**3.4. Anti-cancer activity of 9-(2,3-Dihydro-1,4-benzoxathiin-3-ylMethyl)-9H-purines**

Compounds **1-6** may be considered as drugs with their own entity and anti-tumour activity independent of that of 5-FU. If the previously described compounds are not prodrugs, it is not necessary to maintain the *O,N*-acetalic characteristic with the corresponding weakness of the *O,N*-acetalic bond. Therefore, molecules are being designed in which both structural entities (such as the benzo-heterocyclic ring and the purine base) are linked by a heteroatom-C-C-base-N-atom bond. The design, synthesis and biological evaluation of a series of 2- and 6-disubstituted 9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9*H*-purine derivatives **11-13** were described (Figure 4, Table 4) [121].

**Figure 4.** Several non-acetalic purine derivatives reported by us [121].

inducing agents has proved to be difficult.

similar results. The data are means ± SEM of three independent determinations.

derivatives **11-13** were described (Figure 4, Table 4) [121].

O

S

**Figure 4.** Several non-acetalic purine derivatives reported by us [121].

breast cancer cell line after treatment for 48 h for the three most active compounds.

the IC50 concentrations of **8**, **9** and **10** showed important differences in cell cycle progression compared with DMSO-treated control cells. The cell cycle regulatory activities can be divided into the following two groups: (a) the breast cancer cells showed an accumulation in the S-phase, up to 37.00 ± 2.00 of the cells, mainly at the expense of the G0/G1-phase population that decreased to a percentage of 55.63 ± 1.57 of the cells; (b) compounds **9** and **10** accumulated the cancerous cells in the G2/M-phase (11.08 ± 1.01 and 19.16 ± 0.56, respectively) at the expense of the S-phase cells (26.82 ± 1.26 and 22.73 ± 0.37, respectively). In response to **9** (and **10**), the percentage of apoptotic cells increased, from 0.22 ± 0.31 in control cells to a maximum of 73.37 ± 0.12 (and 65.28 ± 1.92) apoptotic cells at a concentration equal to their IC50 against the MCF-7 cell line. This is a remarkable property because the demonstration of apoptosis in MCF-7 breast cancer cells by known apoptosis-

**Compound Cell cyclea Apoptosisb IC50 (μM) G0/G1 S G2/M Control** 58.62 ± 0.74 33.82 ± 0.72 7.55 ± 1.34 0.22 ± 0.16 **8** 5.04 ± 1.68 55.63 ± 1.57 37.00 ± 2.00 7.37 ± 0.43 44.47 ± 2.98 **9** 7.12 ± 0.46 59.10 ± 1.28 26.82 ± 1.26 11.08 ± 0.01 73.37 ± 0.12 **10** 8.40 ± 0.91 58.10 ± 0.19 22.73 ± 0.37 19.16 ± 0.56 65.28 ± 1.92 a Determined by flow cytometry. 3 bApoptosis was determined using an Annexin V-based assay [120]. The data indicate the percentage of cells undergoing apoptosis in each sample. All experiments were conducted in duplicate and gave

**Table 3.** Anti-proliferative activity, cell cycle distribution and apoptosis induction in the MCF-7 human

**3.4. Anti-cancer activity of 9-(2,3-Dihydro-1,4-benzoxathiin-3-ylMethyl)-9H-purines** 

Compounds **1-6** may be considered as drugs with their own entity and anti-tumour activity independent of that of 5-FU. If the previously described compounds are not prodrugs, it is not necessary to maintain the *O,N*-acetalic characteristic with the corresponding weakness of the *O,N*-acetalic bond. Therefore, molecules are being designed in which both structural entities (such as the benzo-heterocyclic ring and the purine base) are linked by a heteroatom-C-C-base-N-atom bond. The design, synthesis and biological evaluation of a series of 2- and 6-disubstituted 9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9*H*-purine

N

R2

**11** R1 = H, R2 = Cl **12** R1 = H; R2 = Br **13** R1 = Cl; R2 = Cl

N N

N

R1

Compounds **11-13** were subjected to cell cycle and apoptosis studies on the MCF-7 human breast cancer cell line (Table 4). The following two consequences can be stated: (a) in contrast to 5-FU, the six-membered compounds **11-13** provoked a G0/G1-phase cell cycle arrest when the MCF-7 cells were treated during 48 h with the IC50 of the compounds, mainly at the expense of the S-phase populations. The fact that at similar doses the novel derivatives exhibit different sequences of cell cycle perturbations in comparison with 5-FU indicates that these compounds act by different pathways [12]. In the case of **12** it is worth pointing out that, moreover, there is an increase in the G2/M-phase of the cancerous cells; and (b) the apoptotic indices of the target compounds are very important, especially for **13** (58.29% for **11**, 63.05% for **12**, and 76.22% for **13**). Up to now and according to our knowledge, compound **13** is the most important apoptotic inducer against the MCF-7 human breast cancer cell line so far reported.


aDetermined by flow cytometry [12]. bApoptosis was determined using an Annexin V-based assay [12]. The data indicate the percentage of cells undergoing apoptosis in each sample. c Data were taken from [117]. All experiments were conducted in duplicate and gave similar results. The data are means ± SEM of three independent determinations.

**Table 4.** Anti-proliferative activity, cell cycle distribution and apoptosis induction in the MCF-7 human breast cancer cell line after treatment for 48 h for the three most active compounds as anti-proliferative agents.
