Synthesis, Hydrolytic Activation and Cytotoxicity of Etoposide Prodrugs Wolf Wrasidlo, a, * Ulrike Schro¨der, a Kathrin Bernt, a Nicole Hu¨bener, a Doron Shabat, b Gerhard Gaedicke a and Holger Lode a a Charite ´ Children’s Hospital, Humboldt University, Augustenburger Platz 1, 13353 Berlin, Germany b School of Chemistry, Tel-Aviv University, Tel-Aviv 69978, Israel Received 6 June 2001; accepted 26 November 2001 Abstract—Two 4 0 -propylcarbonoxy derivatives (2,3) of etoposide (1), a topoisomerase II inhibitor, were synthesized and evaluated as potential prodrugs for anticancer therapy. Their activation via hydrolysis mechanisms was determined as a function of pH in buffer solutions, in human serum and in the presence of carboxyl ester hydrolase. Cytotoxicity was determined on various tumor cell lines and compared to the parent compound. On cell lines exhibiting resistance to etoposide we observed an enhanced cyto- toxicity of the prodrugs of up to three orders of magnitude. # 2002 Elsevier Science Ltd. All rights reserved. Etoposide (1) is a widely used, highly effective anti- cancer drug against a broad spectrum of tumors including pediatric cancers such as acute lymphatic lymphomas, rhabdomyosarcomas and neuroblastomas, as well as in most common adult cancers. 1,2 It is also used in bone marrow transplantation conditioning regi- mens. However, the therapeutic use of etoposide is lim- ited by toxicity involving mainly myelosuppression. 3 A major problem for the use of this drug (and other topoisomerase inhibitors) is the development of multi- drug resistance. 4,5 Efforts are presently made to synthe- size new derivatives of the natural podophyllotoxin to overcome some of these shortcomings. 6 8 An alternative approach to new analogues of therapeutic agents is the synthesis of prodrugs. In previous work 9 12 we synthe- sized prodrugs of various antitumor agents to improve their bio-availability, phamacokinetics and aqueous solubility. For paclitaxel 11,12 we established that pro- drugs incorporating hydrolytically cleavable moieties are effective in lowering the systemic toxicity of the drug in animal models and most importantly also in patients. 13 Furthermore these prodrugs of paclitaxel revealed a dramatically improved pharmacokinetics and could be formulated for intravenous delivery with a minimum of toxic organic vehicles. Based on these findings the hydrolytic activation approach was applied to etoposide, an important drug in pediatrics and adult oncology. Synthesis Compounds 2 and 3 were prepared as follows. Treat- ment of a solution of 1 (Sigma, Germany) in dichloro- methane, with one equivalent of soketal chloroformate at 70  C followed by the addition of one equivalent of pyridine and reacting for 6 h, lead to 2. The workup consisted of extraction with brine, drying over magne- sium sulfate, centrifugation and concentrating the organic layer to one fifth of the reaction volume. The product was isolated by HPLC purification (C 18 reverse- phase column with 50/50 vol% acetonitrile–water), followed by lyophilization of the eluate (yield 83.5%, purity 99.2%). Compound 3 was prepared from a THF solution of 2 by the addition of 2 N HCl and reacting for 2 h at ambient temperature. Workup consisted of washing with brine, drying over magnesium sulfate, reducing the volume to 25% of the reaction volume and purification via HPLC under the conditions given for 2. The yield of purified product was 85.5% and the purity by HPLC was greater than 99%. The structures of 2 and 3 were established by 1 H NMR, 13 C NMR and MS data. 14,15 Solution stability The chemical stability of solutions of 2 and 3 were determined by dissolving the compounds in 50/50 vol% chromophore–ethanol (1 mg/10 mL solvent mixture) 0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(01)00801-0 Bioorganic & Medicinal Chemistry Letters 12 (2002) 557–560 *Corresponding author. Fax: +49-30-450-559917; e-mail: wolf. wrasidlo@charite.de