**3.2. 1-[2-(2-hydroxymethylphenoxy)-1-methoxyethyl]-5-fluorouracils 7a-f: antiproliferative activity, cell cycle dysregulation and apoptotic induction against breast cancer cells**

Acyclic 5-FU *O,N*-acetals **7a-f** were previously reported by us (Figure 2) [17]. The aminalic bond is established through *N*-1 of the 5-FU moiety.

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 121

emphasizing is that **7e** (the only compound tested) induced neither toxicity nor death in mice after one-month treatment when administered intravenously twice a week, with a 50

**Compound IC50 (M)a Cell cycle (48 h)b Apoptosis (h)c**

 G0/G1 G2/M S 24 48 **Control** 68.39 12.04 19.57 1.24 1.24

**7a** 18.5 ± 0.95 67.18 4.67 28.16 59.90 40.23 **7b** 29.0 ± 1.63 62.72 1.59 35.69 33.35 37.87 **7c** 18.0 ± 0.85 71.01 28.99 0.00 44.36 50.64 **7d** 16.0 ± 1.18 51.45 20.66 27.88 42.24 36.37 **7e** 5.42 ± 0.26 46.92 2.84 50.24 40.73 48.22 **7f** 21.0 ± 1.02 67.32 9.40 23.28 41.15 37.81

The data indicate the percentage of cells undergoing apoptosis in each sample. All experiments were conducted in

**Table 2.** Anti-proliferative activitiy, cell cycle dysregulation, and apoptosis induction in the MCF-7

The anti-tumour potential of the target molecules is stated against the MCF-7 human breast cancer cell line (Table 3). The most active compound (**8**), that presents an allyloxy group as substituent at position 6 of the purine ring, shows an IC50 = 5.04 ± 1.68 μM nearly equipotent as 5-FU. Compounds, **9** and **10**, present bulky substituents as the phenylthio and 2,4-

To study the mechanisms of the anti-tumour and anti-proliferative activities **8-**1**0**, the effects on the cell cycle distribution were analyzed by flow cytometry (Table 3). DMSO-treated cell cultures contained a 58.62 ± 0.74 of the G0/G1-phase cells, a 33.82 ± 0.72 of the S-phase cells and a 7.55 ± 1.34 of the G2/M-phase cells. In contrast, MCF-7 cells treated during 48 h with

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

**3.3. Benzo-Fused Seven-Membered Derivatives Linked To Purines** 

human breast cancer cell line after treatment for 24 and 48 h for the compounds.

R **8** R = OCH2CH=CH2  **9** R = SC6H5

Compounds **8-10** (Figure 3) were synthesized as reported.

3.00 ± 0.11 45.62 0 54.38 52.20 58.06

Apoptosis was determined using an annexin V-based assay [113].

O

N

<sup>N</sup> <sup>R</sup>

N

N

**10** R = SC6H3-Cl2-(2,4)

O

mg/kg dose each time.

O 5-FU

**Ftorafur** 

O

N

N

diclorophenylthio ones, respectively.

**Figure 3.** Several cyclic *O,N*-acetals reported by us [119].

<sup>N</sup> <sup>N</sup>

O

aSee [117]. bDetermined by flow cytometry: see [16]. c

**Figure 2.** Several acyclic 5-FU *O,N*-acetals previously reported by us [17].

The IC50 values of compounds are shown in Table 2. The most active compound is **7e** (5.42 ± 0.26 μM), with an anti-proliferative activitiy in the same order as that of Ftorafur (3.00 ± 0.11 μM). Cell cycle regulation has attracted a great deal of attention as a promising target for cancer research and treatment. The use of cell-cycle-specific treatments in cancer therapy has greatly benefited from the major advances that have been recently made in the identification of the molecular actors regulating the cell cycle and from the better understanding of the connections between cell cycle and apoptosis. As more and more 'cell cycle drugs' are being discovered, their use as anticancer drugs is being extensively investigated [118]. To study the mechanisms of the anti-tumour and anti-proliferative activities of the compounds, the effects on the cell cycle distribution were analyzed by flow cytometry. DMSO-treated cell cultures contained 68.39% G0/G1-phase cells, 12.04% G2/M-phase cells and 19.57% S-phase cells. In contrast, MCF-7 cells treated during 48 h with the IC50 concentrations of **7a–f** showed important differences in cell cycle progression compared with DMSO-treated control cells. The treatment with Ftorafur showed a decrease of the G0/G1-phase cells and a corresponding accumulation of S-phase cells (45.62% G0/G1-phase cells and 54.38% S-phase cells). Moreover, there was an almost total disappearance in the G2/M population of the cells treated with this drug. In general the cell cycle regulatory activities for the newly synthesized compounds can be divided into the following three groups: (a) **7c** and **7d** accumulated the cancerous cells in the G2/M-phase, in the former compound at the expense of the S-phase cells, and (b) **7e** induced a S-phase cell cycle arrest (50.24%) in a similar percentage to that caused by Ftorafur (54.28%, Table 2). Therefore, it can be affirmed that the nitro derivative (**7e**) may act as a 5-FU prodrug. Nevertheless, this hypothesis needs to be corroborated by further assays. In response to **40a**, the percentage of apoptotic cells increased, from 1.24% in control cells to a maximum of 59.9% apoptotic cells (24 h) at a concentration equal to its 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 apoptosisinducing agents has proved to be difficult and only few cytotoxic agents act preferentially through an apoptotic mechanism in human breast cancer cells [113,114]. Finally, a fact worth


emphasizing is that **7e** (the only compound tested) induced neither toxicity nor death in mice after one-month treatment when administered intravenously twice a week, with a 50 mg/kg dose each time.

aSee [117]. bDetermined by flow cytometry: see [16]. c Apoptosis was determined using an annexin V-based assay [113]. The data indicate the percentage of cells undergoing apoptosis in each sample. All experiments were conducted in duplicate and gave similar results. The data are means ± SEM of three independent determinations.

**Table 2.** Anti-proliferative activitiy, cell cycle dysregulation, and apoptosis induction in the MCF-7 human breast cancer cell line after treatment for 24 and 48 h for the compounds.

#### **3.3. Benzo-Fused Seven-Membered Derivatives Linked To Purines**

Compounds **8-10** (Figure 3) were synthesized as reported.

120 Apoptosis and Medicine

**against breast cancer cells** 

bond is established through *N*-1 of the 5-FU moiety.

OH R1

**3.2. 1-[2-(2-hydroxymethylphenoxy)-1-methoxyethyl]-5-fluorouracils 7a-f: antiproliferative activity, cell cycle dysregulation and apoptotic induction** 

<sup>O</sup> OMe

R2 5-FU

**Figure 2.** Several acyclic 5-FU *O,N*-acetals previously reported by us [17].

Acyclic 5-FU *O,N*-acetals **7a-f** were previously reported by us (Figure 2) [17]. The aminalic

The IC50 values of compounds are shown in Table 2. The most active compound is **7e** (5.42 ± 0.26 μM), with an anti-proliferative activitiy in the same order as that of Ftorafur (3.00 ± 0.11 μM). Cell cycle regulation has attracted a great deal of attention as a promising target for cancer research and treatment. The use of cell-cycle-specific treatments in cancer therapy has greatly benefited from the major advances that have been recently made in the identification of the molecular actors regulating the cell cycle and from the better understanding of the connections between cell cycle and apoptosis. As more and more 'cell cycle drugs' are being discovered, their use as anticancer drugs is being extensively investigated [118]. To study the mechanisms of the anti-tumour and anti-proliferative activities of the compounds, the effects on the cell cycle distribution were analyzed by flow cytometry. DMSO-treated cell cultures contained 68.39% G0/G1-phase cells, 12.04% G2/M-phase cells and 19.57% S-phase cells. In contrast, MCF-7 cells treated during 48 h with the IC50 concentrations of **7a–f** showed important differences in cell cycle progression compared with DMSO-treated control cells. The treatment with Ftorafur showed a decrease of the G0/G1-phase cells and a corresponding accumulation of S-phase cells (45.62% G0/G1-phase cells and 54.38% S-phase cells). Moreover, there was an almost total disappearance in the G2/M population of the cells treated with this drug. In general the cell cycle regulatory activities for the newly synthesized compounds can be divided into the following three groups: (a) **7c** and **7d** accumulated the cancerous cells in the G2/M-phase, in the former compound at the expense of the S-phase cells, and (b) **7e** induced a S-phase cell cycle arrest (50.24%) in a similar percentage to that caused by Ftorafur (54.28%, Table 2). Therefore, it can be affirmed that the nitro derivative (**7e**) may act as a 5-FU prodrug. Nevertheless, this hypothesis needs to be corroborated by further assays. In response to **40a**, the percentage of apoptotic cells increased, from 1.24% in control cells to a maximum of 59.9% apoptotic cells (24 h) at a concentration equal to its 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 apoptosisinducing agents has proved to be difficult and only few cytotoxic agents act preferentially through an apoptotic mechanism in human breast cancer cells [113,114]. Finally, a fact worth

**7a** R1 = R2 = H

**7b** R1 = H; R2 = OMe **7c** R1 = Cl; R2 = H **7d** R1 = Br; R2 = H **7e** R1 = NO2; R2 = H **7f** R1 = NH2; R2 = H

**Figure 3.** Several cyclic *O,N*-acetals reported by us [119].

The anti-tumour potential of the target molecules is stated against the MCF-7 human breast cancer cell line (Table 3). The most active compound (**8**), that presents an allyloxy group as substituent at position 6 of the purine ring, shows an IC50 = 5.04 ± 1.68 μM nearly equipotent as 5-FU. Compounds, **9** and **10**, present bulky substituents as the phenylthio and 2,4 diclorophenylthio ones, respectively.

To study the mechanisms of the anti-tumour and anti-proliferative activities **8-**1**0**, the effects on the cell cycle distribution were analyzed by flow cytometry (Table 3). DMSO-treated cell cultures contained a 58.62 ± 0.74 of the G0/G1-phase cells, a 33.82 ± 0.72 of the S-phase cells and a 7.55 ± 1.34 of the G2/M-phase cells. In contrast, MCF-7 cells treated during 48 h with 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 apoptosisinducing agents has proved to be difficult.

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 123

Compound Cell cyclea Apoptosisb

 4.32 ± 0.02 58.07 ± 0.11 39.38 ± 0.98 2.10 ± 0.12 52.81 ± 1.05 11 10.6 ± 0.66 69.71 ± 1.50 23.73 ± 1.65 6.56 ± 0.17 58.29 ± 0.75 12 6.18 ± 1.70 62.85 ± 0.87 26.71 ± 1.25 10.43 ± 0.38 63.05 ± 0.26 13 8.97 ± 0.83 70.30 ± 0.32 23.67 ± 2.40 6.06 ± 2.72 76.22 ± 2.02

Data were taken from [117]. All experiments

Control 58.62 ± 0.74 33.82 ± 0.72 7.55 ± 1.34 0.22 ± 0.16

aDetermined by flow cytometry [12]. bApoptosis was determined using an Annexin V-based assay [12]. The data

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

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

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

IC50 (μM) G0/G1 S G2/M

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.

indicate the percentage of cells undergoing apoptosis in each sample. c

5-FUc

agents.

**and 5-FU** 


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

**Table 3.** 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.
