Antitumoural properties of benzannelated seven-membered 5-fluorouracilderivatives and related open analogues. Molecular markers for apoptosis

and cell cycle dysregulation

Antonio Espinosa a,*, Juan A. Marchal b, A. Aránega c, Miguel Á. Gallo a,Stefania Aiello d, Joaquín Campos a

a Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, c/ Campus de Cartuja, s/n, 18071 Granada, Spainb Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales y de la Salud, Paraje de las Lagunillas s/n, 23071 Jaén, Spain

c Departamento de Anatomía y Embriología Humana, Facultad de Medicina, Avenida de Madrid s/n, 18071 Granada, Spaind Dipartimento Farmacochimico, Tossicologico e Biologico, via Archirafi 32, 90123 Palermo, Italy

Received and revised 26 July 2004; accepted 29 December 2004

Abstract

Attention is increasingly being focussed on the cell cycle and apoptosis as potential targets for therapeutic intervention in cancer. Weprepared a series of bioisosteric benzannelated seven-membered 5-FU O,N-acetals to test them against the MCF-7 human breast cancer cellline. Benzo-fused seven-membered O,O-acetals or their acyclic analogues led to the expected 5-FU O,N-acetals (or aminals), in addition tosix- and 14-membered aminal structures and acyclic compounds. All the cyclic aminals provoked a G0/G1-phase cell cycle arrest, whereasFtorafur, a known prodrug of 5-FU, and 1-[2-(2-hydroxymethyl-4-nitrophenoxy)-1-methoxyethyl]-5-fluorouracil (11) induced an S-phasecell cycle arrest. Although breast cancer is most often treated with conventional cytotoxic agents it has proved difficult to induce apoptosis inbreast cancer cells, but improved clinical responses may be obtained by identifying therapies that are particularly effective in activatingapoptosis. 1-(2,3-Dihydrobenzoxepin-2-yl)-5-fluorouracil (5) may be particularly useful in stimulating apoptosis in breast cancer.© 2005 Elsevier SAS. All rights reserved.

Keywords: Antitumour drugs; Breast cancer; Cell cycle; Programmed cell death; Benzodioxepins

1. Introduction

The pathologies related to viral and/or neoplastic affec-tions are one of the primary causes of death in the world.Natural nucleosides have been the primary models for thedesign of the most important antitumoural and/or antiviralantimetabolites [1,2].

Breast cancer remains the most common cancer in womenand is the most frequent cause of cancer death [3]. In 1990,an estimated 178,904 new cases of breast cancer were reportedin the European Union—representing 28% of all female can-cers [3]. Over the past 20 years, the 5-year survival for patientswith breast cancer has increased by around 10%, largely as a

result of earlier diagnosis and better treatment [4]. Neverthe-less, 5-year survival rates for people with breast cancer rangebetween 60% and 70%, and many patients still die from thedisease [4].

Early detection and definitive surgical management are thefoundation of curative strategies in women with breast can-cer [5]. In addition, most patients with operable breast cancernow receive postoperative medical treatment in the form ofadjuvant chemotherapy, hormone manipulation or both [6].

Although 5-fluorouracil (5-FU, Fig. 1) was first intro-duced in 1957, it remains an essential part of the treatment ofa wide range of solid tumours. 5-FU has antitumour activityagainst epithelial malignancies arising in the gastrointestinaltract, breast, and also in the head and neck. Although thisantimetabolite is toxic, its efficacy makes it one of the mostwidely used agents against solid tumours [7]. One of the chal-lenges of cancer research is the development of prodrugs of

* Corresponding author. Departamento de Química Farmacéutica yOrgánica, Facultad de Farmacia, c/Campus de Cartuja, s/n, 18071 Gra-nada, Spain. Tel.: +34 958 243850; fax: +34 958 243845.

E-mail address: aespinos@ugr.es (A. Espinosa).

Il Farmaco 60 (2005) 91–97

http://france.elsevier.com/direct/FARMAC/

0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.doi:10.1016/j.farmac.2004.12.004

5-FU that diminish or circumvent some of its disadvantages:reduction in toxicity by avoiding certain routes of degrada-tion (prodrug not being a substrate for the enzymes of degra-dation) or by targeting the tumour site (prodrugs that liberatethe active principle selectively in tumour cells). The main ben-efit of 5-FU prodrugs is that of oral administration. They aredesigned to leave the gastrointestinal tract and subsequentlybe enzymatically converted into 5-FU in the liver or withinthe tumour itself in order to expose the tumour to 5-FU for alonger time but at lower concentrations than those observedafter an i. v. bolus, hence minimizing toxicity. Ftorafur (Fig. 1)is the first prodrug of 5-FU. Campos et al. [8] synthesized anovel class of 5-FU-containing acyclonucleosides that are ableto induce myogenic differentiation in rhabdomyosarcomacells, suggesting that these drugs might be useful for differ-entiation therapy in this type of tumour. Such a therapy is anattractive approach to cancer treatment, which assumes thatneoplastic transformation reveals the inability of a cell popu-lation to couple proliferation and differentiation signals.Induced differentiation modulates the cell programme bytransforming malignant cells into mature cells with no pro-liferative potential.

As part of their action on neoplastic cells, many antican-cer drugs activate apoptosis (programmed cell death). Apop-tosis may be a primary mechanism of antineoplastic agents[9]. Although breast cancer is most often treated with con-ventional cytotoxic agents it has proved difficult to induceapoptosis in breast cancer cells using these drugs [10].Improved clinical response may be obtained by identifyingtherapies that are particularly effective in activating apopto-sis and determining how those therapies may be modified toeffect maximum apoptosis induction. The cell cycle appara-tus and apoptosis have recently attracted the attention of

researchers intent on developing new types of anticancertherapy [11,12]. On the other hand, the MCF-7 human breastcancer cell line has been used as an excellent experimentalmodel to improve the efficacy of different therapies before itsuse in patients [13,14].

2. Results and discussion

2.1. Anticancer activities in the human breast cancerMCF-7 cell line of 5-FU O,N-acetals

Design, synthetic procedures and mechanistic aspects ofthe cyclic (Fig. 1) and acyclic (Fig. 2) 5-FU O,N-acetals havebeen recently reported [15,16].

The antiproliferative activities [17] and apoptosis induc-tion [18] in the MCF-7 human breast cancer cell line werefollowed in accordance with the protocols previously reported.

2.1.1. Antiproliferative activities of cyclic O,N-acetalsThe IC50 values of the 5-FU cyclic O,N-acetals are shown

in Table 1 (entries 4–10). The most active compounds are 2,3 and 7 (entries 5, 6 and 11). Compound 1 (entry 4) showsthe least antiproliferative activity (IC50 = 23 ± 0.88 µM) with-out considering 6. The lipophilicity in this structure has beenincreased by means of a fused benzene ring, and an unsatura-

Fig. 1.

Fig. 2.

92 A. Espinosa et al. / Il Farmaco 60 (2005) 91–97

tion has been introduced to give 5. An increase has beenobtained in its antiproliferative activity (IC50 = 14 ± 1.02 µM,entry 8). On comparing structures 5 and 2, it is worth empha-sizing that the bioisosteric change of carbon for oxygen andthe saturation of the double bond in compound 2 doubles theantiproliferative activity (IC50 = 7 ± 0.61 µM, entry 5). Theintroduction of a methoxy group into the benzene ring of 2provokes different influences on the antiproliferative activi-ties. Thus, the C-7 substitution produces an increase of theantiproliferative activity (3, IC50 = 4.5 ± 0.33 µM, entry 6),whilst if C-9 is the substituted position it gives rise to adecrease in the antiproliferative activity (4, IC50 = 22 ±0.93 µM, entry 7).

2.1.1.1. Apoptosis induction of cyclic O,N-acetals. Apopto-sis has been studied in terms of cancer development and treat-ment with attempts made to identify its role in chemothera-peutic agent-induced cytotoxicity.Apoptosis is a form of deathwith morphological properties including cell shrinkage, lossof cell–cell contact, chromatin condensation, intranucleoso-mal degradation of DNA, which are apparently different fromnecrosis. Apoptosis is an essential phenomenon for maintain-ing normal development and homeostasis [19], and also playsimportant roles to prevent the development of malignanttumours [20,21]. Cytotoxic agents often induce only a frac-tion of the cells to become apoptotic. To fully exploit apop-tosis as a mechanism of antineoplastic agent response, a largerproportion of cells needs to be recruited into apoptosis. Pacli-taxel (Taxol®), cyclophosphamide and cytosine arabinosideare the only commonly used cytotoxic agents shown to elicitapoptosis in breast cancer cells [22,23]. Quantitation of apo-ptotic cells was done by monitoring the binding of fluores-

cein isothiocyanate (FITC)-labelledannexinV(aphosphatidyl-serine-binding protein) to cells in response to our titlecompounds as described [18]. The apoptosis study shows thatcompounds 1, 4, 5 and 6, at their IC50 concentrations, pro-voke early apoptosis in the cells treated for 24 and 48 h. It isworth pointing out that compound 4 (entry 7) induces greaterapoptosis at 48 h (46.73%) than at 24 h (40.08%) and so doescompound 1 [48 h (53.92%) and 24 h (46.63%), entry 4]. Thecompounds that show the most important apoptotic indexesat 24 h are 5 (57.33%, entry 8) and 6 (54.33%, entry 9),whereas at 48 h are 1 (53.92%, entry 4) and 5 (51.37%, entry8). These compounds are more potent as apoptosis inductorsagainst the MCF-7 human breast cancer cells than paclitaxel(Taxol®), which induces programmed cell death of up to 43%of the cell population [24]. Accordingly, the early apoptoticinductions and the low IC50 values give rise to a significantantitumour activity.

2.1.1.2. Cell cycle distribution of cyclic 5-FU O,N-acetals.Cell cycle regulation has attracted a great deal of attention asa promising target for cancer research and treatment [25,26].The use of cell-cycle-specific treatment in cancer therapy hasgreatly benefited from the major advances that have beenrecently made in the identification of the molecular actorsregulating the cell cycle and from the better understanding ofthe connections between cell cycle and apoptosis. As moreand more “cell cycle drugs” are being discovered, their useas anticancer drugs is being extensively investigated [26]. Tostudy the mechanisms of the antitumour and antiproliferativeactivities of the compounds, the effects on the cell cycle dis-tribution were analyzed by flow cytometry. Control DMSO-treated cell cultures contained 68.39% G0/G1-phase cells,

Table 1Antiproliferative activities, cell cycle dysregulation, and apoptosis induction in the MCF-7 human breast cancer cell line after treatment for 24 and 48 h for thecompounds

Entry Compound IC50 (µM) a Cell Cycle 48 (h) b Apoptosis c

G0/G1 G2/M S 24 (h) 48 (h)1 Control 68.39 12.04 19.57 1.24 1.242 5-FU 2.75 58.07 2.10 39.38 56.75 52.813 Ftorafur 3 ± 0.11 45.62 0.00 54.38 52.20 58.064 1 23 ± 0.88 50.99 18.51 30.49 46.63 53.925 2 7 ± 0.61 74.41 15.77 9.82 8.45 12.176 3 4.5 ± 0.33 73.41 13.15 13.44 1.50 3.507 4 22 ± 0.93 71.76 10.08 18.16 40.08 46.738 5 14 ± 1.02 86.14 1.60 12.26 57.33 51.379 6 69 ± 2.31 68.61 9.60 21.79 54.33 35.4910 7 5.5 ± 0.58 82.48 5.13 12.40 14.37 19.0511 8 18.5 ± 0.95 67.18 4.67 28.16 59.90 40.2312 9 29 ± 1.63 62.72 1.59 35.69 33.35 37.8713 10 18 ± 0.85 71.01 28.99 0.00 44.36 50.6414 11 16 ± 1.18 51.45 20.66 27.88 42.24 36.3715 12 5.42 ± 0.26 46.92 2.84 50.24 40.73 48.2216 13 21 ± 1.02 67.32 9.40 23.28 41.15 37.81

a See Ref. [17].b Determined by flow cytometry: see Ref. [15].c Apoptosis was determined using an annexin V-based assay [18]. The data indicate the percentage of cells undergoing apoptosis in each sample. All expe-

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

93A. Espinosa et al. / Il Farmaco 60 (2005) 91–97

12.04% G2/M-phase cells and 19.57% S-phase cells. Cyclic5-FU O,N-acetals 2-7 (entries 5–10) provoke a G0/G1-phasecell cycle arrest whereas Ftorafur [1-(2-tetrahydrofuranyl)-5-fluorouracil], a known prodrug of 5-FU, induces a S-phasecell cycle arrest.

2.1.1.3. Molecular markers. After the study of the modifica-tions that the cyclic compounds provoke in the cell cycle wewere interested in explaining why such compounds halt orslow down the progression of the tumour cells through thecell cycle apparatus. The purpose of this research is to under-stand which are the molecular mechanisms responsible fortheir antitumour activities and then to find similarities anddifferences with the 5-FU behaviour. The studies have beenperformed using flow cytometry techniques and immunohis-tochemistry with specific antibodies for these markers. Veryrecently, we have reviewed the complex relationship that existsbetween growth, differentiation, neoplastic transformation,and the expression of genes and tumour suppressor genes [27].

2.1.1.3.1. Modification of the molecular markers caused bythe cyclic 5-FU O,N-acetals. Because of the fact that thecyclic O,N-acetals accumulate the cells in the G1-phase theexpression pattern of cyclin D1 was studied. This cyclin isone of the CDK activator subunits, specifically to CDK4,being responsible of the progression of the cell through theG1-phase. Compounds 2 and 7 gave rise to a spectacular inhi-bition of cyclin D1 up to its total disappearance. This fact didnot take place with 5-FU because the cyclin D1 level increasedin relation to those of the parental MCF-7 cells. On one hand,this would explain why these compounds accumulate the cellsin the G1-phase (on inhibiting cyclin D1 the cell cannotprogress to the S-phase) and on the other, they show a differ-ent mechanism of action from the one shown by 5-FU: inshort, they are not prodrugs. 5-FU increases the cyclin D1 pro-duction so that cells pass in most cases toward the S-phasewhere they are held back. It has been recently reported [28]that cyclin D1 works as an active “switch” in the progressionof the cellular cycle and that high levels of cyclin D1 pro-mote the entry of the cell into the S-phase. Moreover, com-pound 7 increases the expression of proteins p21 or p27 evenas far as double that of the control. These proteins belong tothe family INK2 of the CDK inhibiting proteins that work byhindering the association and activation of cyclins with theircomplexes [29] and hence they halt cells in the G1 and G2/Mphases. Compounds also affect the cdc2 activity that, regu-lated by their corresponding cyclins A or B, is essential forthe entrance into mitosis during the cellular cycle [30]. Allcompounds (2, 3 and 7), with the exception of 5 and 5-FU,significantly decrease the cdc2 activity. cdc2 is needed dur-ing the cellular cycle in the final phase of G1, in the controlpoint named “start” to be committed to the mitotic cycle. Thisis because at the end of G2 (at the beginning of the mitosis)[31], the inhibition of cdc2 by the O,N-acetals implies thehalting of the cycle in G1 and the non-entrance of the tumourcells in mitosis. The increase of the cdc2 expression caused

by 5-FU is due to the fact that this higher activity is necessaryfor the cells to pass rapidly to the S-phase, where cells arestopped by this fluoropyrimidine. Finally, compound 5 alsosignificantly increases the cdc2 levels, which may be becauseits premature activation is one of the requirements for apop-tosis [32]; in fact this compound is the one that induces ahigher proportion of programmed cellular death in the MCF-7 treated cells.

2.1.1.3.2. Apoptosis markers. Since the synthesized com-pounds induce very important apoptosis, we have carried outstudies of the expression of some of the genes that intervenein this phenomenon, among which p53 and the family bcl-2 are outstanding. p53 is a protein with a molar weight of53-kilodalton (kDa) and was discovered in 1979. p53 gene islocated on chromosome 17 (p13) and had been recognized asan oncogene. However, it has been demonstrated that onlymutant-type p53 gene allows abnormal cell growth, and thatwild-type p53 gene acts as a tumour suppressor [33]. Thetumour suppressor gene p53 protects the integrity of thegenome so that if the DNA of the cell is damaged by an agent,an overexpression of this gene is produced inducing the stop-ping in G1 for the repair of the damage. If this is not possible,p53 induces the cell to enter in apoptosis [34]. Therefore, ifp53 gene is mutated and the normal function of p53 is dam-aged, apoptosis is difficult to occur and the proliferation oftumour cells is facilitated [35]. On the other hand, the mem-bers of the family of proteins Bcl-2 work as regulators ofapoptosis, Bcl-2, and Bcl-XL protecting against apoptosis.Bax, Bak, and Bad induce such a phenomenon [36].

The treatment of the MCF-7 cells (wild-type p53) withthese compounds provoked in general an increase in the pro-tein expression of p53, mainly for 5-FU and 5, and a markeddecrease of the levels of bcl-2 for all of them. These datashow that p53 activity is restored with the compounds, allow-ing the entrance of the tumour cells in apoptosis, which per-mits their elimination by this mechanism. In the same waybcl-2 inhibition facilitates the entrance of cells into the pro-grammed cell death.

2.1.1.4. Morphological changes in MCF-7 treated cells. Allthe cyclic O,N-acetals show a similar behaviour and in con-sequence molecule 5 was selected to explain the morphologi-cal modifications produced in the treated cells. Light micro-scope observations showed modifications in the morphologyof MCF-7 cells after treatment with the IC50 values of com-pound 5 at 6, 24 and 48 h DMSO-treated control MCF-7 cells grew as irregular confluent aggregates with a roundedand polygonal cell morphology. At 6 and 24 h of treatment,all compounds induced the appearance of polygonal cells thatbegan to shrink and become spherical in shape. The cellshrinkage increased progressively and a significant propor-tion of the cells became dislodged from their flasks 48 h aftertreatment [36]. In the cell cultures treated with 5-FU a higheramount of cells lost adherence to the flask and were detached(data not shown).

94 A. Espinosa et al. / Il Farmaco 60 (2005) 91–97

2.1.2. Antiproliferative activities and apoptosis inductioncaused by acyclic 5-FU O,N-acetals

The IC50 values of 5-FU Acyclic O,N-acetals 8-13 areshown in the Table 1 (entries 11–16). The most active com-pound is 12 (5.42 ± 0.26 µM, entry 15) with an antiprolifera-tive activity in the same order as that of Ftorafur (3 ± 0.11 µM,entry 3).

It is worth pointing out that in response to 8, the percent-age of apoptotic cells increased from 1.24% in control cellsto a maximum of 59.90% apoptotic cells (24 h) at a concen-tration equal to its IC50 against the MCF-7 cell line (entry11). This is a remarkable property because it has been diffi-cultto demonstrate apoptosis in MCF-7 breast cancer cells byknown apoptosis-inducing agents. Only few cytotoxic agentsact preferentially through an apoptotic mechanism in humanbreast cancer cells [10,18,24].

2.1.2.1. Cell cycle distribution and modifications inthe regulatory molecules of the cell cycle caused bythe acyclic 5-FU O,N-acetals. In contrast to cyclic O,N-acetals, MCF-7 cells treated during 48 h with the IC50 con-centrations of 8–13 (entries 11–16) showed important differ-ences in cell cycle progression compared with DMSO-treated control cells. Ftorafur treatment showed a decrease ofthe G0/G1-phase cells and a corresponding accumulation ofS-phase cells (45.62% G0/G1-phase cells and 54.38% S-phasecells). Moreover, there was an almost total disappearance inthe G2/M population of the cells treated with this drug. Ingeneral the cell cycle regulatory activities for the newly syn-thesized compounds can be divided into the following threegroups: (a) the exposure of MCF-7 cells during 48 h to deriva-tive 7, caused strong differences in the cell cycle progressionin comparison with Ftorafur: the breast cancer cells treatedwith the IC50 doses showed a significant accumulation in theG0/G1-phase, up to 82.48% of the cells (entry 10), mainly atthe expense of the G2/M-phase population that decreased to apercentage of 5.13% of the cells; (b) compounds 10 and 11accumulated the cancerous cells in the G2/M-phase (entries13 and 14), in the former compound at the expense of theS-phase cells, and (c) compound 12 induced a S-phase cellcycle arrest (50.24%) in a similar percentage to that causedby Ftorafur (54.28%, entry 15).

The activity of the acyclic O,N-acetals analyzed (10 and12) provokes a series of modifications in the regulatory mol-ecules of the cellular cycle, including a drastic decrease inthe expression of cyclin D1 with an increment of the activityof the tumour supressor gene p21. On one hand, all theseevents imply the halting of the cells in G1 with the conse-quent blockade of the activity of cyclin complexes D/CDK4[37] and on the other, a specific action of these compoundsdirected towards the inhibition of the formation or the destruc-tion of the existing cyclin D1. These compounds also modu-late the expression of cdc2 with a significant decrease in theactivity of this kinase up to levels practically undetectable forthe MCF-7 cells treated with 10. This fact prevents the bind-ing to their corresponding cyclins and the pRb phosphoryla-

tion, giving rise to the non-progression of cells towards divi-sion [38].

2.1.2.2. Apoptosis markers. In a different way the acyclic5-FU O,N-acetals 10 and 12 modulate the expression of thesemolecules. Thus, 10 markedly decreases the bcl-2 expressionand not so much that of p53, which would indicate an apop-tosis induction non-dependent of p53 as has been demon-strated for other antitumour agents [39]. Otherwise, 12 doessignificantly increase the expression levels of p53 and doesnot modify those of bcl-2, inducing a p53-dependent apopto-sis in the treated cells.

2.2. Toxicity of 5-FU and the novel compounds in mice

Once a novel compound with potent antiproliferative activ-ity was identified using in vitro systems, the next step was toestablish appropriate in vivo toxicity. All compounds wereadministered intravenously twice a week (with a 50 mg/kg)for 6 weeks. 5-FU administration resulted in an excessiveweight loss and a death rate of 50% after 3 weeks. In con-trast, treatment with the new compounds resulted in null tox-icity, allowing drug administration for 6 weeks.

It was particularly interesting to find that the antiprolifera-tive activity of the new compounds was not accompanied bya systemic toxicity as happened with the 5-FU. At a dosehigher than 30 mg/kg body weight, 5-FU is very toxic in mice[40]. These effects are promising, because further improve-ment in the antiproliferative activity of the molecules isallowed without necessarily an increase in adverse effects.

3. Conclusions

In short, this study has investigated the antitumour activ-ity, the cell cycle arrest and apoptotic properties of novel lipo-philic benzene-fused seven-membered 5-FU analogues onhuman breast cancer cells in vitro. Our results demonstrateda dose-dependent activity against MCF-7 and IC50 values inthe low micromolar range. The novel cyclic O,N-acetals slowdown cell cycle progression, as indicated by the decrease inthe %S and the increase in the %G0/G1. Moreover, because alarger proportion of cells (>60%) is being recruited into earlyapoptosis and DNA strand breaks are produced in the MCF-7 cells [36], a cell line where the induction of DNA fragmen-tation is very difficult [29], we suggest that these novel 5-FUderivatives can be considered as specific apoptotic inducers.

On one hand, all these data show the selective activity ofthese new compounds over the regulatory molecules of theG1-phase of the cell cycle, as also the modulator action onp53 and bcl-2 for the induction of apoptosis. Therefore, suchcompounds may be considered as drugs with their own entityand antitumour activity independent from that of 5-FU. Onthe other, these experimental findings provide evidence of spe-cific anticancer activity of these new substances having 5-FUmoiety and warrant further evaluation in in vivo models ofbreast cancer for future clinical applications.

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Acknowledgements

This study was supported by the Instituto de Salud CarlosIII (Fondo de Investigación Sanitaria) through project no.PI03225.

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