**3.5. Anti-cancer activity of cyclic and acyclic O,N-acetals derived from purines and 5-FU**

We have recently published two *O,N*-acetals with outstanding anti-proliferative activities. The most potent antiproliferative agent against the MCF-7 adenocarcinoma cell line belongs to the benzoxazepine *O,N*-acetalic family is 9-[1-(9*H*-fluorenyl-9-methoxycarbonyl)-1,2,3,5 tetrahydro-4,1-benzoxazepine-3-yl]-6-chloro-9*H*-purine (**14**, IC50 = 0.67 ± 0.18 �M), whilst 7-{2- (*N*-hydroxymethylphenyl)-2-nitrobenzenesulfonamido]-1-methoxyethyl} -6-chloro-7*H*-purine (**15**) shows the lowest IC50 value among the family of acyclic *O,N*-acetals (IC50 = 3.25 ± 0.23 �M) (Figure 5). The global apoptotic cells caused by **14** and **15** against MCF-7 were 80.08% and 54.85% of cell population after 48 h, respectively. cDNA microarray technology reveals potential drug targets, which are mainly centred on positive apoptosis regulatory pathway genes, and the repression of genes involved in carcinogenesis, proliferation and tumour invasion [21]. We demonstrated later on that, when the anthranilic alcohol-derived acyclic 5 fluorouracil *O,N*-acetal **16** was administered to human breast cancer cells MCF-7, had no activity against classic pro-apoptotic genes such as *p53*, and even induced the down-regulation of anti-apoptotic genes such as *Bcl-2*. In contrast, several pro-apoptotic genes related with the endoplasmic reticulum (ER)-stress-induced apoptosis, such as *BBC3* and *Noxa*, appeared upregulated. These results seem to show that the mechanism of action and selectivity of **16** was via the activation of the ER-stress-induced apoptosis. The linkage between the 5-FU moiety and the chain of **16** is through the *N*-1 atom of the pyrimidine [20] (Figure 5).

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 125

allowing the regulation of several cell death effectors and modulators, playing a key role in cellular survival. c-IAP1 and c-IAP2 are able to interact with TNFα-receptor-associated factors -1 and -2 (TRAFs) trough their association in a complex with TNFα receptor 2. The TNFα receptor can mediate survival and death cell signals. In this case, c-IAP1 and c-IAP2 have been proposed to reduce the level of caspase-8 activation and protect cells from

**Figure 6.** Identifying some of the structural motifs of IAPs. The baculoviral IAP repeat (BIR) domains; 70–80-amino-acid cysteine- and histidine-rich domains that chelate zinc ions. The presence of at least one BIR domain is the defining characteristic of the IAP family. The number of BIR domains in a given IAP varies from one to three. Another motif is the really interesting new gene (RING) zinc finger, a caspase recruitment domain and ubiquitin-associated (UBA) domains are found in individual IAPs. Finally, the CARD motif (Caspase Recruitment Domain) is a protein–protein interaction domain that mediates oligomerization with other CARD-containing proteins and is found in a number of proteins

There is another family member of the IAPs, called Livin, which are expressed in high levels in melanoma and colon cancer, in embryonic tissues and transformed cells. The overexpression of Livin isoforms α and β blocks apoptosis induced via the extrinsic death

Survivin is involved in the control of cell proliferation and cell death and gene expression is regulated in a cell cycle dependent manner in mitosis. The spliced transcription of the survivin gene gives rise to wild-type survivin, survivin-2α, survivin-2B, survivin-ΔEx-3 and survivin-3B. This protein is stabilized by phosphorylation thanks to a p34cdc2–cyclin B1 complex during mitosis. The anti-apoptotic function of survivin has been linked with its interaction with Smac/DIABLO, with the stabilization of XIAP protein through its binding to

apoptosis in a TNFα relationship [10].

involved in the regulation of cell death.

receptor pathway [125].

**Figure 5.** Several *O,N*-acetalic purine derivatives reported by us [20,21]. Fmoc is the fluorenylmethyloxycarbonyl group; *o*Ns is the -SO2-C6H4-*o*NO2 group.
