Synthesis and antiproliferative activity of retroetoposide

Article abstract of DOI:10.1016/j.bmcl.2003.08.044

Retro-4′-demethyl-4-epipodophyllotoxin 6 was synthesized in eight steps and 10% overall yield from 4′-demethyl-4-epipodophyllotoxin 12. Subsequent coupling of 22 with 1-O-trimethylsilyl-4,6-O-ethylidene-β-D- glucoside 26 afforded retroetoposide 5 which is

Full text of DOI:10.1016/j.bmcl.2003.08.044

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4107–4109
Synthesis and Antiproliferative Activity of Retroetoposide
Philippe Meresse, Prokopios Magiatis, Emmanuel Bertounesque* and Claude Monneret
Laboratoire de Pharmacochimie, UMR 176 CNRS-Institut Curie, Section Recherche, 26 rue d’Ulm, 75248 Paris cedex 05, France
Received 16 June 2003; revised 4 August 2003; accepted 11 August 2003
Abstract—Retro-40-demethyl-4-epipodophyllotoxin 6 was synthesized in eight steps and 10% overall yield from 40-demethyl-4-epi-
podophyllotoxin 12. Subsequent coupling of 22 with 1-O-trimethylsilyl-4,6-O-ethylidene-b-d-glucoside 26 afforded retroetoposide 5
which is 10-fold less cytotoxic than etoposide against L1210 cell line.
# 2003 Elsevier Ltd. All rights reserved.
Natural lignan podophyllotoxin 1 and etoposide 2, a
semi-synthetic analogue, have attracted considerable
application1 as a result of the potent antitumour activity
of the latter compound (Fig. 1), alone or in combi-
nation, for the treatment of refractory testicular cancers
and small cell lung cancers.
analogues9 or of 2-aza-podophyllotoxin.10 For our part,
we recently proposed a third strategy based upon the
synthesis of less epimerizable d-lactone-containing ana-
logues.11
Futhermore, we have focused on the acquirement of
information about the structure–activity relationship
The mechanism of action for 2 involves topoisomerase
II inhibition resulting in DNA strand scission and cell
death.2 Recently, a two-drug model for etoposide
action against human topoisomerase IIa has been
reported by Osheroff and co-workers with important
consequences for topo II-based cancer chemotherapy.3
The development of drug resistance, myelosuppression
and poor oral bioavailability have stimulated further
syntheses of analogues of 2 for better pharmacological
profiles. Therefore, about 100 new analogues have
been synthesized and reported,4 and among them,
etopophos 3, a water-soluble prodrug has been intro-
duced on the market5 whereas GL 331 4 in under
phase II clinical trial.6
(SAR) of retro-40-demethyl-4-epipodophyllotoxin
having the lactone carbonyl group at C-11.
6
Such functionality is present in arylnaphthalene lig-
nans12 like justicidin E13 7, retrojusticidin B 8 and
retrochinensin 9, and in aryltetraline lignans like formo-
solactone 1014 and a-conidendrin (ACON) 1115 (Fig. 2).
Retroarylnaphthalene lactones and retroaryltetraline
lactones have been less studied.16 Antimicrobial and
anti-platelet activating factor activities have been
reported in this former sub-group, and retrojusticidin B
8 has been shown to inhibit HIV-1 reverse tran-
scriptase.17 As for 11, it was shown to display no rele-
vant cytotoxic effect. However, this result may be due to
the lack of some structural determinants as the methyl-
ene dioxy on the A-ring, the 4-hydroxy group on the C-
ring, and the tri- or dimethoxy groups on the E-ring. In
this communication, we report the synthesis and anti-
proliferative activity of retro-40-demethyl-4-epipodo-
phyllotoxin 6 and the corresponding retroetoposide 5.
Besides these problems, the metabolism of etoposide
results in inactivation by hydrolysis and epimerization
to the corresponding 2,3-cis-hydroxyacid and picroeto-
poside which are 500- and 100-fold less active, respec-
tively.7 In order to avoid or minimize the C-2
epimerization and the lactone ring opening, two main
strategies have been followed. The first included repla-
cement of the g-lactone with heterocycles.8 The second
took advantage of the preparation of C-2 substituted
The synthesis of retrolactone 6 was achieved from 40-
demethyl-4-epipodophyllotoxin 1218 (Scheme 1). Bis-
silylation of 12, followed by LAH reduction, led to the
trans-diol 14.19 Treatment of the latter with 2.5 equiva-
lents of pivaloyl chloride furnished a mixture of 15, 16,
and 17 in 65, 27 and 8% yields, respectively.
*Corresponding author. Tel.: +33-1-4234-6659; e-mail: emmanuel.
bertounesque@curie.fr
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.044

4108
P. Meresse et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4107–4109
The prospect of utilizing 16 in place of 15 appeared
attractive for the small-scale preparation of 6 (Scheme
2). Dess–Martin oxidation20 of 16 and subsequent
sodium chlorite oxidation gave the acid derivative 19
(74%, two steps). Following removal of the pivaloyl
group with LiEt3BH, cyclization of the resultant g-
hydroxy carboxylic acid 20 by treatment with DCC in
the presence of 4-DMAP provided the expected g-lac-
tone 21. Final deprotection of 21 was carried out in the
presence of tetrabutylammonium fluoride, affording 6.21
Prior to the glycosidation-step, 6 was selectively pro-
tected at C-40 as the benzyloxycarbonyl derivative 22.
Scheme 1. (i) TBDMSOTf, 2,6-lutidine, CH2Cl2, 0 ꢀC, 1 h, 81%; (ii)
LiAlH4, THF, 0 ꢀC to rt, 30min, 92%; (iii) PivCl, Et 3N, 4-DMAP,
CH2Cl2, rt, 40min, 15 (65%), 16 (27%) and 17 (8%).
On the other hand, the sugar moiety was prepared from
glucose in five steps via standard procedures (Scheme 3).
Hydroxyl protection of the 4,6-O-ethylidene-d-gluco-
pyranose 2322 as the chloroacetyl esters furnished 24.
Selective anomeric deprotection (MeOH, pyridine)
afforded 25 as a mixture (a/b=70:30) which was then
converted into the silylated glucoside donor 26 by
treatment with trimethylsilyl chloride in the presence of
Scheme 2. (i) Periodinane, CH2Cl2, rt, 50min, 90%; (ii) NaClO ,
2
NH2SO3H, t-BuOH/H2O (2:1) rt, 1 h, 82%; (iii) LiEt3BH, THF, 0 ꢀC,
1 h, 97%; (iv) DCC, 4-DMAP, CH2Cl2, rt, 1 h, 83%; (v) TBAF, THF,
rt, 4 h, 80%; (vi) PhCH2OCOCl, Et3N, CH2Cl2, 0 ꢀC, 1 h, 85%.
Figure 1. Structures of podophyllotoxin 1 and its derivatives 2–5.
Scheme 3. (i) ClCH2COCl, CH2Cl2, pyridine, À20to 0 ꢀC then 3.5 h,
80%; (ii) MeOH, pyridine, 40 ꢀC, quant yield; (iii) Me3SiCl, Et3N,
CH2Cl2, 50%; (iv) 26 (0.85 equiv) 22 (1 equiv), BF3–Et2O, CH2Cl2,
À60to À30 ꢀC for 3 h then À10 ꢀC for 2 h, 27 (40%) and 28 (10%); (v)
Zn(OAc)2, MeOH, reflux for 1.15 h, 80%; (vi) H2, 10% Pd/C, MeOH,
rt, 98%.
Figure 2. Retrolactones 6–11.

P. Meresse et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4107–4109
4109
Et3N. The b-anomer was obtained in pure form by
crystallization of the crude reaction mixture.
Prod. Rep. 1999, 16, 75. (b) Ward, R. S. Nat. Prod. Rep. 1997,
14, 43.
13. (a) Wada, K.; Munakata, K. Tetrahedron Lett. 1970, 23,
2017. (b) Holmes, T. L.; Stevenson, R. J. Org. Chem. 1971, 36,
3450.
14. Kuo, Y.-H.; Yu, M.-T. Heterocycles 1993, 36, 529.
15. Dantzig, A.; LaLonde, R. T.; Ramdayal, F.; Shepard,
R. L.; Yanai, K.; Zhang, M. J. Med. Chem. 2001, 44, 180.
16. Subbaraju, G. V.; Pillai, K. R. Indian J. Chem. Sect. B
1996, 35, 1233.
17. (a) Kawazoe, K.; Yutani, A.; Tamemoto, K.; Yuasa, S.;
Shibata, H.; Higuti, T.; Takaishi, Y. J. Nat. Prod. 2001, 64,
588. (b) Tanabe, Y.; Seko, S.; Nishii, Y.; Yoshida, T.; Utsumi,
N.; Suzukamo, G. J. Chem. Soc., Perkin Trans. 1 1996, 2157.
(c) Chang, C. W.; Lin, M. T.; Lee, S. S.; Liu, K. C. S. C.; Hsu,
F. L.; Lin, J. Y. Antiviral Res. 1995, 27, 367.
Kuhn-like coupling23 (BF3–Et2O, À30 ꢀC) of 22 with 26
produced a mixture of the desired glucoside 27 in 40%
yield with high stereoselectivity and the unsaturated
retrolactone 28 (10%), both resulting from elimination
(E1 vs SN1) due to the acidity of the a-proton, and acid-
catalyzed dehydration of 22. Finally, deprotection of
the sugar by methanolysis, followed by hydrogenation,
generated retroetoposide 5.21
Compounds 6 and 5 were tested against L1210cell line:
retrolactone 6 was almost devoid of cytotoxic effect with
an IC50=27.4 mM. For its part, retroetoposide 5 was
18. Kuhn, M.; Keller-Ju´ slen, C.; von Wartburg, A. Helv.
Chim. Acta 1969, 52, 944.
less active (IC50=6.74 mM) than etoposide
2
(IC50=0.834 mM), whereas it remains active as an inhib-
itor of topoisomerase II24 and blocks the L1210cells at
the G2/M transition (66% at 50 mM). Unlike several 4-
b-O-N-[2(N0,N0-dialkylamino)alkyl] carbamate deriva-
tives25 of 6, retroetoposide 5 displayed poor activity. We
suggest that specific intramolecular interactions between
the sugar moiety and the carbonyl group at C-11 due to
their proximity may affect the conformation, and thus,
account for the activity of 5. Further syntheses of retro-
40-demethyl-4-epipodophyllotoxin analogues will be
reported in due course.
19. Zhou, X.-M.; Lee, K. J.-H.; Cheng, J.; Wu, S.-S.; Chen,
H.-X.; Guo, X.; Cheng, Y.-C.; Lee, K.-H. J. Med. Chem.
1994, 37, 287.
20. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277.
21. All newly-synthesized compounds gave satisfactory ana-
lytical and spectroscopic data.
20
6. Mp 129–131 ꢀC (acetone/hexane); ½aꢁD À16 (c 1, CHCl3);
IR (CDCl3) 3685, 3539, 1771 cmÀ1 1H NMR: (300 MHz,
;
CDCl3) d 6.94 (s, 1H); 6.50(s, 1H), 6.03 (s, 2H), 5.99 and 5.95
(2 d, 1H, J=1.2 Hz), 5.48 (br s, 1H), 5.14 (t, J=3.3 Hz), 4.47
(t, 1H, J=8.2 Hz), 4.35 (d, 1H, J=7.2 Hz), 3.80(s, 6H), 3.62
(dd, 1H, J=11.2, 8.2 Hz), 3.34 (m, 1H), 2.79 (dd, 1H, J=14,
3.3 Hz), 2.60(d, 1H, J=3.3 Hz). MS (DCI/NH3) m/z 418
(M+NH4)+. Anal. calcd for C21H20O8: C, 63.00; H, 5.03.
Found: C, 62.86; H, 5.05.
Acknowledgements
20
5. Mp 161 ꢀC; ½aꢁD À17.5 (c 1, CHCl3); H NMR (300 MHz,
CDCl3) d 6.87 (s, 1H,), 6.47 (s, 1H), 5.97–5.94 (m, 4H), 5.22
(d, 1H, J=2.2 Hz), 4.70–4.68 (m, 2H), 4.55 (d, 1H, J=7.8 Hz),
4.36–4.33 (m, 2H), 4.19 (dd, 1H, J=4.6, 10.3 Hz), 3.76 (s, 6H),
3.67–3.61 (m, 2H), 3.54–3.39 (m, 3H), 3.32–3.20(m, 2H), 1.33
(d, 3H, J=5 Hz); MS (DCI/NH3) m/z 606 (M+NH4)+. Anal.
calcd for C29H32O13: C, 59.18; H, 5.43; O, 35.34. Found: C,
59.31; H, 5.40; O, 35.27.
1
We thank CNRS and Institut Curie for financial sup-
port. Laboratoires Servier are gratefully acknowledged
for a doctoral grant to P. Meresse and for biological
evaluation by S. Leonce.
References and Notes
22. Hiral, N.; Sakashita, S.; Sano, T.; Inoue, T.; Ohigashi, H.;
Premasthira, C.-U.; Asakawa, Y.; Harada, J.; Fujii, Y. Phy-
tochemistry 2000, 55, 131.
23. Allevi, P.; Anastasia, M.; Ciuffreda, P.; Bigatti, E.; Mac-
Donald, P. J. Org. Chem. 1993, 58, 4175.
24. Topoisomerase II-mediated DNA relaxation assay:
Supercoiled pBS DNA (0.1 mg) was incubated for 15 min at
30 ꢀC, in a 50mM Tris–HCl buffer, pH 7.5, containing 1 mM
1. Stahelin, H. F.; von Wartburg, A. Cancer Res. 1991, 51, 5.
2. (a) Wilstermann, A. M.; Osheroff, N. Curr. Top. Med.
Chem. 2002, 3, 1349. (b) Meresse, P.; Bertounesque, E.;
Dechaux, E.; Monneret, C. Curr. Med. Chem. In press.
3. Bromberg, K. D.; Burgin, A. B.; Osheroff, N. J. Biol.
Chem. 2003, 278, 7046.
4. Zhang, Y.; Lee, K.-H. Chinese Pharm. J. 1994, 46, 319.
5. Saulnier, M. G.; Langley, D. R.; Kadow, J. F.; Senter,
P. D.; Oknipe, J.; Tun, M. M.; Vyas, D. M.; Doyle, T. W.
Bioorg. Med. Chem. Lett. 1994, 4, 2569.
6. Liu, J. M.; Chen, L. T.; Chao, Y.; Li, A. F. Cancer
Chemother. Pharmacol. 2002, 49, 425.
7. van Maanen, J. M. S.; Retel, J.; de Vries, J.; Pinedo, H. M.
J. Natl. Cancer Inst. 1988, 1526, and references cited therein.
8. Ramos, A. C.; Pelaez-Lamamie de Clairac, R.; Medarde,
´
M. Heterocycles 1999, 51, 1443, and references cited therein.
9. (a) Glinski, M. B.; Freed, J. C.; Durst, T. J. Org. Chem.
1987, 52, 2749. (b) Van Vliet, D. S.; Lee, H.-K. Tetrahedron
Lett. 1999, 40, 2259.
10. Pearce, H. L.; Back, N. J.; Cramer, T. L.; Zang, Y.; Lee,
K.-H. Tetrahedron Lett. 1989, 30, 907.
11. Roulland, E.; Bertounesque, E.; Huel, C.; Monneret, C.
Tetrahedron Lett. 2000, 41, 6769.
12. For recent reviews of lignans, see: (a) Ward, R. S. Nat.
ATP, 120mM KCl, 10mM MgCl , 0.5 mM DTT, 0.1 mM
2
EDTA and 30 mg BSA, in the presence of the drug at 50 mM
(total reaction volume 10 mL). Two units of human DNA
topoisomerase II were added to the duplex, ꢀpreincubated as
described, and incubated for 30min at 30 C. The DNA–
topoisomerase II cleavage complexes were dissociated by
addition of SDS (final concentration 0.5%) and of proteinase
K (Sigma) to 500 mg/mL, followed by incubation for 30min at
55 ꢀC. DNA samples were then added to the electrophoresis
dye mixture (5 mL) and electrophoresed (35 V/cm) in a 1%
agarose gel in TBEx1, containing ethidium bromide (1 mg/
mL), at room temperature for 2 h. Gels were washed and
photographed under UV light.
25. Monneret, C. Antitumor Activity of Podophyllotoxin D-
ring Analogue Derivatives. Oral communication. XVIIth
International Symposium on Medicinal Chemistry, Barcelona,
Sept. 1–5, 2002.