Synthesis and cytotoxic activity of tetracenomycin d and of saintopin analogues

Article abstract of DOI:10.1016/S0968-0896(01)00273-5

Regiospecific synthesis of title compounds is based either on cycloaddition of ketene acetals derived from Hagemann's ester or of homophthalic anhydrides. Thus, tetracenomycin D and 3,8-di-O-methyl saintopin have been prepared in few steps. New derivatives of 10-deoxysaintorpin have been also obtained. Evaluation of their cytotoxicity against L1210 leukemia cells are reported. Copyright

Full text of DOI:10.1016/S0968-0896(01)00273-5

Bioorganic & Medicinal Chemistry 10 (2002) 253–260
Synthesis and Cytotoxic Activity of Tetracenomycin D and of
Saintopin Analogues
Philippe Martin,a Stephane Rodier,a Martine Mondon,a Brigitte Renoux,a
Bruno Pfeiffer,b Pierre Renard,b Alain Pierrec and Jean-Pierre Gessona,*
a
` ´ ´ ´
Laboratoire de Synthese et Reactivite des Substances Naturelles, Universite de Poitiers et CNRS, 40 avenue du Recteur Pineau,
86022 Poitiers, France
´
b
ADIR, 1 rue Carle Hebert, 92415 Courbevoie Cedex, France
cInstitut de Recherche Servier, 11 rue des Moulineaux, 92450 Suresnes, France
Received 27 March 2001; accepted 25 July 2001
Abstract—Regiospecific synthesis of title compounds is based either on cycloaddition of ketene acetals derived from Hagemann’s
ester or of homophthalic anhydrides. Thus, tetracenomycin D and 3,8-di-O-methyl saintopin have been prepared in few steps. New
derivatives of 10-deoxysaintopin have been also obtained. Evaluation of their cytotoxicity against L1210 leukemia cells are repor-
ted. # 2001 Elsevier Science Ltd. All rights reserved.
Topotecan,9 it may be of interest to search for other
specific topo I inhibitors based on the tetracyclic core of
Introduction
A large number of naturally occuring polycyclic qui-
nones are known, and some of them such as the
anthracyclines are largely used in cancer chemo-
therapy.1 In addition to the latter which are tetrahydro
derivatives, fully aromatized naphthacenequinones have
been obtained from anthracycline producing strains or
from other organisms. Among these, saintopin 1,2 sain-
topin E 2,3 UCE 1022 34 and UCE 6 4,5 isolated from
Actinomycete fermentation broths, have been shown to
be cytotoxic, 4 being equipotent to camptothecin
against several cancer cell lines.5 Saintopin is a dual
inducer of topoisomerase I- and topisomerase II-medi-
ated DNA cleavage6 while UCE 6 and UCE 1022 are
specific inducers of topoisomerase I. All these anti-
biotics seem to act by trapping the reversible topoi-
somerase I/DNA cleavable complex.7 Interaction of
saintopin with DNA has been studied by means of sur-
face-enhanced Raman scattering spectroscopy8 and it
has been shown that only partial intercalation (A and B
rings) occurs and that the peripheral ring D hydroxyl
groups are buried in DNA. Taking into account the
interesting pharmacological profile of the recently
introduced camptothecin derivatives Irinotecan and
saintopin and UCE-6. In this respect, it is worthy to
note that the closely related tetracenomycins A2 5, B1 6,
B2 7, B3 8, D (or D1) 9 and D3 10 (Fig. 1) have been
isolated from Streptomyces glaucescens TU 4910 and
Streptomyces olivaceus TU 235311 but no cytotoxic
activity was reported for these compounds. To our
knowledge, only one total synthesis of tetracenomycins
A2, B1, B2 and D has been described in 1992 by
Cameron12 based on two Diels–Alder cycloadditions
and a regiospecific deoxygenation in eight steps from
chloronaphthazarin.
We now describe a new synthesis of tetracenomycin D
and preparation of saintopin analogues and a pre-
liminary evaluation of their cytotoxic activity.
Results
Chemistry
The first approach to these tetracyclic targets was based
on our previous synthesis of 11-deoxy anthracyclinones
starting from Hagemann’s ester 11.13 It was anticipated
that easy aromatization of intermediate 9,10-dihydro
tetracyclic derivatives should afford naphthacenequi-
nones in few steps. Thus, the known ketene acetal 1213
*Corresponding author. Fax: +33-5-49-45-35-01; e-mail: jean-pierre.
gesson@univ-poitiers.fr
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00273-5

254
P. Martin et al. / Bioorg. Med. Chem. 10 (2002) 253–260
Figure 1.
was condensed with quinone 13 (prepared from 2,5-
dichloro benzoquinone and 3-methoxy-1-ethoxy-1-tri-
methylsilyloxy-buta-1,3-diene)14 to give a mixture of
14a and of naphthacenequinone 15 (a trace of 14b was
also noticed). This mixture was treated with DDQ15 to
afford 15 which upon deprotection with BBr3 gave 10-
deoxy saintopin 16 (28%) as a dark red solid. The same
reaction sequence was then applied to a synthesis of the
corresponding 3-O-methyl derivative 20 (as in tetra-
cenomycin B1). Since selective deprotection of the
ethoxy group failed,16 replacement of the latter by a silyl
ether protecting group was then considered. 17 was
obtained by the method of Mander17 (TBSOTf, Et3N,
90%) from 11 without noticeable formation of the more
stable endocyclic dienol silyl ether. After conversion to
ketene acetal 18, condensation with quinone 13 gave the
fully aromatized tetracycle 19 (albeit isolated in a low
8% yield) together with a mixture of this material with
the expected 9,10-dihydro derivative, which could not
be aromatized to 19 upon treatment with DDQ or Pd/
C. Fluoride induced desilylation of 19 afforded 3-O-
methyl-10-deoxy saintopin 20.
before, only formation of silyl enol ether 22 was detec-
ted (TBSOTf, Et3N, CH2Cl2, 86%). 22 was then con-
verted to 23 (LDA, THF, TMSCl, 71%) and condensed
with 13 to give the fully aromatized derivative 24 (29%)
and, after deprotection, 25 (91%) (Fig. 2).
Then, application of the above methodology to the
synthesis of tetracenomycin D was attempted. The
required 6-methyl derivative 26 was prepared as repor-
ted from acetaldehyde and ethyl acetoacetate.18 After
conversion to enol ether 27, the sensitive ketene acetal
28 was prepared as usual (LDA-TMSCl) and condensed
with 13. There was obtained a disappointing low 13%
yield of tetracycle 29 which could not be aromatized to
3,8-di-O-methyl tetracenomycin D 30 either in presence
of Pd/C (diglyme at reflux) or DDQ.
The rather unexpected failure to aromatize 29 led us to
use the base catalyzed condensation of a homophthalic
anhydride with a quinone, a strategy first introduced by
Tamura19 for tetracyclic compounds synthesis. Enol
ether 27 was converted to 31 (90%) upon heating with
Pd/C in diglyme (trace amounts of 3,5-dimethyl anisole
were also isolated). Homophthalic anhydride 3219 was
then prepared in three steps: ethoxycarbonylation to
33,20 hydrolysis to 34 and dehydration (Fig. 3).
Although the last two steps resulted in high yields, the
first one turned out to be difficult. After an extensive
study of reaction conditions, including the correspond-
ing acid, 33 was obtained in 30% yield (42% based on
recovered starting material). Upon deprotonation with
Taking into account potential functionalization of
Hagemann’s ester, the preparation of analogues bearing
side chains at C-7 or C-10 was then investigated. First, a
3-hydroxypropyl side chain at C-7 was chosen by ana-
logy with the UCE-6 side chain (which is indeed located
at C-10). The sodium enolate of 11 (NaH, DMF) was
treated with 1 equivof the TPS ether of 3-bromo-1-
propanol to give 21 in 29% isolated yield. As shown

P. Martin et al. / Bioorg. Med. Chem. 10 (2002) 253–260
255
Figure 2.
Figure 3.

256
P. Martin et al. / Bioorg. Med. Chem. 10 (2002) 253–260
Table 1. Cytotoxicity and cell cycle effects of the tetracyclic compounds
Compound
Cytotoxicity
IC50 L1210 (mM)
Percent of L1210 cells in the cell cycle phasesa
G1
S
G2M
> G2M
Conc.mM
9
15
16
20
25
30
39
40
22.1
> 10
2.8
14.7
40.3
13.3
13.9
2.3
6
7
—
9
25
—
28
28
15
83
—
75
39
—
24
24
21
3
—
8
1
—
1
50
—
5
100
—
20
20
5
NEb
7
34
NEb
41
41
63
1
1
aPercent of untreated control L1210 cells in the phases of the cell cycle: 41% (G1), 28% (S), 24% (G2M), 1% (8N). NE, not evaluated.
LDA, 32 was condensed with 13 to afford 30 (60%).
Final demethylation with excess BBr3 at ꢀ78 ꢁC gave
tetracenomycin D 9 (43%) as a dark red unsoluble solid.
1H NMR data and UV spectra of this material were
identical to those reported by Zeek10 and by Cameron.12
into account the cytotoxicity data observed above, fur-
ther modifications are underway to increase the potency
of these quinones.
Experimental
Application of this route to a synthesis of saintopin was
then attempted. The known homophthalic anhydride
3521 was prepared from ethyl 2,4-dimethoxy-6-methyl
benzoate 36 (Fig. 3): ethoxycarbonylation to 37 (34%),
hydrolysis to 38 (98%) and dehydration (quant). Con-
densation of 35 with quinone 13 afforded 3,8,10-tri-O-
methyl saintopin 39 (72%). Attempted perdemethyl-
ation with excess BBr3 of this material afforded an
untractable complex mixture of dark red solids. This
result is not surprising in view of the recently reported
instability of saintopin which easily affords artefacts
upon extraction from the culture broth.22However 3,8-
di-O-methyl saintopin 40 resulting from easy demethyl-
ation of the chelated ether at C-10 could be isolated if
the reaction is rapidly stopped after addition of BBr3.
Melting points are uncorrected. 1H NMR and 13C
NMR were recorded on a 300 MHz spectrometer, using
CDCl3 as solvent with TMS as internal standard.
Assignment of H NMR and 13C NMR spectra were
1
achieved using DEPT (Multiplicity by DEPT: s=C,
d=CH, t=CH2, q=CH3). High resolution MS were
performed by the ‘Service Central de Microanalyse’
(CNRS, Lyon). All reactions were run under an inert
atmosphere. THF was dried over and distilled from
sodium/benzophenone and CH2Cl2 was distilled from
P2O5. Organic extract mixtures were dried over anhy-
drous Na2SO4 and filtered and the solvent was then
removed under reduced pressure. All separations were
done under flash chromatography (MPLC) conditions
on silica gel (25–40 mm) completed, if necessary, by
preparative thin-layer chromatography (TLC) per-
formed on silica gel plates (60 GF254).
Cytotoxic activity
Tetracyclic derivatives were tested in vitro against
L1210 leukemia and for their effect on the L1210 cell
cycle.23 Results are reported in Table 1.
9,10-Dihydro-8-ethoxy-1,11-dihydroxy-3-methoxy-naph-
thacene-5,12-dione (14a) and 9,10-dihydro-8,11-diethoxy
-1-hydroxy-3-methoxy-naphthacene-5,12-dione (14b). To
a solution of 13 (270 mg, 1.16 mmol) in anhydrous THF
(23 mL) maintained at 0 ꢁC was added ketene acetal 12
(359 mg, 1.1 equiv) in THF (10 mL). After stirring at rt
for 48 h, hydrolysis with 10% HCl was followed by
extraction with CH2Cl2. Chromatography over silica gel
using CH2Cl2 as eluent afforded 14a and 15 (142 mg,
39%) together with a mixture of 14a and 14b (30 mg,
8%). Preparative TLC afforded almost pure 14a and
14b contaminated by ca. 20% of 14a.
These data show that 10-deoxy saintopin 16 and 3,8-di-
O-methyl saintopin 40 are the most active compounds
prepared in this study.
Etherification at C-3 or C-8 or introduction of a side
chain at C-10 (tetracenomycin D) or C-7 (25) resulted in
lower cytotoxicity. However, introduction of a hydroxyl
at C-10 (40 vs 15) enhanced cytotoxicity. Finally, it is
interesting to note that 16 and 40 have different effects
on the cell cycle: 75% of the cells in the G2M phase for
16 compared with 24% for control and 63% of the cells
in the G1 phase for 40 compared with 41% for control.
1
14a. H NMR (300 MHz, CDCl3) d 1.41 (t, 3H, J=7
Hz, CH3), 2.48 (t, 2H, J=8.6 Hz, H-9), 3.01 (t, 2H,
J=8.6 Hz, H-10), 3.93 (s, 3H, OCH3), 3.99 (q, 2H, J=7
Hz, OCH2), 5.61 (s, 1H, H-7), 6.67 (d, 1H, J=2.5 Hz,
H-2), 7.35 (d, 1H, J=2.5 Hz, H-4), 7.42 (s, 1H, H-6),
12.46 (s, 1H, OH) and 12.49 (s, H, OH).
Conclusion
We have shown that substituted naphthacenequinones
related to tetracenomycin D and saintopin can be
obtained in few steps by cycloaddition of silyl ketene
acetals or homophthalic anhydrides with a naphthoqui-
none. Following these synthetic schemes, and taking
1
14b. H NMR (300 MHz, CDCl3) d 1.41 (t, 3H, J=7
Hz, CH3), 1.51 (t, 3H, J=7 Hz, CH3), 2.43 (t, 2H,

P. Martin et al. / Bioorg. Med. Chem. 10 (2002) 253–260
257
J=8.3 Hz, H-9), 3.06 (t, 2H, J=8.3 Hz, H-10), 3.91 (s,
3H, OCH3), 4.02 (q, 2H, J=7 Hz, OCH2), 5.66 (s, 1H,
H-7), 6.68 (d, 1H, J=2.5 Hz, H-2), 7.28 (d, 1H, J=2.5
Hz, H-4), 7.63 (s, 1H, H-6), 13.46 (s, 1H, OH).
anhydrous ether and filtered. After evaporation ketene
acetal 18 (440 mg, 96%) was obtained as a pale yellow
oil which was rapidly used in the next step. H NMR
(300 MHz, CDCl3) d 0.06–0.23 (m, 15H, Si(CH3)2 and
Si(CH3)3), 0.92 (s, 9H, C(CH3)3), 1.25 (t, 3H, J=7 Hz,
CH3), 2.13 (t, 2H, J=6.5 Hz, H-5), 2.43 (t, 2H, J=6.5
Hz, H-6), 3.85 (q, 2H, J=7 Hz, OCH2), 4.79 (broad s, 1H,
C¼CH), 5.04 (broad s, 1H, C¼CH), 5.41 (s, 1H, H-3).
1
8-Ethoxy-1,11-dihydroxy-3-methoxy-naphthacene-5,12-
dione (15). To a solution of the above mixture of 14a,b
and 15 (171 mg) in toluene (13 mL) was added DDQ
(106 mg, 0.467 mmol). After stirring at reflux for 6 h,
toluene was removed in vacuo and the crude reaction
mixture was extracted with CH2Cl2. Recristallization
from CH2Cl2 afforded 15 (170 mg, 95%). Mp 264 ꢁC.
1H NMR (300 MHz, CDCl3) d 1.52 (t, 3H, J=7 Hz,
CH3), 3.95 (s, 3H, OCH3), 4.21 (q, 2H, J=7 Hz,
OCH2), 6.72 (d, 1H, J=2.5 Hz, H-2), 7.25 (s, 1H, H-7),
7.32 (d, 1H, J=9 Hz, H-9), 7.43 (d, 1H, J=2.5 Hz, H-
4), 8.17 (s, 1H, H-6), 8.39 (d, 1H, J=9 Hz, H-10), 12.56
(s, 1H, OH) and 13.83 (s, 1H, OH).MS (EI) m/z 364
(100), 336 (73). HRMS (EI) m/z calcd (C21H16O6):
364.0946, obsd : 364.0927.
8-tertButyldimethylsilyloxy-1,11-dihydroxy-3-methoxy-
naphthacene - 5,12 - dione (19). Same procedure as for
14a,b. After chromatography 19 (6%) was obtained
together with a mixture of the corresponding 9,10-dihy-
dro (11-OH and 11-OEt) derivatives (35%). Attempted
aromatization of the latter with DDQ failed. 19: mp
200–210 ꢁC (dec); 1H NMR (300 MHz, CDCl3) d 0.30 (s,
6H, Si(CH3)2), 1.03 (s, 9H, Si(CH3)3), 3.95 (s, 3H,
CH3), 6.71 (d, 1H, J=2.5 Hz, H-2), 7.22 (dd, 1H,
J=2.3 and 9 Hz, H-9), 7.30 (d, 1H, J=8.3 Hz, H-7),
7.43 (d, 1H, J=2.5 Hz, H-4), 8.14 (d, 1H, J=2.3 Hz,
H-6), 8.39 (d, 1H, J=9 Hz, H-10), 12.55 (s, 1H, OH)
and 13.86 (s, 1H, OH); MS (EI) m/z 450 (77), 393 (100),
197 (44), 73 (32), 57 (34). HRMS (EI) m/z calcd
(C25H26O6Si): 450.1498, obsd: 450.1503.
1,3,8,11-Tetrahydroxy-naphthacene-5,12-dione (10-deoxy
saintopin) (16). BBr3 (0.1 mL, 1.09 mmol) was added to
a well stirred suspension of 15 (80 mg, 0.22 mmol) in
CH2Cl2 (1.5 mL) maintained at ꢀ78 ꢁC under nitrogen.
After stirring for 16 h at rt, the crude reaction mixture
was poured into iced water and extracted with CH2Cl2
and the unsoluble fraction was filtered. A dark red solid
was obtained which was dried and triturated with ether
1,8,11-Trihydroxy-3-methoxy-naphthacene-5,12-dione (3
-O-methyl 10-deoxy saintopin) (20). Desilylation of 19
(27 mg, 0.06 mmol) to 20 was carried out by treatment
with Bu4NF (31 mg, 0.12 mmol) in THF (0.25 mL).
Extraction as usual with CH2Cl2 afforded 20 (14 mg,
69%) as a red solid after precipitation from EtOAc/Pet
to give 16 (22 mg, 28%). Mp > 250 ꢁC (dec). H NMR
1
(300 MHz, CDCl3) d 6.58 (s, 1H, H-2), 7.14 (s, 1H, H-
7), 7.26 (d, 1H, J=9 Hz, H-9), 7.36 (s, 1H, H-4), 8.01 (s,
1H, H-6), 8.21 (d, 1H, J=9 Hz, H-10), 10.75 (s, 1H,
OH), 11.30 (s, 1H, OH), 12.29 (s, 1H, OH) and 13.78 (s,
1H, OH). HRMS (EI) m/z calcd (C18H10O6): 322.0477,
obsd: 322.0463.
ether: mp 190 ꢁC (dec); H NMR (300 MHz, CDCl3) d
1
3.92 (s, 3H, CH3), 6.68 (d, 1H, J=2.5 Hz, H-2), 7.05–
7.35 (m, 2H, H-7 and H-9), 7.36 (d, 1H, J=2.5 Hz, H-
4), 7.92 (s, 1H, H-6), 8.20 (d, 1H, J=9 Hz, H-10). MS
(EI) m/z 336 (25), 142 (90), 100 (100), 58 (70); HRMS
(EI) m/z calcd (C19H12O6): 336.0633, obsd: 336.0637.
Ethyl 2-Methylene-4-tertbutyldimethylsilyloxy-cyclohexa
-3-ene carboxylate (17). To a solution of Hagemann’s
ester 11 (250 mg, 1.37 mmol) in CH2Cl2, was added
Et3N (0.28 mL, 2.06 mmol, 1.5 equiv) and tert-butyldi-
methylsilyl triflate (0.41 mL, 1.78 mmol, 1.3 equiv).
After stirring for 40 min at rt, dilution with CH2Cl2 and
extraction as usual, there was obtained a crude oil
which was rapidly filtered over florisil (eluent: EtOAc/
pet ether 2.5:97.5). The unstable oily enol ether 17 (367
Ethyl 2-methyl-3-(3-tertbutyldiphenylsilyloxypropyl)-cy-
clohex-2-en-4-one carboxylate (21). To a cooled (0 ꢁC)
solution of Hagemann’s ester 11 (250 mg, 1.37 mmol) in
DMF (7.5 mL) was slowly added a 60% suspension of
NaH (55 mg, 1.375 mmol). After stirring for 20 min, 3-
bromo-1-tertbutyldiphenylsilyloxypropane (52 mg, 1.38
mmol) was added and the mixture was stirred for 24 h
at rt. After hydrolysis with 1 N HCl and extraction as
usual with ether, flash chromatography over silica gel
(eluent EtOAc/Pet ether) afforded 21 as an oil (189 mg,
1
mg, 90%) was rapidly used in the next step. H NMR
(300 MHz, CDCl3) d 0.15 (s, 6H, Si(CH3)2), 0.91 (s, 9H,
C(CH3)3), 1.26 (t, 3H, J=7 Hz, CH3), 1.85–2.3 (m, 4H,
H-5 and H-6), 3.24 (t, 1H, J=5 Hz, H-1), 4.17 (q, 2H, J=7
Hz, OCH2), 4.65 (broad s, 1H, C¼CH), 4.76 (broad s,
1H, C¼CH), 5.50 (s, 1H, H-3).
1
29%). H NMR d 0.15 (s, 6H, Si(CH3)2), 1.05 (s, 9H,
C(CH3)3), 1.26 (t, 3H, J=7 Hz, CH3), 1.57 (m, 2H,
CH2), 1.99 (s, 3H, C¼CCH3), 2.22-255 (m, 6H, H-5, H-
6 and C¼CCH2), 3.26 (t, 1H, J=5 Hz, H-1), 3.67 (t,
2H, J=6 Hz, CH2OSi), 4.18 (q, 2H, J=7 Hz, OCH2),
7.38 (m, 6H, H-arom), 7.67 (m, 4H, H-arom); 13C NMR
(75.5 MHz, CDCl3) d 14.2 (CH3), 19.1, 34.7, 47.6 (C-4),
61.15 (OCH2), 63.55 (COSi), 127.6, 129.5, 133.95,
135.45 (C-arom), 137.3 (C-2), 150.1 (C-3), 172.3 (COO),
197.3 (CO); MS (FAB) m/z 479 (43, MH+), 422 (33),
421 (100), 401 (29), 223 (59). HRMS (FAB) m/z calcd
(MH+, C29H39O4Si): 479.2617, obsd: 479.2634.
1-(1-Ethoxy-1-trimethylsilyloxy-methylene)-2-methylene-
4- tertbutyldimethylsilyloxy -cyclohex -3-ene (18). To a
cooled (ꢀ78 ꢁC) solution of LDA prepared from diiso-
propylamine (0.38 mL, 2.73 mmol) and 1.6 N nBuLi in
hexane (1.7 mL, 2.72 mmol) in THF (1.5 mL), under
N2, was added a solution of 17 (367 mg, 1.24 mmol) in
THF (0.3 mL). After stirring for 1 h at ꢀ78 ꢁC, TMSCl
(0.36 mL, 2.85 mmol) was added and the mixture was
stirred overnight at rt. After removal of excess TMSCl
and solvents under vacuo, the residue was diluted with
Ethyl 2-methylene-4-tertbutyldimethylsilyloxy-3-(3-tert-
butyldiphenylsilyloxypropyl)-cyclo-hex-3-ene carboxylate

258
P. Martin et al. / Bioorg. Med. Chem. 10 (2002) 253–260
(22). Same procedure as for 17, starting from 21. Com-
pound 22 was isolated as a colorless oil (86%) which was
rapidly used in the next step. 1H NMR (300MHz, CDCl3)
d 0.12 (s, 6H, Si(CH3)2), 0.89 (s, 9H, C(CH3)3), 1.04 (s, 9H,
C(CH3)3), 1.24 (t, 3H, J=7 Hz, CH3), 1.55–2.35 (m, 8H,
H-5, H-6, CH2, C¼CCH2), 3.26 (t, 1H, J=5 Hz, H-1),
3.64 (t, 2H, J=6 Hz, CH2OSi), 4.16 (q, 2H, J=7 Hz,
OCH2), 4.71 (broad s, 1H, C¼CH), 4.98 (broad s, 1H,
C¼CH), 7.38 (m, 6H, H-arom), 7.67 (m, 4H, H-arom).
mmol) then dimethyl sulfate (12.6 mL, 133 mmol). After
refluxing for 48 h, the mixture was cooled, filtered and
concentrated under reduced pressure. Separation by
flash chromatography (EtOAc/pet ether, 5:95) afforded
27 (13.7 g, 64%) and starting material 26 (3.6 g, 18%).
1
27: H NMR (300 MHz, CDCl3) d 1.00 (d, 3H, J=6.6
Hz, CH3), 1.30 (t, 3H, J=7.3 Hz, CH3), 1.94 (broad d,
1H, J=16 Hz, H-5a), 2.20 (s, 3H, CH3), 2.64 (dd, 1H,
J=16 and 8 Hz, H-5b), 2.92 (m, 1H, H-6), 3.66 (s, 3H,
OCH3), 4.18 (q, 2H, J=7.3 Hz), 4.94 (d, 1H, J=1.4 Hz,
H-3). 13C NMR (75.5 MHz, CDCl3) d 14.3 q, 17.8 q,
21.8 q, 29.3 d, 34.7 t, 54.8 q, 59.3 t, 98.8 d, 119.6 s, 144.7
s, 162.0 s, 168.0 s. HRMS (EI) m/z calcd (C12H18O3):
210.1255, obsd: 210,1237.
1-(1-Ethoxy-1-trimethylsilyloxy-methylene)-2-methylene-
3-(3-tertbutyldiphenylsilyloxypropyl)-4-tertbutyldimethyl-
silyloxy-cyclohex-3-ene (23). Same procedure as for 19,
starting from 22. Compound 23 was isolated as a pale
yellow oil (71%) which was rapidly used in the next
1
step. H NMR (300 MHz, CDCl3) d 0.05–0.25 (m, 15H,
1-(1-Ethoxy-1-trimethylsilyloxy-methylene)-4-methoxy-6
-methyl-2-methylene-cyclohex-3-ene (28). Same proce-
dure as for 18, starting from 27. Compound 28 was
obtained (quant) as a pale yellow oil which was rapidly
used in the next step. 28: 1H NMR (300 MHz, CDCl3) d
0.2 (s, 9H, Si(CH3)3), 1.00 (d, 3H, J=7 Hz, CH3), 1.26
(t, 3H, J=7 Hz, CH3), 1.87 (broad d, 1H, J=17 Hz, H-
5a), 2.50 (broad dd, 1H, J=17 and 7 Hz, H-5b), 3.14
(m, 1H, H-6), 3.59 (s, 3H, OCH3), 3.87 (q, 2H, J=7 Hz,
OCH2), 4.92 (d, 1H, J=2 Hz,=CH), 5.10 (d, 1H, J=2
Hz;=CH), 5.27 (s, 1H,=CH).
Si(CH3)2 and Si(CH3)3), 0.90 (s, 9H, C(CH3)3), 1.06 (s,
9H, C(CH3)3), 1.24 (t, 3H, J=7 Hz, CH3), 1.80 (m, 2H,
CH2), 2.21 and 2.39 (2m, 6H, C¼CCH2, H-5, H-6), 3.69
(t, 2H, J=6 Hz, CH2OSi), 3.87 (q, 2H, J=7 Hz, OCH2),
5.13 (broad s, 2H, C¼CH2), 7.39 (m, 6H, H-arom), 7.70
(m, 4H, H-arom).
8-tertButyldimethylsilyloxy-7-(3-tertbutyldiphenylsilylox-
ypropyl)-1,11-dihydroxy-3-methoxy-naphthacene-5,12-
dione (24). Same procedure as for 14a,b, starting from
23. Compound 24 (29%) was obtained as a red solid. 1H
NMR (300 MHz, CDCl3) d 0.28 (s, 6H, Si(CH3)2), 0.99
(s, 9H, C(CH3)3), 1.08 (s, 9H, C(CH3)3), 1.89 (m, 2H,
CH2), 3.13 (t, 2H, J=8 Hz, CH2Ar), 3.80 (t, 2H, J=6.5
Hz, CH2OSi), 3.94 (s, 3H, OCH3), 6.69 (d, 1H, J=2.5
Hz, H-2), 7.16 (d, 1H, J=9 Hz, H-9), 7.36 (m, 6H, H-
arom), 7.43 (d, 1H, J=2.5 Hz, H-4), 7.69 (m, 4H, H-
arom), 8.26 (d, 1H, J=9 Hz, H-10), 8.43 (s, 1H, H-6),
12.55 (s, 1H, OH), 13.80 (s, 1H, OH); 13C NMR
(75.5 MHz, CDCl3) d ꢀ3.9 (Si(CH3)2), 18.3 and 19.25
(C(CH3)3), 22.5 (CH2), 25.8 and 27.0 (C(CH3)3), 33.35
(ArCH2), 55.95 (OCH3), 64.15 (CH2OSi), 106.6 d,
107.05 s, 107.45 d, 111.1 s, 118.25 d, 121.9 d, 122.8 s,
124.1 d, 127.5 d, 128.3 s, 129.5 d, 129.8 s, 133.9 s, 135.6
d, 136.2 s, 136.7 s, 155.7 s, 163.4 s, 164.95 s, 166.1 s,
170.1 s 181.6 and 189.6 (CO). MS (EI) m/z 746 (12), 731
(17), 717 (28), 703 (86), 689 (100); HRMS (EI) m/z calcd
(C44H50O7Si2): 746.3095, obsd: 746.3081.
4.151,11-Dihydroxy-3,8-dimethoxy-10-methyl-9,10-dihy-
dronaphthacene-5,12-dione (29). Same procedure as for
14a,b starting from 28. Compound 29 was isolated
1
(13%) as a red solid. H NMR (300 MHz, CDCl3) d
1.16 (d, 3H, J=7 Hz, CH3), 2.18 (broad d, 1H, J=16
Hz, H-9a), 2.80 (broad dd, 1H, J=16 and 7 Hz, H-
9b), 3.56 (m, 1H, H-10), 3.78 (s, 3H, OCH3), 3.92 (s,
3H, OCH3), 5.62 (s, 1H, H-7), 6.65 (d, 1H, J=2 Hz,
H-2), 7.32 (d, 1H, J=2 Hz, H-4), 7.43 (s, 1H, H-6),
12.43 (s, 1H, OH) and 12.47 (s, 1H, OH). HRMS (EI)
m/z calcd (C21H16O6): 364.0947, obsd: 364.0961.
Ethyl 4-methoxy-2,6-dimethyl benzoate (31). A mixture
of 10% Pd/C (100 mg) and 27 (500 mg, 2.37 mmol) was
heated at 220 ꢁC for 14 h. After dilution with EtOAc
and filtration, flash chromatography (eluent EtOAc/pet
ether 5:95) afforded 31 as a colorless oil (446 mg, 90%).
1H NMR (300 MHz, CDCl3) d 1.36 (t, 3H, J=7 Hz,
CH3), 2.32 (s, 6H, CH3), 3.76 (s, 3H, OCH3), 4.35 (q,
2H, J=7 Hz, OCH2), 6.56 (broad s, 2H, H-3, H-5); 13C
NMR (75.5 MHz, CDCl3) d 14.2 (OCH2CH3), 20.2 (2
CH3), 55.1 (OCH3), 60.6 (OCH2), 113.2 (C-3, C-5),
126.6 (C-1), 137.2 (C-2, C-6), 160.0 (C-4), 169.7 (COO).
MS (EI) m/z 208 (M), 163 (100); HRMS (EI) m/z calcd
(C12H16O3): 208.1099, obsd: 208.1095.
1,8,11-Trihydroxy-7-(3-hydroxypropyl)-3-methoxy-naph-
thacene-5,12-dione (25). A solution of 24 (150 mg, 0.2
mmol) and nBu4NF (190 mg, 0.73 mmol) in THF (1.2
mL) was stirred for 5 h at rt, poured over satd NH4Cl
and extracted with CH2Cl2. The insoluble fraction was
dissolved in CH2Cl2/MeOH and 25 was precipitated with
pet ether as a dark red solid (72 mg, 91%). Mp
190 ꢁC (dec); 1H NMR (300MHz, CDCl3) d 1.66 (m, 2H,
CH2), 3.01 (m, 2H, ArCH2), 3.50 (t, 2H, J=6.5 Hz,
CH2O), 3.93 (s, 3H, CH3), 6.86 (d, 1H, J=2.2 Hz, H-2),
7.21 (d, 1H, J=2.2 Hz, H-4), 7.36 (d, 1H, J=9 Hz, H-9),
8.18 (d, 1H, J=9 Hz, H-10), 8.25 (s, 1H, H-6). MS (EI) m/
z 394 (24), 350 (43), 142 (100), 100 (59), 57 (31); HRMS
(EI) m/z calcd (C22H18O7): 394.1052, obsd: 394.1058.
4.17Ethyl 2-Ethoxycarbonylmethyl-4-methoxy-6-methyl
benzoate (33). To a cooled (ꢀ78 ꢁC) solution of 31 (1 g,
4.26 mmol) in anhydrous THF (4 mL) was added a
1.5 M solution of LDA in hexane–THF (1:2 v/v, 3.52
mL, 5.28 mmol), and, after 5 min, diethyl carbonate
(2.38 mL, 19.2 mmol). Stirring was continued under N2
for 1 h and the reaction mixture was then poured over
satd NH4Cl. After extraction with CH2Cl2 as usual,
flash chromatography (eluent EtOAc/pet ether 5:95 to
10:90) afforded diester 33 as a colorless oil (386 mg,
Ethyl 2,6-dimethyl-4-methoxy-cyclohexa-1,3-diene car-
boxylate (27). To a solution of 2618 (20 g, 102 mmol),
in anhydrous acetone, was added K2CO3 (21.1 g, 154

P. Martin et al. / Bioorg. Med. Chem. 10 (2002) 253–260
259
30%), together with ethyl 2-diethoxycarbonylmethyl-4-
methoxy-6-methyl benzoate (105 mg, 8%) and starting
material 31 (278 mg, 28%). 33: H NMR (300 MHz,
pension of 30 (25 mg, 0.069 mmol) in CH2Cl2 (0.8 mL)
was added BBr3 (33 mL, 0.34 mmol). After stirring for
48 h at rt followed by addition of water (10 mL), the
insoluble fraction was filtered and purified by pre-
parative TLC (eluent CH2Cl2/MeOH, 95:5) to afford
1
CDCl3) d 1.24 (t, 3H, J=7 Hz, CH3), 1.36 (s, 3H, CH3),
2.39 (s, 3H, CH3), 3.71 (s, 2H, ArCH2), 3.80 (s, 3H,
OCH3), 4.14 (q, 2H, J=7 Hz, OCH2), 4.35 (q, 2H, J=7
Hz, OCH2), 6.64 (broad s, 1H, H-arom), 6.66 (broad s, 1H,
H-arom); 13C NMR (75.5MHz, CDCl3) d 14.0 (2 C,
OCH2CH3), 21.0 (ArCH3), 40.0 (ArCH2), 55.0 (OCH3),
60.6 (2C, OCH2), 114.1 (C-3, C-5), 125.5 (C-1), 134.6
and 139.0 (C-2, C-6), 160.0 (C-4), 168.6 and 170.9
(COO). MS (EI) m/z 280 (M, 25), 234 (55), 206 (55), 179
(100), 162 (58); HRMS (EI) m/z calcd (C15H20O5):
280.1310, obsd: 280.1313.
1
tetracenomycin D (10 mg, 43%) as a dark red solid. H
NMR (300 MHz, methanol-d4) d 2.78 (s, 3H, CH3), 6.35
(d, 1H, J=2 Hz, H-2), 6.82 (d, 1H, J=2 Hz, H-9), 6.91
(d, 1H, J=2 Hz, H-7), 7.03 (d, 1H, J=2 Hz, H-4), 7.72
(s, 1H, H-6); UV (methanol) l (e) 219 (22,400), 240
(28,800), 276 (26,200), 292 (20,200), 307 (19,100), 338
(15,100), 493 (12,700). MS (EI) m/z 336 (M, 100).
1,11 - Dihydroxy - 3,8,10- trimethoxy- naphthacene -5,12 -
dione (39). Same procedure as for 30, starting from 13
and 3521 (prepared as above from 36 in three steps).
Compound 39 was isolated as a dark red solid (72%).
2-Carboxymethyl-4-methoxy-6-methyl benzoic acid (34). A
solution of 33 (200 mg, 0.71 mmol), NaOH (1 g) in
ethanol (5 mL) and water (1 mL) was heated under
reflux for 16 h. After extraction with CH2Cl2, diacid 34
Mp 284–285 ꢁC; H NMR (300 MHz, CDCl3) d 3.92 (s,
1
3H, OCH3), 3.94 (s, 3H, OCH3), 4.03 (s, 3H, OCH3),
6.63 (d, 1H, J=2 Hz, H-9), 6.71 (d, 1H, J=2.5 Hz, H-
2), 6.87 (d, 1H, J=2 Hz, H-10), 7.40 (d, 1H, J=2.5 Hz,
H-4), 8.06 (s, 1H, H-6), 12.50 (s, 1H, OH), 14.80 (s, 1H,
OH). HRMS (EI) m/z calcd (C21H16O7): 380.0896,
obsd: 380.0887.
1
was obtained as a white solid (130 mg, 82%). H NMR
(300 MHz, acetone d6) d 2.42 (s, 3H, CH3), 3.80 (broad
s, 5H, OCH3 and ArCH2), 6.76 (broad s, 2H, H-3 and
H-5), 10 (broad s, 2H, COOH); 13C NMR (75.5 MHz,
acetone-d6)
d 21.5 (ArCH3), 40.3 (ArCH2), 55.7
(OCH3), 115.4 and 115.5 (C-3, C-5), 127.0 (C-1), 136.5
(C-2), 139.7 (C-6), 161.2 (C-4), 170.5 and 172.6 (COO).
MS (EI) m/z 224 (M, 13), 180 (50), 178 (55), 162 (88),
149 (80), 91 (100); HRMS (EI) m/z calcd (C11H12O5):
224.0685, obsd: 224.0675.
4.233,8-Di-O-methyl saintopin (1,10,11-trihydroxy-3,8-
dimethoxy-naphthacene-5,12-dione (40). Same procedure
as for tetracenomycin D (5 min, 0 ꢁC), starting from 39.
Compound 40 was obtained as a dark red solid (unde-
terminated yield). 1H NMR (300 MHz, acetone-d6) d
3.93 (s, 6H, 2 OCH3), 6.80 (d, 1H, J=2 Hz), 6.82 (d,
1H, J=2 Hz), 7.21 (d, 1H, J=2 Hz), 7.33 (d, 1H, J=2
Hz), 8.10 (s, 1H), 12.50 (s, 1H, OH). HRMS (EI) m/z
calcd (C20H14O7): 366.0739, obsd: 366.0732.
6-Methoxy-8-methyl-isochroman-1,3-dione (32). A solu-
tion of acetyl chloride (84 mg, 1.07 mmol) in anhydrous
acetone (0.1 mL) was added to a diacid 34 (60 mg, 0.27
mmol) in acetone (0.5 mL). After stirring for 2 h at rt,
concentration under reduced pressure led to anhydride
1
32 (quant) as a white solid. H NMR (300 MHz, ace-
tone-d6) d 2.63 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 4.22 (s,
2H, Ar–CH2), 6.90 (d, 1H, J=2 Hz, H-arom), 6.92 (d,
1H, J=2 Hz, H-arom). MS (EI) m/z 206 (M, 30), 162
(100), 149 (30), 134 (31), 119 (24), 91 (60); HRMS (EI)
m/z calcd (C11H10O4): 206.0579, obsd: 206.0587.
Acknowledgements
We thank ADIR and the ‘‘Ligue Nationale contre le
Cancer’’, Comite de Charente-Maritime for financial
support.
1,11-Dihydroxy-3,8-dimethoxy-10-methyl-naphthacene-
5,12-dione (30). A solution of 32 (75 mg, 0.36 mmol) in
anhydrous THF (6 mL) was treated with 60% NaH
(17.5 mg, 0.44 mmol) at 0 ꢁC. After stirring for 5 min at
0 ꢁC, a solution of 13 (86 mg, 0.36 mmol) in anhydrous
THF (4.5 mL) was added and the resulting mixture was
stirred for 24 h at rt, quenched with satd NH4Cl and
then with 1 N HCl. After extraction as usual with
CH2Cl2, and evaporation, 30 was obtained by pre-
cipitation from CH2Cl2 by addition of pet ether as a red
solid (80 mg, 60%). Mp 240 ꢁC (dec); 1H NMR
(300 MHz, CDCl3) d 2.96 (s, 3H, CH3), 3.94 (s, 6H,
OCH3), 6.71 (d, 1H, J=2 Hz, H-2), 7.01 (d, 1H, J=2
Hz, H-9), 7.09 (d, 1H, J=2 Hz, H-7), 7.40 (d, 1H, J=2
Hz, H-4), 8.10 (s, 1H, H-6), 12.50 (s, 1H, OH), 14.60 (s,
1H, OH). MS (EI) m/z 364 (M, 100), 350 (7), 321 (12),
303 (8); HRMS (EI) m/z calcd (C21H18O6): 364.0947,
obsd: 364.0961.
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