Enantiomerically Pure Substituted Benzo-Fused Heterocycles — A New Class of Anti-Breast Cancer Agents

The starting material ( RS )-2,3-dihydro-2 H -1,4-benzoxathiin-2-methanol ( 35 ) was prepared as previously reported (Díaz-Gavilán et al., 2008a) whilst ( RS )-(2,3-dihydro-1,4-benzodioxin-2-yl)methanol ( 36 ) was synthesized by the reaction of cathecol with epichlorohydrin in NaOH and water (Díaz-Gavilán et al., 2007).


Introduction
With more than 10 million new cases each year cancer is at present one of the most devastating diseases worldwide with an immense affliction burden not only for affected individuals, their relatives and friends but also representing heavy challenges to health care systems (Steward & Kleihues, 2003). In the year 2000, cancer was responsible for 12% of nearly 56 million deaths worldwide and in many countries this percentage is even higher with more than a quarter of deaths attributable to cancer. Moreover, it is expected that cancer rates further increase by 50% to 15 million new cases in the year 2020, mainly due to steadily ageing populations in both developed and developing countries (Fresco et al., 2010).
In recent years, many studies have shown an association between cell cycle regulation and cancer inasmuch as the cell cycle inhibitors are being considered as a weapon for the management of cancer (Hajduch et al., 1999). Ultimately a great level of interest has arisen in the G 0 /G 1 phase regulatory molecules such as cyclin D1, CdkIs, and p53 as potential therapeutic targets in diseases where control of inappropriate cellular proliferation would be a therapeutic benefit (Sherr, 1996).
Apoptosis is an essential physiological process throughout the life of multi-cellular organisms important in the development and in the maintenance of tissue homeostasis. Apoptosis is involved in controlling the cell number and proliferation during embryogenesis, deletion of activated lymphocytes at the end of the immune response, elimination of self-reactive lymphocytes, in controlled destruction of damaged, aged, infected, transformed, and other harmful cells (Nagata, 1997;Testa, 2004). Zivny et al. have recently reviewed the apoptotic pathways, molecules involved in the cross-talk between individual apoptosis pathways, apoptosis regulation as well as mechanisms of action of conventional anticancer drugs and new promising agents, which trigger directly or indirectly apoptosis of hematologic cancer cells (Zivny et al., 2010).
We report herein the synthesis and antiproliferative activities of purine derivatives 1-11 (Chart 2) against the cancerous MCF-7 and MDA-MB-231 human breast cancer cell lines and the corresponding normal one (MCF-10A) to define the in vitro therapeutic index (TI) as a measure of the selectivity. From a structural point of view, the compounds studied differ from others previously reported (Díaz-Gavilán et al., 2008b) by the addition of an extra halogen or PhSgroups on the purine ring. Finally the most active racemic compound (1) was resolved and the antiproliferative activity of its enantiomers was measured (López-Cara et al., 2011). Modern drug discovery relies on high speed organic synthesis. Microwave-assisted organic synthesis is proving to be instrumental for the rapid synthesis of compounds with new and improved biological activities (Al-Obeidi et al., 2003; Kappe & Dallinger, 2006). We previously investigated the Vorbrüggen condensation in microwave-assisted organic synthesis (Conejo-García et al., 2008). Microwave advantage is chiefly the quick access to the target molecules as well as the better yield obtained in the only isomer formed making the purification processes much easier.

The chiral switch from the benzo-fused seven-membered O,N-acetal (1)
Preparation of the O,N-acetals 1-4 was achieved by the microwave-assisted Vorbrüggen onepot condensation of the cyclic acetals 12 and 13 (Díaz-Gavilán et al., 2004) and the commercially available purine bases 6-chloro-, 6-bromo-and 2,6-dichloro-purines, using chlorotrimethylsilane (TMSCl), 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and tin(IV) chloride as the Lewis acid in anhydrous acetonitrile. The reaction mixture was microwave-irradiated at a temperature of 140 °C or 160 °C for 5 min (Scheme 1 Compounds 14 and 15 were isolated from the reactions and the acyclic O,N-acetal 10 was also obtained in the synthesis of 1. Traces of the N-7' regioisomer 11 were detected in the synthesis of 2. The following modifications were carried out on 2: a) selective nucleophilic substitution of the chorine atom at position 6 of the purine ring using NaI and trifluoroacetic acid (TFA) to yield 5; b) reduction of the nitro group with SnCl 2 to give rise to 6 and 7; and c) the treatment with the PhSH to produce 8 and 9.
Compounds 14 and 15 were obtained along with the cyclic and acyclic O,N-acetals in the reaction of purines with 12 and 13, respectively. Their importance lies in the information that they provide of the mechanism of the reaction with purines (López-Cara et al., 2011).

Resolution of (RS)-1 into its eantiomers: Biological activities
The issue of drug chirality is now a major theme in the design and development of new drugs, underpinned by a new understanding of the role of molecular recognition in many pharmacologically relevant events. In general, three methods are utilized for the production of a chiral drug: the chiral pool, separation of racemates, and asymmetric synthesis. Although the use of chiral drugs predates modern medicine, only since the 1980's has there been a significant increase in the development of chiral pharmaceutical drugs. An important commercial reason is that as patents on racemic drugs expire, pharmaceutical companies have the opportunity to extend patent coverage through development of the chiral switch enantiomers with desired bioactivity (Núñez et al., 2009).
(RS)-9-[1-(p-Nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-2,6-dichloro-9H-purine (1) Table 1 shows the antiproliferative activity (IC 50 values) for 1-11 and 5-fluorouracil (5-FU). All the compounds were first assayed as antiproliferative agents against the human breast adenocarcinoma cell line MCF-7 (p53 wild-type and ras mutated). Compounds (1, 2, 5-7, and 10, 11) were selected to be further assayed on the human breast cancer cell line MDA-MB-231, which has high levels of mutant p53, the most commonly mutated gene in human cancer. Additionally, we used a non-cancerous human mammary epithelial cell line (MCF-10A), in order to study the therapeutic index against breast cancer. It must be pointed out that from the twenty IC 50 values against the two cancerous cell lines, the majority of the IC 50 values were below 1 μM. As shown in Table 1, all the compounds were more active as anti-proliferative agents against MDA-MB-231 than against the MCF-7 human breast cancer cell line, except for the acyclic derivative 10, whose anti-proliferative effect remains the same in both cancer cell lines. The  When the homochiral forms were analyzed we found differences in the IC 50 values between (S)-1 and (R)-1 enantiomers, although no differences in activity were found between the two enantiomers against the MDA-MB-231 cell line. However both enantiomers present higher anti-proliferative activity than the racemic compound showing the greatest differences against MCF-7 cells. Enantiomer (S)-1 shows higher anti-tumour activity, up to twice that of (R)-1 in the MCF-7 cell line (Table 3). Studies with other compounds showed similar results with more potency in cytotoxicity in an enantiomer in comparison with the racemate. This enantioselective cytotoxicity indicates that the enantiomers of some chiral drugs may differ both quantitatively and qualitatively in their biological activity ( Table 3. Anti-proliferative activities of (RS)-1 and its enantiomers against the cancerous cell lines MCF-7 and MDA-MB-231.
Once the anti-tumour activity of compounds was determined against the different breast cell lines, we carried out a selection between those that showed a great cytotoxic effect against MCF-7, including (R)-1 and (S)-1, in order to determine their influence on the several cell cycle phases. In this study we have included drugs used in clinic against breast cancer, such 5-FU and paclitaxel, with a known mechanism of action at the level of cell cycle.
In order to analyze if the anti-tumour effects of the drugs involve changes in cell cycle distribution, the non-tumour cell line MCF-10A and the breast cancer cell lines MCF-7 and MDA-MB-231 were treated with the compounds during 48 hours and then analyzed by flow cytometry. The non-accumulation in a specific phase was detected during treatment with the drugs in most of the cell lines analyzed in comparison with control-DMSO-treated cells. Only the (R)-1 enantiomer was able to induce in MDA-MB-231 cells an accumulation in both G 0 /G 1 and G 2 /M phases with the consequently significant decreased in the S phase. Also an accumulation in the phase G 2 /M was detected in MCF-7-5 treated cells. Treatment with 5-FU and paclitaxel, as has been described previously (Grem et al., 1999), induced accumulation in the S or G 2 /M phases depending on the cell line analyzed. Similar data were obtained when cell lines were treated for 24 hours with 0.5 mM mimosine to synchronize the cells in the G 1 /S phase (data not shown). These results indicate that compounds inhibited all phases of the cell cycle, probably through the inhibition of protein synthesis as has been proved with other anti-tumour drugs (Duncan et al., 2009).
Finally, to determine if the observed growth inhibition was due to apoptosis, both flow cytometry and confocal microscopy studies were carried out. Cells were treated with the IC 50  The effects of compounds on the pattern of cell death were also confirmed by confocal microscopy after staining with FITC-conjugated annexin V and the nuclear non-vital stain PI. MCF-7 cells treated with compounds showed several staining patterns. Some cells displayed an intense FITC staining located at the plasma membrane and a nucleus with intensely PIlabelled marginated chromatin, suggesting that they were in the course of apoptosis. Other cells showed a peculiar staining pattern, because they exhibited nuclei with the same features observed in true apoptotic cells and, at the same time, cytoplasm homogeneously stained for annexin V. In fact, the FITC staining was located not only at the cell surface, but also within the cytoplasm. Therefore, these cells were considered as aponecrotic cells as has been previously established (Formigli et al., 2002). In addition, patches of localised partially condensed chromatin were found in other cells abutted along the inner part of the nuclear membrane. In the control cultures, most of the cells turned out to be negative for both staining except for some dying cells with the staining features of apoptosis (data not shown). The present data support the effect of the compounds in some
The anti-carcinogenic potential of the target molecules is reported against the MCF-7 human breast cancer cell line (Table 4). In general, (RS)-9-(2,3-dihydro-1,4-benzoxathiin-2-ylmethyl)-9H-purines 27-29 (series A) show a better activity than their isosteres (RS)-9-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-9H-purines 31-33 (series B  In recent years, many studies have shown an association between cell cycle regulation and cancer inasmuch as the cell cycle inhibitors are being considered as a weapon for the management of cancer (Hajduch et al., 1999). To study the mechanisms of the anti-tumour activity of the compounds (27-29 and 32), the effects on the cell cycle distribution were analysed by flow cytometry ( Apoptosis was determined using an Annexin V-based assay . The data indicate the percentage of cells undergoing apoptosis in each sample. c All experiments were conducted in duplicate and gave similar results. The data are means ± SEM of three independent determinations.   (Table 5). Apoptosis is a major form of cell death characterized by changes in signalling pathways that lead to the recruitment and activation of caspases, a family of cysteine-containing, aspartate-specific proteases. Caspases exist as inactive proenzymes in cells, and are activated through their processing into two subunits in response to apoptotic stimulation. Activated caspases cleave a variety of important cellular proteins, other caspases, and Bcl-2 family members, leading to a commitment to cell death. Caspase-9 is involved in one of the relatively well-characterized caspase cascades. It is triggered by cytochrome C release from the mitochondria, which promotes the activation of caspase-9 by forming a complex with Apaf-1 in the presence of dATP. Once activated, caspase-9 initiates a caspase cascade that finally induces cell death (Altieri, 2003). Western blot assays showed that compounds 27-29 induced activation of caspase 9 at late times (16 h and 36 h of treatment) similarly to paclitaxel used as control compound. These data confirm that levels of apoptosis showed by annexin V assays that are dependent of intrinsic pathway of cell death. p53 was not activated by the compounds which indicate that apoptosis was induced in a p53 independent manner (Conejo-García et al., 2011).

Different apoptosis modulation in breast cancer cells of enantiomers of benzo-fused six-membered heterocycles linked to purines
The intrinsically chiral and non-racemic nature of the living world often results in its different interactions with the enantiomers of a given substance. If this substance is a drug, it might well be that only one of the two isomers is capable of exerting the desired therapeutic effect. The other may be inert, harmful or responsible for possibly undesirable side effects.    The following can be stated from Tables 6 and 7: a. In the MCF-7 cell line, compounds are more potent as programmed cell-death inducers than in SKBR-3, and more specifically, (R)-29 and (S)-18 are the more effective apoptotic inducers (77% and 60% at their IC 50 , respectively) in the MCF-7 cell line.

García
b. In the SKBR-3 cell line the best apoptotic values are observed at their 3 × IC 50 concentrations.
Previous works scarcely reports a different pattern in apoptosis levels between enantiomers. An exception is D-( _ )-lentiginosine, the non-natural enantiomer of the iminosugar indolizidine alkaloid that acts as an apoptosis inducer on different tumour cells in contrast to its natural enantiomer (Macchi et al., 2010). All homochiral compounds included in this study show a different apoptosis effect between the two enantiomers. Apoptotic defects in cancer cells are the primary obstacle that limits the therapeutic efficacy of anticancer agents, and hence the development of novel agents targeting novel canonical and non-canonical programmed cell death pathways has become an imperative mission for clinical research (Cummings et al., 2004). Compounds 27-29, and 16-18 induce strong levels of cell death measured by citotoxicity analysis and by phosphatidylserine externalization (Annexin V binding) (Tables 6 and 7) even in the MCF-7 breast cancer cells that have shown deficiency in the caspase-activation mechanisms (Kagawa et al., 2001).
Whereas compound (S)-27 activates the canonical intrinsic caspase-8/caspase-3 apoptotic pathway on the MCF-7 cell line, compound (RS)-29 induces caspase-2 activation. However, a strong apoptosis induction is also detected in the rest of the compounds analysed. The caspase-independent apoptosis in cells exposed to different drugs with diverse cellular effects has been previously described (Macchi et al., 2010). While caspase-2 activation could induce cell death through cytochrome c/mitochondria damage (Robertson et al., 2002), non-caspase-mediated increase in phosphatidylserine externalization can occur in response to high intracellular Ca 2+ levels that alters scramblase and translocase (Vanags et al., 1996;26, Kagan et al., 2000). Additionally, non-caspase proteases may activate and cleave the cytoskeleton proteins attached to phospholipids, including focal adhesion kinase and the actin-capping protein αadducin (van de Water, 1999). To further confirm the involvement of caspases, including caspase-3, in the apoptosis induced by the most apoptotic compounds in the caspase-3 wild type SKBR-3 cell line, cells were pre-treated with the pan-caspase inhibitor z-VAD-fmk for

Conclusion
Cancer continues to be a major health problem in developing as well as undeveloped countries. Although major advances have been made in the chemotherapeutic management of some patients, the continued commitment to the laborious task of discovering new anticancer agents remains critically important, in the course of identifying various chemical substances, which may serve as leads for designing novel anti-tumour agents.
The ever-increasing use of asymmetric syntheses over many years has been manifested by the biological importance of enantiomerically pure single compound entity factors and further has been strongly guided by drug regulatory bodies because of strict rules and regulations about single isomers. A contributing factor to this effect has been, and continues to be the development of new, novel and efficient methods for accessing single isomers. In general, the binomial enantiomers → different biological activities and in particular, enantiomers → different antiproliferative activities are rarely known, in spite of their great importance. It seems that in the future this topic will receive increasing attention and will help better understanding of the molecular recognition between drugs and biological targets.