Synthesis and Cytotoxic Evaluation of Novel Spirohydantoin Derivatives of the Dihydrothieno[2,3-b]naphtho-4,9-dione System Isabel Gomez-Monterrey, † Giovanni Santelli, ‡ Pietro Campiglia, † Daniela Califano, ‡ Fabiano Falasconi, ‡ Claudio Pisano, § Loredana Vesci, § Teresa Lama, † Paolo Grieco, † and Ettore Novellino* ,† Dipartimento di Chimica Farmaceutica e Tossicologica, University of Naples “Federico II”, Napoli, Italy, Oncologia Sperimentale E, Istituto dei Tumori Fondazione “G. Pascale”, Napoli, Italy, and Area di Ricerca OncologicasR&S, Sigma-Tau S.p.A., Pomezia, Roma, Italy Received June 24, 2004 The synthesis and cytotoxic evaluation of 3-(alkyl)(alkyl-substituted)spiro[(dihydroimidazo- 2,4-dione)-5,3′-(2′,3′-dihydrothieno[2,3-b]naphtho-4′,9′-dione)]derivatives are described. Evalu- ation of these analogues against the MCF-7 human breast carcinoma and SW 620 human colon carcinoma cell lines uncovered for most of the compounds a cytotoxic potency comparable to or greater than that of doxorubicin. Compound 15 exhibited remarkable cytotoxic activity against several other human solid tumor cell lines. Interestingly, only a partial cross-resistance to compound 15 in selected tumor cell sublines known to be resistant to doxorubicin (MCF-7/Dx and A2780/Dx) was observed, whereas a total absence of cross-resistance in a tumor cell subline selected for resistance to cisplatin was found (A2780/DDP). Introduction The quinone group forms the basis of a number of clinical and experimental anticancer drugs, notably the natural antibiotic doxorubicin and the synthetic com- pound mitoxantrone. Although these two drugs in particular occupy a prominent position in the chemo- therapeutic control of a number of human cancers, they have markedly reduced efficacy in resistant disease and against slowly growing tumors. 1 The clinical importance of this class of antitumor agents has stimulated the development of new agents that, retaining the “core” quinonic moiety, could exhibit different spectra of potency with an improved tolerance profile. 2-6 In general, the structural characteristics of the quino- ne derivatives are characterized by two common fea- tures which seem to determine their antineoplastic activity: a planar, polycyclic nucleus capable of binding to the DNA by intercalation and one or two side chains containing different substituents in a defined orienta- tion to the chromophore. It has been also suggested that the side chain has the function of additionally stabilizing a drug-DNA complex, binding electrostatically to the phosphate backbone of the polynucleotide. 7,8 In the course of a medicinal chemistry program aimed at discovering new quinone derivatives endowed with antitumor activity, we have recently developed a series of 3-amino-3-ethoxycarbonyl-2,3-dihydrothieno[2,3-b]- naphtho-4,9-dione derivatives substituted at the posi- tion 3 with different amino acids. 9 The rationale for using such amino acids has been to extend the overall conjugation of the planar chromophore by amide groups, as a side arm, thereby leading to more effective target- binding activity. 10,11 The most active of these deriva- tives, the 3-(glycyl)amino-3-ethoxycarbonyl-2,3-dihy- drothieno[2,3-b]naphtho-4,9-dione, showed remarkable cytotoxic activity at submicromolar concentration not only against several human leukaemia and solid tumor cell lines but also toward resistant human cell lines. The results obtained on dihydrothieno[2,3-b]naphtho-4,9- dione derivatives showed that the stereochemistry at C-3 of these compounds is not an essential feature for cytotoxic activity. 9 These results have also highlighted the potential value of the DTNQ as template in the development of more effective antitumoral agents (DTNQ ) 3-amino-3-ethoxycarbonyl-2,3-dihydrothieno[2,3-b]- naphtho-4,9-dione). Herein, we designed a set of spirohydantoin deriva- tives in which the planar chromophore (DTNQ) was combined, through the hydantoin ring, to an alkyl side chain. To determine how the steric and electronic properties of the side chain may influence the cytotoxic activity, we examined several derivatives containing different alkyl (methyl, ethyl, propyl, butyl, sec-butyl, and tert-butyl) and alkyl-substituted chains (hydroxy- ethyl, hydroxypropyl, aminoethyl, aminopropyl, ami- nobutyl, (N,N-dimethyl)aminoethyl, (N,N-dimethyl)- aminopropyl, (N,N-diethyl)aminoethyl, and (N,N-diethyl)- aminopropyl) in position 3 of the hydantoin ring. The cytotoxic activity was evaluated toward the human breast carcinoma (MCF-7) and human colon carcinoma (SW 620) cell lines. Moreover one of the most active compounds was tested against several other human tumor derived cell lines as well as against the catalytic activity of human topoisomerase II. Chemistry The new 3-(alkyl)(alkyl-substitued)spiro[(dihydroimi- dazo-2,4-dione)-5,3′-(2′,3′-dihydrothieno[2,3-b]naphtho- 4′,9′-dione)] derivatives (3-17) were prepared applying the synthetic route shown in Scheme 1. * To whom correspondence should be addressed. Address: Dipar- timento di Chimica Farmaceutica e Tossicologica, Universita ` di Napoli “Federico II”, Via D. Montesano 49, 80131, Naples, Italy. Phone: +39- 081-678643. Fax: +39-081-678644. E-mail: novellin@unina.it. † University of Naples “Federico II”. ‡ Istituto dei Tumori Fondazione “G. Pascale”. § Sigma-Tau S.p.A. 1152 J. Med. Chem. 2005, 48, 1152-1157 10.1021/jm0408565 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/22/2005