potent antiviral and cytotoxic activities [2b].
Among these, those analogues modified at the C- and
D-rings with the general structure showed at Fig. 1 are
particularly worthy of note [4]. These derivatives have
an interesting selectivity against the human colon
tumoural cell line HT-29, in spite of the absence of the
lactone moiety which has always been considered an
important factor for high cytotoxic activity [5].
In our previous studies, the A and E rings were
untouched, and also there are few references about
modifications to these and the influence of such changes
on the cytotoxic activity of cyclolignans.
Regarding the A ring, several N-heterocyclic A-ring
podophyllotoxin derivatives have been prepared [6] and
the biological evaluation indicates no antimitotic activ-
ity, either weak inhibition of topoisomerase II in vitro.
Nonetheless, one of them exhibited an improved cyto-
toxic profile compared to that of etoposide, so a diferent
mechanism of action was postulated.
* Corresponding author.
E-mail address: macg@usal.es (A. Castro).
´
0223-5234/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(02)00007-7

66
A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 65ꢀ74
/
lacking the methylenedioxy group and/or with different
functionalization on the A ring.
2. Chemistry
The preparation of the analogues 3ꢀ
/
7 and 9ꢀ10,
/
which lacked the methylene group on the dioxole ring is
shown in Fig. 2 and summarized in Table 1.
The starting materials for the transformation pre-
sented here were the natural products deoxypodophyl-
Fig. 1. Structures of podophylotoxin and related compounds. (A)
Cyclolignans in clinical use. (B) Selective cytotoxic cyclolignans
lacking the lactone ring.
lotoxin
and
podophyllotoxin
isolated
from
podophyllum resin [10]. Podophyllotoxin was also
transformed into the isoxazole 8 as previously described
by us [11].
The ring A-opened compounds have been found in
naturally occurring podophyllotoxin congeners such as
diphyllin and sikkimotoxin which have been shown to
be less cytotoxic than podophyllotoxin [7]. Also a series
of 3,4-O,O-demethylene-7b-substituted podophyllotox-
ins and their 4?-O-demethyl analogues have been
synthesized and evaluated for their cytotoxicity and
their ability to inhibit human DNA topoisomerase II.
All the tested compounds were found to be less potent
than the methylenedioxy-bearing analogues [8].
However we have shown in previous work [9] that
analogues without the methylenedioxy group have a
very interesting immunosuppressive activity. All these
facts made us to think that more work is necessary to
understand the influence of that group on the cytotoxi-
city. Thus, in this paper we present our studies directed
towards the preparation of podophyllotoxin analogues
Boron trichloride was used to attain the cleavage of
the methylenedioxy group selectively, in the presence of
the aromatic methoxyl groups of the trimethoxyphenyl
moiety. The best yields of demethylenated compounds
were obtained when the reaction mixture was kept one
hour at low temperature and one hour under reflux in
the presence of calcium carbonate. After longer reaction
periods, 3,4-demethylenepodophyllotoxin (4) was ac-
companied by two other compounds, its 7-epimer 5
and the dehydration product 6, likely formed by
dehydratation followed by double bond isomerization
to the more stable a,b-unsaturated enone system.
Compound 6 was also the major product of the acid-
catalyzed cleavage of the methylenedioxy ring of 2
according to Schreier’s method [12].
With the aim of analyzing the influence on cytotoxi-
city, we have considered simultaneous modifications in
Fig. 2. Preparation of the 3,4-O,O-demethylenated cyclolignans.

A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 65ꢀ
/74
67
Table 1
Preparation of compounds 3ꢀ7
/
a
Substrate
Method
Temperature (8C)
Time (iꢁ
/
ii) (h)
Products
1
1
2
2
2
8
A
B
ꢂ
150
/
78
1ꢁ
3
/
1
3 (67%)
3 (41%)ꢁ
4 (80%)
/
7 (10%)
A
A
B
ꢂ/
65 to ꢂ
/
70
1ꢁ1
/
ꢂ
/
78
2.5ꢁ
3
/4
4 (11%)ꢁ
6 (43%)
9ꢁ10 (70%)ꢁ
/
5 (16%)ꢁ
/6 (14%)
150
A
0
1ꢁ
/
1.5
/
/8? (6%)
a
Method A: (i) BCl3ꢂCH2Cl2, low temperature; (ii) H2O, acetone, CaCO3, reflux. Method B: H3PO4, phenol, AcOH.
/
A ring and other parts of the cyclolignan skeketon that
also shown immunosuppressive activity[10]. Thus, the
isoxazole 8 was treated with boron trichloride to afford
a 9:1 mixture of 9 and 10 as it can be deduced from the
1H-NMR spectra. At this point, it was not possible the
purification of 10; this was achieved later on, after
reaction with dihalogenated derivatives. It has been
reported that cleavage of the methoxy groups usually
requires either higher temperatures or longer reaction
times[13]. However, when 8 was demethylenated at low
temperature, there was a large proportion of starting
material, and when the reaction was performed at 0 8C,
the major product was 9 together with a small propor-
tion of 10. Even the compound 8?, which had the
methylenedioxy but demethylated at C-4?, was also
isolated.
signals in their espectra. During the recycling of the
dioxole, epimerization of the lactone ring occurred due
to the basic media used in the reaction. The purification
of carboxylic acid was difficult and it was isolated as its
methyl ester 11a, obtained after treatment of the
reaction product with diazomethane. The same anolo-
gue 11a was obtained from demethylenedeoxypicropo-
dophyllin 7 under the same conditions. When 4 was
treated with methyl dichloroacetate, the only isolated
product was 13, as a result of the dehydratation and
aromatization under the reflux conditions of the reac-
tion.
Treatment of 3 with 1,2-dibromoethane gave a reac-
tion product from which the dioxane derivatives 14 and
15 were isolated. In this case only a partial isomerization
of the lactone ring occurred.
Once the two phenolic groups were obtained, they
were transformed into other five- or six-membered rings
with different substituents by condensation with diha-
logenated substrates under basic conditions (Figs. 3 and
4). Thus, condensation of 3 with methyl dichloroacetate
and dichloromethyl methyl ether yielded the analogous
compounds 11 and 12 as a mixture of epimers at C-10
position, as it can be deduced from several duplicated
The mixture of 9 and 10 was also treated with 1,2-
dibromoethane under the same conditions and the
corresponding dioxanes 20 and 21 were obtained. In
order to obtain the corresponding substituted dioxole
analogues, the mixture of 9 and 10 was treated with the
corresponding dihalogenated derivatives, giving 16 and
17 when methyl dichloroacetate was used; the mixture of
epimers is clearly observed in the NMR 13C spectrum of
Fig. 3. Formation of new dioxole/dioxane rings from 3 and 4. (i) Cl2CHꢀ
/
COOCH3 or Cl2CHꢀ
/
Oꢀ
/
CH3, K2CO3, DMF, 90ꢀ
/
100 8C. (ii) Brꢀ
/
CH2 ꢀ
/
CH2 ꢀBr, K2CO3, NaI, acetone, reflux.
/

68
A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 65ꢀ74
/
Fig. 4. Formation of new dioxole/dioxane rings from 9 and 10. (i) Cl2CHꢀ
/
COOCH3, K2CO3, DMF, 90ꢀ
/
100 8C. (ii) CH2N2, ether. (iii) Cl2CHꢀ
/
Oꢀ
/
CH3 or BrꢀCH2 ꢀCH2 ꢀBr, K2CO3, NaI, acetone, reflux.
/
/
/
derivative 17, in which several signals are duplicated
(Table 6). In the case of dichloromethyl methyl ether,
instead of the corresponding dioxole analogues, the
acetonides 18 and 19 ware obtained as the result from
the reaction between the phenolic groups and the
acetone used as solvent.
vs 2). Epimerization of the hydroxyl group at C-7
decreased the cytotoxic potency (5 vs. 4) four times.
Acetylation of the catechol groups also led to derivatives
about one order of magnitude less cytotoxic.
The derivatives with the isoxazole ring were among
the least cytotoxic compounds prepared, although it is
worth noticing that derivative 19 was ten times more
potent against P-388 than the other tested lines.
3. Biological results and discussion
The compounds thus prepared were evaluated in vitro
to establish their cytotoxicity[14] against cell cultures of
P-388 murine leukaemia, A-549 human lung carcinoma,
HT-29 human colon carcinoma and MEL-28 malignant
human melanoma. The results obtained are shown in
Table 2.
The derivatives tested showed cytotoxicity levels two
or three orders of magnitude lower than those of the
parent compounds podophyllotoxin and deoxypodo-
phyllotoxin. However, the cytotoxicity remained at the
micromolar level and some general observations can be
made.
For the most part, there are no differences among the
IC50 values for the four cell lines; only compounds 4, 4a,
5 and 8 are about twice as potent against P-388, A-549
and MEL-28 as against HT-29.
Elimination of the methylenedioxy group led, in
general, to less cytotoxic compounds, the decrease in
potency being larger in the case of deoxypodophyllo-
toxin (3 vs. 1) than in the podophyllotoxin derivatives (4
Table 2
Cytotoxic activity of cyclolignans modified at ring A (IC50 mM)
Compound
P-388
A-549
HT-29
MEL-28
1
0.003
0.005
0.005
ꢀ/
2
0.012
0.55
1.3
0.012
0.55
1.3
0.012
0.55
1.3
ꢀ
0.55
1.3
/
2a
3
4
0.62
2.0
0.62
2.0
1.2
5.1
0.62
2.0
4a
5
2.5
20.6
2.5
20.6
12.4
2.5
20.6
5a
6
20.6
5.2
5.2
21.4
25.9
2.3
5.2
21.4
51.8
5.7
ꢀ/
21.4
6a
7
21.4
51.8
11
ꢀ
/
ꢀ/
8
5.7
23.4
23.3
5.5
8?
10
11a
12
17
19
23.4
23.3
2.2
ꢀ
ꢀ/
/
23.4
23.3
5.5
ꢀ
/
23.4
23.3
5.5
ꢀ
/
ꢀ/
ꢀ
/
0.23
23
2.2
0.23
0.23
0.23
ꢀ/
ꢀ/
23
22.0
ꢀ/
23
22.0
ꢀ/
23
22.0

A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 65ꢀ
/74
69
Also, the epimerization of the lactone ring (7 vs. 3) led
to much less cytotoxic derivatives, although the cyto-
toxicity was partially recovered when a new dioxole ring
was formed (11a and 12 vs. 7). This result clearly
indicates the importance of the methylenedioxy ring,
which is a common moiety in several antimitotic natural
products.
mg, 67%) was separated after crystalization from
dichloromethane: m.p. 250ꢀ254 8C. Physical data is
according with those described previously [15]. [a]D22
/
ꢂ100.28 (c, 0.5%). UV lmax(o): 216 (20800), 288 (3500).
/
IR (cmꢂ1): 3550, 3020, 1780, 1600, 1510, 1250, 1140,
1
1010. H-NMR (Table 3). 13C-NMR (Table 5). HRMS
(FABꢁ
/
) Calc. for C21H23O7 [Mꢁ
H]ꢁ 387.1443, Found
387.1417. Anal. C21H22O7 (C, H).
/
Acetylation of 3 with acetic anhydride in pyridine
afforded 3a. IR (cmꢂ1): 3010, 1775, 1600, 1500, 1200,
4. Experimental
1
1130, 1010. H-NMR (Table 3). 13C-NMR (Table 5).
4.1. Chemistry
4.1.2.2. Method B. A solution of 1 (500 mg, 1.25 mmol)
and phenol (400 mg) in 85% H3PO4 (15 mL) and glacial
acetic acid (5 mL) was heated for 3 h at 150 8C. The
reaction mixture was diluted with water and extracted
with EtOAc. The organic layer was washed with 5%
aqueous NaHCO3 and water and then evaporated in
vacuo and fractionated on a silica gel column (Cl2CH2/
EtOAc 1:1) to afford 3 (183 mg, 38%) and a mixture of
Melting points were determined by heating in an
external silicone bath and were uncorrected. Optical
rotations were recorded on a Perkin-Elmer 241 polari-
meter in chloroform solution and UV spectra on a
Hitachi 100-60 spectrophotometer in ethanol solution.
IR spectra were obtained on a Beckmann (Acculab
VIII) spectrophotometer in chloroform solution. EIMS
and HRMS were run in a VG-TS-250 spectrometer
working at 70 eV. NMR spectra were recorded at 200
MHz for 1H and 50.3 for 13C in deuterochloroform
using TMS as internal reference, on a Bruker WP 200
SY. Chemical shift values are expressed in ppm followed
by multiplicity and coupling constants (J) in Hz. Flash
chromatography was performed on silica gel (Merck no.
9385). Elemental analysis were obtained with a LECO
3ꢁ7 (120 mg). That mixture was acetylated and
/
chromatographed on silica gel (Cl2CH2/EtOAc 9:1) to
yield 3a (15 mg, 3%) and 7a (60 mg, 10%). Compound
7a. IR (cmꢂ1): 2950, 1780, 1600, 1510, 1230, 1140, 1020.
1H-NMR (Table 3). 13C-NMR (Table 5).
4.1.3. 3,4-O,O-Demethylenepodophyllotoxin (4)
By use of the same procedure A outline for the
preparation of 3, compound 4 (80%) was prepared from
2. Physical data is in accord with those described
CHNS-932 and were within 9
/
0.4% of the theoretical
values.
previously [15]. EM (m/z): 402 [Mꢁ]. [a]D22
ꢂ92.98 (c,
/
4.1.1. Isolation of lignans 1 and 2
0.5%). UV lmax(o): 212 (29100), 285 (2900). IR (cmꢂ1):
3420, 1780, 1590, 1510, 1250, 1130, 1010. 1H-NMR
(Table 3). 13C-NMR (Table 5).
The resin (50 g) of Podophyllum emodi was extracted
with hot CHCl3. The soluble fraction was chromato-
graphed on neutral alumina (activity II), and the
following cyclolignans were eluted with CHCl3: deox-
ypodophyllotoxin (1, 3.7%), podophyllotoxin (2, 36%)
and 4?-demethylpodophyllotoxin (1.3%).
Acetylation of 4 yielded 4a. [a]D22
ꢂ70.48 (c, 0.9%).
/
UV lmax(o): 222 (15700), 268 (1400). IR (cmꢂ1): 3000,
1780, 1745, 1600, 1510, 1250, 1130, 1010. 1H-NMR
(Table 3). 13C-NMR (Table 5).
4.1.2. 3,4-O,O-Demethylenedeoxypodophyllotoxin (3)
4.1.4. 3,4-O,O-Demethyleneepipodophyllotoxin (5)
Procedure A was applied to 2 (500 mg) with the
followed modifications. The reaction mixture was
4.1.2.1. Method A. To a solution of boron trichloride in
dichloromethane (1 M, 1 mL) precooled at ꢂ
/
70 to
keeped at ꢂ78 8C for 2.5 h, followed by 4 h reflux
/
ꢂ
/
65 8C was added dropwise 1 (100 mg, 0.25 mmol) in
with calcium carbonate. The mixture was worked up as
described before for 3. The reaction product was a
mixture of 4, 5 and 6. Purification of this product by
flash column chromatography with Cl2CH2/EtOAc (1:1)
gave 6 (65 mg, 14%), 4 (50 mg, 11%) and 5 (75 mg, 16%).
dichloromethane (5 mL) over 15 min. After the mixture
was stirred at the same temperature for an additional 1
h, the mixture was poured into ice-water and extracted
with ethyl acetate. The combined organic layers were
washed with brine until neutral pH, dried over anhy-
drous sodium sulfate and filtered. The filtrate was
evaporated to give a white solid which was put into a
mixture of acetoneꢀ
mLꢀ0.3 g) and refluxed for 1 h. The white suspension
was diluted with water, neutralized with 2N HCl until
pH 2ꢀ3 and extracted with ethyl acetate. Evaporation of
the solvent afforded a reaction product from which 3 (65
Compound 5: EM (m/z): 402 (Mꢁ). [a]D22
ꢂ77.88 (c,
/
0.5%). UV lmax(o): 220 (17500), 285 (3100). IR (cmꢂ1):
3490, 1780, 1750, 1600, 1510, 1240, 1125, 1010. 1H-
NMR (Table 3). 13C-NMR (Table 5).
/
waterꢀ
/
calcium carbonate (1 mLꢀ1
/
/
Acetylation of 5 afforded 5a. [a]2D2 ꢂ
71.08 (c, 0.63%).
/
UV lmax(o): 209 (25000), 267 (1900). IR (cmꢂ1): 1780,
1
1745, 1600, 1510, 1230, 1135, 1015. H-NMR (Table 3).
/
13C-NMR (Table 5).

70
A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 65ꢀ74
/
Table 3
1H-NMR data of compounds 3ꢀ
/15
a
a
a
a
H
3
3a
4
4a
5
5a
6
6a
2
5
7
8
9
6.72 s
6.49 s
7.09 s
6.94 s
7.19 s
6.47 s
7.21 s
6.98 s
6.94 s
6.49 s
7.34 s
6.98 s
6.76 s
6.70 s
3.10 m
7.16 s
7.03 s
3.80 m
2.70ꢀ
/
3.10 m
3.10 m
2.60ꢀ/3.20 m
4.75 d (9.0)
3.00 m
4.50 m
4.12 dd
(10.1;8.8)
6.45 s
5.93 d (8.9)
2.88 m
4.85 d (3.3)
2.92 m
6.20 d (3.8)
3.00 m
4.38 dd (8.5;8.0) 4.93 m
2.70ꢀ
/
2.60ꢀ/3.20 m
ꢀ/
ꢀ/
4.41 dd (8.3;6.1) 4.48 m
3.95 dd (9.4;8.3) 3.90 m
4.39 dd (9.4;6.7) 4.31 d (9.3)
4.23 t (9.4)
4.90 m
3.97 dd
(10.6;8.5)
6.21 s
ꢀ/
ꢀ/
2?,6?
7?
8?
6.40 s
4.50 d (4.5)
6.30 s
4.71 d (3.5)
6.33 s
6.35 s
6.48 s
4.69 bs
6.37 s
4.90 m
4.50 m
3.00 m
4.72 d (4.2)
2.98 dd
(14.4;4.2)
3.73 s
4.51 d (5.3)
3.32 dd
(14.1;5.3)
3.66 s
4.78 d (5.0)
3.30 dd
(14.0;5.0)
3.70 s
2.70ꢀ
/
3.10 m
2.60ꢀ/3.20 m
ꢀ/
ꢀ/
CH3O-3?,5?
CH3O-4?
OAc
3.67 s
3.66 s
3.72 s
3.79 s
2.23 s
2.29 s
3.68 s
3.67 s
3.72 s
3.64 s
3.77 s
3.79 s
2.25
3.80 s
3.66 s
3.78 s
ꢀ/
ꢀ/
ꢀ/
ꢀ/
2.30
2.24
ꢀ/
ꢀ/
2.28
2.23
ꢀ/
ꢀ/
2.30
ꢀ/
ꢀ/
2.19
ꢀ/
2.10
ꢀ/
ꢀ/
7
7a
11a
12
13
14
15
2
5
7
6.67 s
6.57 s
7.02 s
6.72 s
2.90 m
6.71 s
6.58(6.60) s
2.82 m
6.73 s
6.64 s
2.95 m
7.29 s
7.20 s
7.75 s
6.72 s
6.59 s
3.05 m
6.68 s
6.59 s
2.75 dd
(15.2;6.1)
2.35 dd
(15.2;4.7)
2.99 m
2.82 dd
(15.1;6.0)
2.43 dd
(15.1;6.0)
3.00 m
2.51 dd
(15.4;5.0)
2.90 s
2.48 dd
(15.2;5.6)
3.00 m
2.52 dd
(15.0;5.5)
3.04 m
ꢀ/
2.73 m
2.73 m
8
9
ꢀ/
4.40 dd (9.5;7.8) 4.42 dd (9.4;6.2) 4.43 dd (9.0;7.4) 4.47 dd (9.2;7.1) 5.40 s
4.45 dd (8.8;6.2) 4.42 dd (8.7;7.3)
3.89 dd (9.5;3.3) 4.04 dd
(11.4;9.4)
3.96 dd (9.0;2.8) 4.00 m
ꢀ/
3.90 m
3.95 m
2?,6?
6.29 s
6.38 s
6.31 s 6.33(6.35) s
6.53 d (2.1)
6.55 d (2.1)
6.32 s
6.35 s
ꢀ/
ꢀ/
ꢀ
/
ꢀ/
ꢀ/
ꢀ/
7?
8?
4.28 d (2.8)
4.18 d (5.0)
3.36 dd (9.7;2.8) 3.23 dd (8.8;5.0) 3.30 m
4.31 bs
4.38 bs
3.35 m
3.78 s
3.84 s
ꢀ
ꢀ/
/
4.61 d (3.3)
2.73 m
4.28 d (3.4)
3.32 dd (9.5;3.4)
3.77 s
CH3O-3?,5?
CH3O-4?
OAc
3.68 s
3.78 s
3.77 s
3.82 s
2.24 s
2.20 s
3.76 s
3.80 s
3.87 s, 3.85 s 3.74 s
3.99 s 3.80 s
3.81 s
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
COOCH3
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
3.83 s
6.28 s
ꢀ
/
3.84 s
6.45 s
ꢀ
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
/
Oꢀ
OCH3
CH2 ꢀ
/CHꢀ
/
O
6.80(6.84) s
3.41(3.44) s
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
CH2 ꢀ
/
ꢀ/
4.26 bs
4.26 bs
a
NMR in acetone-d6.
4.1.5. 3,4-O,O-Demethylene-b-apopicropodophyllin (6)
It was eluted from the aforementioned chromatogra-
phy and also by treatment of 2 with phenol and H3PO4
following method B: a solution of 2 (500 mg) and phenol
(200 mg) in 15 mL of H3PO4 and 5 mL of glacial AcOH
was heated for 3 h at 150 8C. The reaction mixture was
diluted with water and extracted with EtOAc. The
organic layer was washed with 5% aqueous NaHCO3
and water and the evaporated in vacuo and fractionated
on a silica gel column (Cl2CH2/EtOAc 1:1) to afford 200
3300, 1730, 1680, 1590, 1500, 1230, 1110, 1010. 1H-
NMR (Table 3). 13C-NMR (Table 5).
Acetylation of 6 afforded 6a. [a]2D2 ꢁ
52.28 (c, 0.64%).
/
UV lmax(o): 207 (28500), 272 (1700). IR (cmꢂ1): 3010,
1765, 1690, 1600, 1500, 1200, 1130, 1010. 1H-NMR
(Table 3). 13C-NMR (Table 5).
4.1.6. 3,4-O,O-Demethylenedeoxypicropodophyllin (7)
Procedure A was applied to deoxypicropodophyllin
(100 mg obtained by basic epimerization of 1 [16]) to
afford 75 mg (75%) of 7. EM (m/z): 386 (Mꢁ). [a]D22
mg (43%) of 6. EM (m/z): 384 [Mꢁ]. [a]D22
0.25%). UV lmax(o): 222 (23900), 286 (4800). IR (cmꢂ1):
ꢁ67.28 (c,
/
ꢁ46.88 (c, 0.96%). UV lmax(o): 222 (23300), 288 (7200).
/

A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 65ꢀ
/74
71
Table 4
1H RMN data of compounds 8?, 9, 16ꢀ
/21
a
H
8?
9
16
17
18
19
20
21
2
5
8
9
7.45 s
6.55 s
7.38 s 7.54 s
6.61 s 6.64 s
3.75 m 3.80 m
7.50 s
6.61 s
3.80 m
7.37 s
6.47 s
3.90 m
7.37 s
6.46 s
3.90 m
7.56 s
6.64 s
3.90 m
7.55 s
6.63 s
3.77 m
3.80 m
4.83 t (7.3)
4.60 m 4.86 t (6.2) 4.83 t (7.7) 4.83 dd (9.0;8.0) 4.82 dd (9.1;8.0) 4.84 dd (9.1;8.1) 4.83 t (7.5)
3.80 m
6.14 s
4.63 d (5.1)
3.80 m 3.80 m
6.22 s 6.13 s
4.60 m 4.66 d (5.1) 4.63 d (5.1) 4.62 d (5.1)
3.80 m
6.12 s
3.90 m
6.15 s
3.90 m
6.15 s
4.60 d (5.1)
3.90 m
6.12 s
4.64 d (5.1)
3.77 m
6.14 s
4.63 d (5.1)
2?,6?
7?
8?
3.20 dd (12.1;5.1) 3.27 m 3.20 m
3.20 m
3.75 s
3.20 dd (12.3;5.1) 3.18 dd (12.3;5.1) 3.20 dd (12.3;5.1) 3.19 dd (11.3;5.1)
CH3Oꢀ
CH3O-4?
COOCH3
/
3?,5?
3.77 s
3.69 s 3.74 s
3.87 s
3.67 s 3.69 s
3.74 s
3.80 s
3.67 s
3.77 s
3.73 s
3.80 s
3.68 s
3.77 s
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
3.66 s
3.65 s
3.66 s
3.67 s
Oꢀ
Oꢀ
OCH3
CH2 ꢀ
/
CH2 ꢀ
/
O
6.00 s
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
/
ꢀ
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
/
/CHꢀ
/O
ꢀ/
ꢀ/
ꢀ/
6.34(6.36) s 6.34(6.35) s
3.86(3.88) s 3.83(3.85) s
ꢀ/
/CH2 ꢀ
/
ꢀ/
ꢀ/
4.27 s
4.27 s
CH3
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
1.67 s
1.68 s
1.66 s
1.67 s
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
a
NMR in acetone-d6.
IR (cmꢂ1): 3400, 1740, 1600, 1510, 1240, 1110, 1010.
1H-NMR (Table 3). 13C-NMR (Table 5).
Na2SO4 and evaporated. The resulting residue was
treated with an ethereal solution of diazomethane and
then chromatographed on silica gel (Cl2CH2/EtOAc
92:8) to give 11a (65 mg, 46%). 11a (25%) was also
obtained by reaction of 7 with methyl dichloroacetate.
4.1.7. Isoxazoles 9 and 8?
Isoxazole 8 (456 mg, 1.03 mmol, obtained by de-
scribed procedures [11]) was demethylenated using the
method A with the following modifications: the tem-
perature of the first step was 0 8C for 1 h and the reflux
time was extended to 90 min. The reaction product was
precipitated in dichloromethane to obtain 300 mg (70%)
of a 9:1 mixture of 9 and 10 and 100 mg of residue which
was chromatographed on silica gel (Cl2CH2/EtOAc
75:25) to afford 28 mg (6%) of 8?.
M.p. 95ꢀ
/
98 8C (MeOHꢀCl2CH2). UV lmax(o): 214
/
(20500), 290 (3600). IR (cmꢂ1): 1770, 1600, 1500,
1240, 1135, 1010. 1H-NMR (Table 3). 13C-NMR (Table
5). Anal. C24H24O9 (C, H).
4.1.8.2. Compound 12. From 3 (150 mg, 0.39 mmol),
dichloromethyl methyl ether (0.1 mL, 1,12 mmol),
anhyd. K2CO3 (1 g) in DMF (3 mL). The mixture was
9: IR (cmꢂ1): 3430, 1730, 1709, 1600, 1510, 1260,
stirred at 90ꢀ
cooling, water (5 mL) was added and it was stirred at
90ꢀ100 8C for 40 min. A reaction product was obtained
/
100 8C under nitrogen for 2 h. After
1
1110. H-NMR (Table 4). 13C-NMR (Table 6). HRMS
(FAB ꢁ
/
) Calc. for C21H22NO8 [Mꢁ
H]ꢁ 416.1345,
/
/
found 416.1301.
8?: [a]2D2
after the same work up described above for 11a. After
chromatography of the reaction product with Cl2CH2/
EtOAc 9:1, 12 (32 mg, 20%) was isolated. UV lmax(o):
214 (20500), 290 (3600). IR (cmꢂ1): 1760, 1590, 1510,
1240, 1125, 1010. 1H-NMR (Table 3). 13C-NMR (Table
ꢂ145.38 (c, 0.5%). UV lmax(o): 215 (26200),
/
274 (11600), 313 (6900). IR(cmꢂ1): 3540, 1740, 1630,
1
1510, 1220, 1130, 1050. H-NMR (Table 4). 13C-NMR
(Table 6).
5). HRMS (FAB ꢁ
/
) calcd for C23H25O8 [Mꢁ
H]ꢁ
429.1549, found 429.1502. Anal. C23H24O8 (C, H).
/
4.1.8. Formation of the new substituted dioxole ring:
compounds 11aꢀ
/
13, 16ꢀ19
/
4.1.8.1. Compound 11a. A mixture of 3 (120 mg, 0.31
mmol), methyl dichloroacetate (0.1 mL, 0.96 mmol) and
anhyd. K2CO3 (300 mg) in DMF (2 mL) was stirred at
4.1.8.3. Compound 13. From 4 (100 mg, 0.25 mmol),
methyl dichloroacetate (0.1 mL, 0.96 mmol), anhyd.
K2CO3 (300 mg) in DMF (2 mL). The mixture was
90ꢀ
mixture, water (5 mL) was added and it was stirred at
90ꢀ100 8C for 40 min. The mixture was cooled, weakly
/
100 8C under nitrogen for 2 h. After cooling the
stirred at 90ꢀ
cooling, water (5 mL) was added and it was stirred at
90ꢀ100 8C for 40 min. A reaction product was obtained
/
100 8C under nitrogen for 2 h. After
/
/
acidified with 2N HCl and then weakly basified with sat.
NaHCO3 and washed with ethyl acetate. The aqueous
layer was acidified with 2N HCl and extracted with ethyl
acetate. The extract was washed with brine, dried over
after the same work up described above for 11a. After
esterification and chromatography (Cl2CH2/EtOAc
92:8) of the reaction product, 13 (20 mg, 18%) was
1
isolated. H-NMR (Table 3). 13C-NMR (Table 5).

Table 5
13C-NMR data of compounds 3ꢀ
15
/
a
a
a
a
C
3
3a
4
4a
5
5a
6
6a
7
7a
11a
12
13
14
15
c
1
128.1
116.2
144.7
145.3
118.0
130.1
32.8
130.6
123.8
141.1
141.3
125.6
133.7
32.8
133.7
114.3
145.5
145.5
117.0
130.1
72.6
133.7
122.6
141.8
142.4
125.5
134.6
73.1
132.3
117.4
145.4
146.3
117.6
130.5
66.5
133.0
125.6
141.7
143.2
126.0
134.3
67.1
127.6
115.6
145.0
145.2
116.6
129.1
29.4
128.0
123.1
141.4
141.4
125.0
129.5
28.8
127.4
115.6
142.7
143.7
116.4
129.0
31.3
134.0
122.7
140.7
140.8
124.0
134.0
31.6
129.1
108.9
146.0
146.0
110.0
131.5
32.0
128.5
108.4
145.2
145.2
109.4
130.6
32.1
130.6
104.2
147.8
149.0
104.4
130.0
119.3
140.3
67.9
127.9
116.9
142.5
142.7
119.0
130.5
32.3
127.8
116.8
142.3
142.5
117.8
130.3
32.1
2
3
4
5
c
c
6
7
8
34.1
72.5
32.7
71.8
41.7
72.0
38.6
71.2
39.7
68.4
36.7
67.3
123.4
71.7
137.4
70.9
33.0
73.4
33.7
72.1
33.1
72.6
33.1
72.7
32.8
72.1
33.1
71.9
9
1?
2?
3?
4?
5?
6?
7?
8?
138.3
110.2
153.5
138.4
153.5
110.2
44.1
135.8
108.8
152.8
136.3
152.8
108.8
43.4
137.9
109.9
153.5
137.3
108.3
152.9
137.5
152.9
108.3
43.3
137.5
109.7
153.5
138.3
153.5
109.7
41.2
137.6
108.4
152.9
137.8
152.9
108.4
41.4
140.7
106.8
154.2
138.0
154.2
106.8
42.9
135.0
106.3
153.5
137.5
153.5
106.3
42.3
138.5
105.2
153.3
136.6
153.3
105.2
45.0
138.0
105.7
153.6
136.5
153.6
105.7
44.5
138.0
105.8
153.6
138.2
153.6
105.8
45.3
138.0
105.0
153.3
138.2
153.3
105.0
45.2
134.7
108.0
153.2
138.6
153.2
108.0
140.3
128.8
169.3
56.4
136.6
108.2
152.4
136.9
152.4
108.2
43.1
138.2
104.9
153.1
136.6
153.1
104.9
44.6
b
ꢀ/
153.5
109.9
44.5
c
47.9
47.4
45.8
45.4
44.2
43.4
160.5
172.8
56.4
156.8
171.9
56.4
46.7
46.2
46.2
46.3
47.5
46.0
9?
175.5
56.5
60.4
174.4
56.4
60.7
175.4
56.5
60.4
173.4
56.2
60.7
175.8
56.4
60.4
173.8
56.3
60.7
179.9
56.2
60.9
177.6
56.3
60.8
178.0
56.4
60.7
178.3
56.1
60.8
175.1
56.2
60.7
178.4
56.0
60.5
CH3O-3?,5?
CH3O-4?
OAc
60.4
60.8
61.0
ꢀ/
ꢀ/
20.5
168.0
ꢀ/
ꢀ/
20.5(2C), 21.0
168.1(2C) 171.2
ꢀ/
ꢀ/
20.5(2C), 20.9
167.8(2C) 170.3
ꢀ/
ꢀ/
20.8
168.1
ꢀ/
ꢀ/
20.5
166.1
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
COOCH3
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
52.8
165.6
103.0
ꢀ
ꢀ/
119.8
50.0
/
53.1
165.0
103.5
ꢀ
ꢀ/
ꢀ/
ꢀ/
64.3
64.4
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
Oꢀ
OCH3
CH2 ꢀ
/CHꢀ
/
O
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
CH2 ꢀ
/
ꢀ/
ꢀ/
64.1(2C)
a
b
c
NMR in acetone-d6.
Signal not observed.
Exchangeable signals.

A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 65ꢀ
/74
73
Table 6
13C-NMR data of compounds 8?, 9, 16ꢀ
/21
a
C
8?
9
16
17
18
19
20
21
1
118.8
103.9
147.5
150.5
109.2
130.5
156.4
43.4
117.4
110.3
145.4
149.0
116.1
131.5
156.5
43.9
119.6
103.4
146.7
149.4
109.6
134.8
156.2
43.4
119.5
103.3
119.2
103.8
150.4
119.1
103.8
147.4
150.4
108.9
134.1
156.5
43.5
118.4
113.1
143.5
146.5
117.9
133.6
156.2
43.6
118.3
113.0
143.3
146.5
117.8
133.8
156.1
43.4
2
3
146.8(146.6)
149.3
b
4
ꢀ/
5
109.5(109.2)
134.2
156.1
108.9
134.3
156.5
43.5
6
7
8
43.3
74.5
9
74.4
73.9
74.6
74.4
74.3
74.4
74.3
1?
2?
3?
4?
5?
6?
7?
135.0
106.1
146.8
134.2
146.8
106.1
47.8
135.6
107.4
147.9
133.7
147.9
107.4
47.2
135.3
106.5
153.1
137.4
153.1
106.5
48.0
130.3
135.1
106.5
153.0
130.6
106.1
146.8
134.5
146.8
106.1
47.8
135.5
106.5
153.0
137.3
153.0
106.5
47.2
131.0
106.0
146.7
134.1
146.7
134.1
47.0
106.1(106.5)
153.0(152.7)
135.5(135.3)
153.0(152.7)
106.1(106.5)
47.8
b
ꢀ/
153.0
106.5
47.9
8?
9?
50.0
171.4
56.3
50.3
172.0
56.2
49.9
171.3
56.1
49.8
171.3
56.3(56.0)
50.0
171.5
56.1
50.3
171.5
56.3
50.1
171.5
56.1
50.1
171.5
56.2
CH3O-3?,5?
CH3O-4?
COOCH3
52.0
ꢀ/
51.6
60.8
53.2
ꢀ/
60.8
52.0
ꢀ/
52.0
118.2
60.8
52.0
ꢀ/
51.9
ꢀ/
101.6
53.1
104.3
52.0
b
Oꢀ
COOCH3
/
Cꢀ
/O
ꢀ/
ꢀ/
104.3
52.1
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
165.1
165.1
ꢀ/
ꢀ/
ꢀ/
ꢀ/
CH3
ꢀ/
ꢀ/
ꢀ/
ꢀ/
25.9
25.8
ꢀ/
ꢀ/
ꢀ/
CH2 ꢀ
/
CH2 ꢀ
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
64.2, 64.5
64.1, 64.5
a
NMR in acetone-d6.
Signal not observed.
b
4.1.8.4. Compounds 16 and 17. From a 9:1 mixture of 9
and 10 (70 mg), methyl dichloroacetate (0.05 mL, 0.48
mmol), anhyd. K2CO3 (140 mg) in DMF (2 mL). The
tographed (Cl2CH2/EtOAc 93:7) to yield 10 mg (9%) of
18 and 45 mg of 19 (39%).
1
18: H-NMR (Table 4). 13C-NMR (Table 6).
mixture was stirred at 90ꢀ
h. After cooling, water (2 mL) was added and it was
stirred at 90ꢀ100 8C for 30 min. A reaction product was
obtained after the same work up described above for
11a. Column chromatography of the reaction product
with Cl2CH2/EtOAc 6:4 yielded 11 mg of 16 (12%) and
50 mg (60%) of 17.
16: UV lmax(o): 224 (27800), 274 (16100), 311 (10500).
IR (cmꢂ1): 1760, 1735, 1590, 1510, 1210, 1127, 1010.
1H-NMR (Table 4). 13C-NMR (Table 6). HRMS
/
100 8C under nitrogen for 1.5
19: M.p. 78ꢀ83 8C. UV lmax(o): 222 (15900), 276
/
(11100), 315 (7500). IR (cmꢂ1): 3447, 1740, 1610, 1510,
1240, 1110, 1010. 1H-NMR (Table 4). 13C-NMR (Table
/
6). HRMS (FABꢁ
/
) Calc. for C24H26O8N [Mꢁ
H]ꢁ
456.1658. Found 456.1649. Anal. C24H25O8N (C, H, N).
/
4.1.9. Formation of the new dioxane ring: compounds 14,
15, 20, 21
) Calc. for C25H26O10N [Mꢁ
H]ꢁ 500.1557.
Found 500.1532. Anal. C25H25O10N (C, H, N).
/
4.1.9.1. Compounds 14 and 15. A mixture of 3 (205 mg,
0.53 mmol), 1,2-dibromoethane (0.1 mL, 1.16 mmol),
K2CO3 (430 mg) and sodium iodide (4 mg) in acetone (5
mL) was refluxed for 40 min. After cooling, water was
added and extracted with ethyl acetate. The organic
layer was washed with brine until pH 7, dried and
evaporated off. Flash chromatography (Cl2CH2/EtOAc
95:5) of the residue afforded: 43 mg (20%) of 14 and 48
mg (22%) of a mixture of 14 and 15.
(FABꢁ
/
17: EM (m/z): 486 (Mꢁ
1). IR (cmꢂ1): 3445, 1760,
/
1730, 1610, 1510, 1214, 1116, 1010. 1H-NMR (Table 4).
13C-NMR (Table 6).
4.1.8.5. Compounds 18 and 19. A 9:1 mixture of 9ꢁ10
/
(100 mg), dichloromethyl methyl ether (0.33 mL, 3.7
mmol), K2CO3 (83 mg) and sodium iodide (2 mg) in
acetone (5 mL) was refluxed for 48 h. After cooling,
water was added and extracted with ethyl acetate. The
organic layer was washed with brine until pH 7, dried
and evaporated off. The reaction product was chroma-
14: UV lmax(o): 213 (98800). IR (cmꢂ1): 1775, 1590,
1
1505, 1247, 1126, 1020. H-NMR (Table 3). 13C-NMR
(Table 5). HRMS (FABꢁ
/
) Calc. for C23H25O7 [Mꢁ
/
H]ꢁ 413.1600. Found 413.1578. Anal. C23H24O7 (C, H).
1
15: H-NMR (Table 3). 13C-NMR (Table 5).

74
A. Castro et al. / European Journal of Medicinal Chemistry 38 (2003) 65ꢀ74
/
4.1.9.2. Compounds 20 and 21. The mixture of 9ꢁ
/
10
References
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