**3.1. Benzo-fused seven-membered derivatives linked to 5-fluorouracil (5-FU)**

We have reported the synthesis and anticancer activities of compounds **1**-**5** [16] and *trans*-**6** [17] (Figure 1). In all cases, the linkage between the 5-FU moiety and the seven-membered ring was carried out through its *N*-1 atom. The structural nature of **5** implies that this compound cannot be considered as a 5-FU prodrug and it was suspected that the remaining compounds (**1**-**6**) would not be 5-FU prodrugs.

**Figure 1.** Several 5-FU derivatives showing interesting anti-tumour activities.

The IC50 values of the 5-FU cyclic *O,N*-acetals are shown in Table 1 (entries 4-9). The most active compounds are **1**, **2** and **6** (entries 4, 5 and 9). On comparing structures **4** and **1**, it is worth emphasizing that the bioisosteric change of carbon for oxygen and the saturation of the double bond in compound **1** increases the anti-proliferative activity two fold in (IC50 = 7.00 ± 0.61 μM, entry 4). The introduction of a methoxy group into the benzene ring of **1** provokes different influences on the anti-proliferative activities. Thus, the C-7 substitution produces an increase of the anti-proliferative activity (**2**, IC50 = 4.50 ± 0.33 μM, entry 5), whilst if C-9 is the substituted position it gives rise to a decrease in the anti-proliferative activity of **3** (IC50 = 22.0 ± 0.93 μM, entry 6).

Apoptosis has been studied in terms of cancer development and treatment with attempts made to identify its role in chemotherapeutic agent-induced cytotoxicity. Cytotoxic agents often induce only a fraction of the cells to become apoptotic. To fully exploit apoptosis as a mechanism of anti-neoplastic agent response, a larger proportion of cells needs to be recruited into apoptosis. Paclitaxel (Taxol®), cyclophosphamide and cytosine arabinoside

the corresponding original references.

compounds (**1**-**6**) would not be 5-FU prodrugs.

5-FU O

Ftorafur

R1

O

5FU

OH

activity of **3** (IC50 = 22.0 ± 0.93 μM, entry 6).

We will concentrate in this part of the review on the evolution of the chemical structures and on the biological properties, whilst the chemical syntheses will be referred to through

**3.1. Benzo-fused seven-membered derivatives linked to 5-fluorouracil (5-FU)** 

O

**5 6**

The IC50 values of the 5-FU cyclic *O,N*-acetals are shown in Table 1 (entries 4-9). The most active compounds are **1**, **2** and **6** (entries 4, 5 and 9). On comparing structures **4** and **1**, it is worth emphasizing that the bioisosteric change of carbon for oxygen and the saturation of the double bond in compound **1** increases the anti-proliferative activity two fold in (IC50 = 7.00 ± 0.61 μM, entry 4). The introduction of a methoxy group into the benzene ring of **1** provokes different influences on the anti-proliferative activities. Thus, the C-7 substitution produces an increase of the anti-proliferative activity (**2**, IC50 = 4.50 ± 0.33 μM, entry 5), whilst if C-9 is the substituted position it gives rise to a decrease in the anti-proliferative

Apoptosis has been studied in terms of cancer development and treatment with attempts made to identify its role in chemotherapeutic agent-induced cytotoxicity. Cytotoxic agents often induce only a fraction of the cells to become apoptotic. To fully exploit apoptosis as a mechanism of anti-neoplastic agent response, a larger proportion of cells needs to be recruited into apoptosis. Paclitaxel (Taxol®), cyclophosphamide and cytosine arabinoside

5-FU

O

**4**

O

O

O O

5FU 5-FU

5-FU

O

R2

**1** R1 = R2 = H R3 = F **2** R1 = OMe; R2 = H R3 = F **3** R1 = H; R2 = OMe R3 = F

**Figure 1.** Several 5-FU derivatives showing interesting anti-tumour activities.

We have reported the synthesis and anticancer activities of compounds **1**-**5** [16] and *trans*-**6** [17] (Figure 1). In all cases, the linkage between the 5-FU moiety and the seven-membered ring was carried out through its *N*-1 atom. The structural nature of **5** implies that this compound cannot be considered as a 5-FU prodrug and it was suspected that the remaining are the only commonly used cytotoxic agents shown to elicit apoptosis in breast cancer cells [111,112]. Quantitation of apoptotic cells was done by monitoring the binding of fluorescein isothiocyanate (FITC)-labelled annexin V (a phosphatidylserine-binding protein) to cells in response to our title compounds as described [113]. The apoptosis study shows that **3**, **4** and **5**, at their IC50 concentrations, provoke early apoptosis in the cells treated for 24 and 48 h. It is worth pointing out that **3** (entry 6) induces greater apoptosis at 48 h (46.73%) than at 24 h (40.08%). The compounds that show the most important apoptotic indexes at 24 h are **4** (57.33%, entry 7) and **5** (54.33%, entry 8), whereas at 48 h is **4** (51.37%, entry 7). These compounds are more potent as apoptosis inductors against the MCF-7 human breast cancer cells than paclitaxel (Taxol®), which induced programmed cell death of up to 43% of the cell population [114]. Accordingly, the early apoptotic inductions and the low IC50 values give rise to a significant anti-tumour activity.

Since the synthesized compounds induce very important apoptosis, we have carried out studies of the expression of some of the genes that intervene in this phenomenon, among which p53 and the family bcl-2 are outstanding. The tumour suppressor gene p53 protects the integrity of the genome so that if the DNA of the cell is damaged by an agent, an overexpression of it is produced inducing the stopping in G1 for the repair of the damage, or if this is not possible, enter apoptosis [115]. On the other hand, the members of the family of proteins Bcl-2 work as regulators of apoptosis, Bcl-2 and Bcl-XL protecting against apoptosis. Bax, Bak and Bad induce such a phenomenon [116]. The treatment of the MCF-7 cells (wild-type p53) with these compounds provoked in general an increase in the protein expression of p53, mainly for 5-FU and **4**, and a marked decrease of the levels of bcl-2 for all of them. These data show that p53 activity is restored with the compounds, allowing the entrance of the tumour cells in apoptosis, which permits their elimination by this mechanism. In the same way bcl-2 inhibition facilitates the entrance of cells into the programmed cell death.


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 1.** 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.
