Novel Prodrugs of Tegafur that Display Improved Anticancer Activity and Antiangiogenic Properties Dikla Engel, †,‡ Abraham Nudelman,* ,† Nataly Tarasenko, ‡ Inesa Levovich, †,‡ Igor Makarovsky, † Segev Sochotnikov, † Igor Tarasenko, ‡ and Ada Rephaeli* ,‡ Chemistry Department, Bar-Ilan UniVersity, Ramat Gan, 52900, Israel, and Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel AViV UniVersity Beilinson Campus, Petach TikVa, 49100, Israel ReceiVed August 8, 2007 New and more potent prodrugs of the 5-fluorouracyl family derived by hydroxymethylation or acyloxy- methylation of 5-fluoro-1-(tetrahydro-2-furanyl)-2,4(1H,3H)-pyrimidinedione (tegafur, 1) are described. The anticancer activity of the butyroyloxymethyl-tegafur derivative 3 and not that of tegafur was attenuated by the antioxidant N-acetylcysteine, suggesting that the increased activity of the prodrug is in part mediated by an increase of reactive oxygen species. Compound 3 in an in vitro matrigel assay was found to be a more potent antiangiogenic agent than tegafur. In vivo 3 was significantly more potent than tegafur in inhibiting 4T1 breast carcinoma lung metastases and growth of HT-29 human colon carcinoma tumors in a mouse xenograft. In summary, the multifunctional prodrugs of tegafur display selectivity toward cancer cells, antiangiogenic activity, and anticancer activities in vitro and in vivo, superior to those of tegafur. 5-Fluoro- 1-(tetrahydro-2-furanyl)-2,4(1H,3H)-pyrimidinedione (tegafur, 1), the oral prodrug of 5-FU, has been widely used for treatment of gastrointestinal malignancies with modest efficacy. The aim of this study was to develop and characterize new and more potent prodrugs of the 5-FU family derived by hydroxymethylation or acyloxymethylation of tegafur. Comparison between the effect of tegafur and the new prodrugs on the viability of a variety of cancer cell lines showed that the IC 50 and IC 90 values of the novel prodrugs were 5–10-fold lower than those of tegafur. While significant differences between the IC 50 values of tegafur were observed between the sensitive HT-29 and the resistant LS-1034 colon cancer cell lines, the prodrugs affected them to a similar degree, suggesting that they overcame drug resistance. The increased potency of the prodrugs could be attributed to the antiproliferative contribution imparted by formaldehyde and butyric acid, released upon metabolic degradation. The anticancer activity of the butyroyloxymethyl-tegafur derivative 3 and not that of tegafur was attenuated by the antioxidant N-acetylcysteine, suggesting that the increased activity of the prodrug is in part mediated by an increase of reactive oxygen species. Compound 3 in an in vitro matrigel assay was found to be a more potent antiangiogenic agent than tegafur. In vivo 3 was significantly more potent than tegafur in inhibiting 4T1 breast carcinoma lung metastases and growth of HT-29 human colon carcinoma tumors in a mouse xenograft. In summary, the multifunctional prodrugs of tegafur display selectivity toward cancer cells, antiangiogenic activity and anticancer activities in vitro and in vivo, superior to those of tegafur. Introduction The synthesis and biological activities of prodrugs of butyric acid having the general formula Me(CH 2 ) 2 COOCH(R)OR 1 , where R ) H, Me, Pr, tert-Bu; R 1 ) OC-alkyl, OC-Ar, and P(O)(OEt) 2 , have been described. 1–3 These acyloxyalkyl pro- drugs serve as molecular carriers for the efficient transport of butyric acid to the cells, leading to a significant increase in its potency. 4 The expected intracellular hydrolytic degradation products of the prodrugs are two carboxylic acids and an aldehyde (Scheme 1). While studying these prodrugs, we discovered that the formaldehyde, released metabolically together with butyric acid, a histone deacetylase (HDAC a ) inhibitor, plays a major and specific role in increasing the antineoplastic activity and is a critical antiproliferative factor in the induction of cancer cell differentiation and death. 1,3,5–7 These compounds specifically affect molecular events in the cancer cell, including changes in gene expression and induction of apoptosis, and thereby contribute significantly to the improvement of the anticancer activity of the parent drugs. While the accepted paradigm views formaldehyde as a highly toxic substance, in living cells it plays a vital role in fundamental biological pathways. Intracellular formaldehyde is formed by oxidative demethylation of N-, O-, and S-methylated compounds, xenobiotics, and by demethylation of DNA and histones. In the cells, formaldehyde is captured by 5′-methyl-tetrahydrofolate, which converts homocysteine to methionine and, by addition of ATP, it is converted into S-adenosyl-L-methionine (SAM), the universal methyl donor participating in synthesis of essential molecules in the cells. 8,9 Formation of adducts between DNA and anthracyclins, mediated by intracellular formaldehyde, has been extensively studied by others and us and has been shown to potentiate the anticancer * To whom correspondence should be addressed. Phone: +972-3-531- 8314(A.N.); +972-3-937-6126(A.R.). Fax: +972-3-531-8314 (A.N.); +972- 3-922-8096 (A.R.). E-mail: nudelman@mail.biu.ac.il (A.N.); adarep@ post.tau.ac.il (A.R.). † Bar-Ilan University. ‡ Tel Aviv University Beilinson Campus. a Abbreviations: HDAC, histone deacetylase; SAM, S-adenosyl-L-me- thionine; AN-1, butyroyloxymenthyl butyrate; AN-7, butyroyloxymethyl- diethyl phosphate; AN-9, pivaloyloxymethyl butyrate; Dox, doxorubicin; tegafur, 5-fluoro-1-(tetrahydro-2-furanyl)-2,4(1H,3H)-pyrimidinedione; 5-FU, 5-fluorouracil; SC, semicarbazide; SSAO, semicarbazide sensitive amine oxidase; HMTA, hexamethylenetetramine; NAC, N-acetylcysteine; ROS, reactive oxygen species; HUVEC, human umbilical vein endothelial cells; CEA, carcinoma embryonic antigen; ES +/- , electron spray; CI + , chemical ionization. J. Med. Chem. 2008, 51, 314–323 314 10.1021/jm7009827 CCC: $40.75  2008 American Chemical Society Published on Web 12/29/2007