Stereoselective Synthesis and Anticancer Activity of 2,6-Disubstituted trans-3-Methylidenetetrahydropyran-4-ones

In this report, we present efficient and stereoselective syntheses of 2,6-disubstituted trans-3-methylidenetetrahydropyran-4-ones and 2-(4-methoxyphenyl)-5-methylidenetetrahydropyran-4-one that significantly broaden the spectrum of the available methylidenetetrahydropyran-4-ones with various substitution patterns. Target compounds were obtained using Horner–Wadsworth–Emmons methodology for the introduction of methylidene group onto the pyranone ring. 3-Diethoxyphosphoryltetrahydropyran-4-ones, which were key intermediates in this synthesis, were prepared by fully or highly stereoselective addition of Gilman or Grignard reagents to 3-diethoxyphosphoryldihydropyran-4-ones. Addition occurred preferentially by axial attack of the Michael donors on the dihydropyranone ring. Relative configurations and conformations of the obtained adducts were assigned using a detailed analysis of the NMR spectra. The obtained methylidenepyran-4-ones were evaluated for cytotoxic activity against two cancer cell lines (HL-60 and MCF-7). 2,6-Disubstituted 3-methylidenetetrahydropyran-4-ones with isopropyl and phenyl substituents in position 2 were more cytotoxic than analogs with n-butyl substituent. Two of the most cytotoxic analogs were then selected for further investigation on the HL-60 cell line. Both analogs induced morphological changes characteristic of apoptosis in cancer cells, significantly inhibited proliferation and induced apoptotic cell death. Both compounds also generated DNA damage, and one of the analogs arrested the cell cycle of HL-60 cells in the G2/M phase. In addition, both analogs were able to inhibit the activity of topoisomerase IIα. Based on these findings, the investigated analogs may be further optimized for the development of new and effective topoisomerase II inhibitors.


Introduction
The tetrahydropyran skeleton is present in a great number of natural products, which show various important biological properties, including anticancer activity. This activity depends on many structural and stereochemical features, and one of the most important is the substitution pattern of the tetrahydropyran ring. In this respect, many compounds containing a trans-2,6-disubstituted tetrahydropyrane moiety show significant cytotoxic activity. Representative examples are irciniastatins A and B 1 isolated from the Indo-Pacific marine sponge Ircinia ramosoa, which are powerful murine and human cancer cell growth inhibitors [1]; and aspergillide C 2 isolated from marine-derived fungus Aspergillus ostianus strain 01F313, which showed cytotoxic activity against mouse lymphocytic leukemia cells (L1210) [2] (Figure 1). Another important feature, which can strongly influence cytotoxic activity of tetrahydropyranes is the presence of exo-methylidene bond vicinal to the carbonyl group. This moiety is a strong Michael acceptor and can react with various bionucleophilesin particular, with mercapto groups in cysteine residues of enzymes or other functional proteins. The exo-methylidene motif is characteristic of a large group of natural products, including sesquiterpene lactones (e.g., vernolepin 3) and is believed to be the reason for their strong and often very specific anticancer properties [3][4][5][6]. Several natural compounds containing the 2,6-disubstituted-3-methylidenetetrahydropyran-4-one skeleton are known. Representative examples are norperovskatone 4, recently isolated from Perovskia atriplicifolia, which possesses remarkable anti-hepatitis B virus (HBV) activity [7] or (+)-okilactomycin 5 [8] which exhibits in vitro cytotoxicity against a number of human cancer cell lines, including lymphoid leukemia L1210 and leukemia P388, and in vivo activity against Ehrlich ascites carcinoma. Very recently, we reported on the synthesis and anticancer properties of a series of 2,2,6-trisubstituted 5-methylidenetetrahydropyran-4-ones (6) [9]. The analysis of the structure-activity relationship of the obtained compounds has revealed that cytotoxicity strongly depends on the nature of the substituent in position 6 (vicinal to the methylidene bond)-butyl and particularly iso-propyl groups being the best. The nature of substituents in position 2 is less important, although, in general, aromatic substituents are more advantageous for cancer growth inhibition than non-aromatic ones. One of the obtained compounds, 6-isopropyl-2,2-dimethyl-5-methylidenetetrahydropyran-4-one, showed very high cytotoxic activity against HL-60 human leukemia cells, induced apoptosis of these cells and caused the arrest of the cell cycle in the G2/M phase. Encouraged by these results, in this paper we present a simple, effective and stereoselective synthesis of 2,6-disubstituted trans-3-methylidenetetrahydropyran-4-ones 13a-o, which significantly broadens the spectrum of the available methylidenetetrahydropyran-4-ones with various substitution patterns. We applied synthetic methodology which proved its effectiveness in the synthesis of 5-methylidenetetrahydropyran-4-ones 6 and used Horner-Wadsworth-Emmons reagents for the olefination of formaldehyde to introduce an exo-methylidene group onto the tetrahydropyranone ring. In designing substituents for the new methylidenetetrahydropyran-4-one analogs, we turned special attention to these, which proved their effectiveness in enhancing the cytotoxicity of 2,2,6-trisubstituted 3-methylidenetetrahydropyra-4-ones 6, i.e., isopropyl and n-butyl groups. Additionally, we decided to introduce the ferrocenyl moiety onto the tetrahydropyran-4-one skeleton. The ferrocenyl group has proved itself to be an excellent choice to design new drugs [10][11][12], most probably because of its small size, aromaticity, hydrophobicity, low cytotoxicity against normal cells and redox behavior [13][14][15][16][17][18]. Several ferrocene derivatives have already been used as anticancer, antimalarial, antiviral and antibiotic agents [14][15][16]19,20].
The obtained methylidenepyran-4-ones 13a-o were evaluated for their cytotoxic activity against two cancer cell lines (HL-60 and MCF-7), and for comparison, against a normal cell line (HUVEC). Additionally, the influences of two selected analogs on proliferation, cell cycle distribution, apoptosis induction, DNA damage and topoisomerase IIα activity were also investigated.

Chemistry
General information NMR spectra were recorded on a Bruker DPX 250 or Bruker Avance II Plus instrument (Bruker, Billerica, MA, USA) at 250.13 MHz or 700 for 1 H; 62.9 or 176 MHz for 13 C; and 101.3 MHz for 31 P NMR. Tetramethylsilane was used as an internal standard and 85% H 3 PO 4 as an external standard. 31 P NMR spectra were recorded using broadband proton decoupling. Melting points were determined in open capillaries and are uncorrected. Column chromatography was performed on silica gel 60 (230-400 mesh) (Aldrich, Sant Louis, MO, USA). Thin-layer chromatography was performed on the pre-coated TLC sheets of silica gel 60 F254 (Aldrich). The purity of the synthesized compounds was confirmed by the combustion elemental analyses (CH, elemental analyzer EuroVector 3018, Elementar Analysensysteme GmbH, Langenselbold, Germany). MS spectra of intermediates were recorded on Waters 2695-Waters ZQ 2000 LC/MS apparatus. EI mass spectra of final compounds were recorded on a GCMS-QP2010 ULTR A instrument (Shimadzu, Kioto, Japan). The mass spectra were obtained using the following operating conditions: electron energy of 70 eV and ion source temperature of 200 • C. Samples were introduced via a direct insertion probe heated from 30 to 300 • C. All reagents and starting materials were purchased from commercial vendors and used without further purification. Organic solvents were dried and distilled prior to use. Standard syringe techniques were used for transferring dry solvents.
In a round-bottomed three-necked flask under an argon atmosphere, NaH (0.51 g, 17.00 mmol, 80% in mineral oil) was suspended in THF (48 mL). The suspension was stirred, and a solution of diethyl 2-oxopropylphosphonate 7 (3.00 g, 15.45 mmol) in THF (3 mL) was added dropwise. The reaction mixture was stirred for 30 min, cooled below −30 • C in dry ice-acetone bath and n-butyllithium (6.80 mL of 2.5 M solution in hexane, 17.00 mmol) was added dropwise. The reaction mixture was stirred for 30 min at this temperature, cooled to −78 • C in a dry ice-acetone bath and solution of aldehyde (18.54 mmol) in THF (6 mL) was added dropwise. Reaction mixture was stirred for 1.5 h at this temperature. After this time, the reaction was quenched by adding saturated solution of ammonium chloride (100 mL). The water layer was extracted with DCM (3 × 100 mL); the organic layers were combined, washed with brine and dried over MgSO 4 . The solvents were evaporated under reduced pressure, and the resulting crude product was purified by column chromatography (eluent ethyl acetate).

Metabolic activity by MTT assay
The MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to assess the cytotoxicity of the investigated compounds. Briefly, HL-60, MCF-7 and HUVEC cells were seeded in 24-well plates at density of 8 × 10 4 /mL. After 24 h incubation, cells were treated with various concentrations of the compounds and incubated for another 24 h or 48 h. Then, the cells were incubated with MTT solution (100 µL; 5 mg/mL in PBS) for 1.5 h and centrifuged. The formazan product was dissolved in DMSO, and the absorption was measured using FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, LLC, San Jose, CA, USA) at 560 nm. Assays were performed twice in triplicate for each analog.
Cell morphology HL-60 cells were treated with compound 13d or 13g at IC 50 and 2IC 50 for 24 h. Then, the cells were examined for morphological changes using a light microscope at 40× magnification and photographed.
Apoptosis assay Apoptotic cells were detected using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA). HL-60 cells (2.0 × 10 5 cells/mL) seeded in 6-well plates and cultured overnight were treated with various concentrations of 13d or 13g for 24 h. Then, the cells were washed with PBS and resuspended in the binding buffer. After staining with FITC Annexin V and propidium iodide (15 min, room temperature) cells were analyzed by flow cytometry using CytoFlex and Kaluza Analysis Software v2 (Beckman Coulter Inc., Brea, CA, USA).

Analysis of apoptosis, DNA damage and cell cycle
Apoptosis, DNA damage, proliferation and cell cycle kinetics were investigated using the Apoptosis, DNA Damage and Cell Proliferation Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol. Briefly, HL-60 cells (2.0 × 10 5 cells/mL) were seeded in 6-well plates and left to grow for 24 h. Then, the cells were treated with 13d or 13g at IC 50 and 2IC 50 concentrations. After 24 h incubation, the cells were treated with BrdU solution for 8 h. Then, the cells were collected by centrifugation, fixed and permeabilized. To expose the incorporated BrdU, the cells were incubated with DNase (300 µg/mL in PBS) for 1 h at 37 • C. Afterwards, the cells were stained with fluorescent antibodies, including Anti-BrdU, Anti-Cleaved PARP (Asp214) and Anti-H2AX (pS139) for 20 min at room temperature. Total DNA for cell cycle analysis was stained using DAPI solution (1 µg/mL in staining buffer). Finally, the cells were analyzed by flow cytometry using CytoFLEX (Beckman Coulter, Inc., Brea, CA, USA) and the data analysis was performed with Kaluza Analysis Software v2 (Beckam Coulter, Inc., Brea, CA, USA).

Statistical analysis
All data are expressed as mean ±SEM. Statistical analyses were carried out using Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was assessed using Student's t-test (for comparison of two groups) or one-way ANOVA followed by a post-hoc multiple comparison Student-Newman-Keuls test (for comparison of three or more groups). * p < 0.05, ** p < 0.01 and *** p < 0.001 were considered significant.

Chemistry
Addition of diethyl 2-oxopropylphosphonate 7 to aliphatic or aromatic aldehydes was performed using conditions described by Wada et al. [21]. Treatment of 7 [22] with 1.1 equivalents of sodium hydride at room temperature and next 1.1 equivalents of n-butyllithium solution (2,5 M in hexane) at the temperature range from -30 to -40 • C gave dianion 8, which was reacted with 1.2 equivalents of aldehyde (Scheme 1). After reaction, work-up and purification by column chromatography produced 4-hydroxy-2-oxoalkylphosphonates 9a-e in good yields ( Table 1). Conversion of 9a-e into 3-diethoxyphosphoryldihydropyran-4-ones 11a-e was realized performing the reaction with N,N-dimethylformamide dimethyl acetal (DMF-DMA, 3 equivalents) followed by treatment of the intermediate dimethylaminovinylphosphonates 10a-e with 2.5 equivalents of BF 3 · Et 2 O for 24 h, at room temperature. Surprisingly, cyclization of 9e with ferrocenyl substituent, which was obtained by reaction of dianion 8 with ferrocenecarboxaldehyde, was inefficient in these conditions. Analysis of the 31 P NMR spectra of the crude reaction mixture showed a number of signals which indicated degradation of the obtained compound. However, when the reaction was performed with three equivalents of DMF-DMA for 3 h at room temperature, without addition of BF 3 · Et 2 O, the expected 3-diethoxyphosphoryl-6-ferrocenyldihydropyran-4-one 11e was obtained. Attempts to purify 11a-e by column chromatography failed due to their decomposition. However, inspection of 1 H, 31 P and 13 C NMR spectra of these compounds showed that their purity can be estimated as 80-95%. Therefore, crude dihydropyran-4-ones 11a-e were used in the next step.
With 3-diethoxyphosphoryldihydropyran-4-ones 11a-e in hand, we decided to introduce various substituents next to the phosphoryl group by performing conjugate addition to these compounds (Scheme 2). The reactions proceeded smoothly with Gilman reagents, Ph 2 CuLi and n-Bu 2 CuLi in the presence of TMSCl at −78 • C in THF, to give the expected 2,6-disubsttituted-3-diethoxyphosphoryltetrahydropyran-4-ones 12a-j as mixtures of two diastereoisomers, r-2-trans-3-trans-6-12a-j and r-2-cis-3-trans-6-12a-j, and enol forms, transenol-12a-j. Yields and isomer ratios are given in Table 2. It is worth noticing that in all cases, yields are significantly higher for the phenyl substituent than for the n-butyl one. Evidently, the phenyl Gilman reagent is a better nucleophile in these reactions than the n-butyl one. Scheme 2. Synthesis of 6-substituted 2-n-butyl-or 2-phenyl-3-diethoxyphosphoryltetrahydropyran-4-ones 12a-j using Gillman reagents, as Michael donors. Table 2.
Yields and isomers ratio of 6-substituted 2-n-butyl-or 2-phenyl-3diethoxyphosphoryltetrahydropyran-4-ones 12a-j. Structures of the obtained 3-diethoxyphosphoryl-2,6-disubstitutedtetrahydropyran-4-ones 12a-j were established using 1 H, 13 C and 31 P NMR spectra, and mass spectrometry. Analysis of NMR spectra allowed us to assign the relative configurations and preferred conformations of the obtained isomers. Figure 2 shows characteristic coupling constants for r-2-trans-3-trans-6and r-2-cis-3-trans-6-2-n-butyl-6-ethyl-3diethoxyphosphoryltetrahydropyran-4-one 12a taken from the NMR spectra of the mixture of both diastereoisomers. Large coupling constants J H5-H6 (10.9 Hz and 8.7 Hz) and small coupling constants J H5 -H6 (2.9 Hz and 4.0 Hz) indicate diaxial and axial-equatorial relationships of these protons, respectively, and prove the equatorial position of the ethyl group in both diastereoisomers. On the other hand, a large coupling constant J C1 (n-Bu)-P = 13.6 Hz in r-2-trans-3-trans-6-12a proves the diaxial relation of n-butyl and phosphoryl groups, whereas J C1 (n-Bu)-P~0 Hz in r-2-cis-3-trans-6-12a indicates the axial-equatorial position of these groups. Signals of the enol form, trans-enol-12a, were also present, including a characteristic doublet of hydroxyl group with chemical shifts 11-12 ppm and coupling constant J H-P~1 Hz. This coupling constant shows the existence of resonance assisted hydrogen bond (RAHB) reported recently for the related organophosphorus compounds [23]. As similar sets of coupling constants were observed for 2,6-disubstituted 3-diethoxyphosphoryltetrahydropyran-4-ones 12b-j, we believe that these assignments are valid for the whole series.
Unfortunately, the introduction of an isopropyl group in position 2 by addition of the appropriate Gilman reagent, i-Pr 2 CuLi, to 3-diethoxyphosphoryldihydropyran-4-ones 11a-e, was inefficient. However, when we performed this addition using Grignard reagent, i-PrMgCl, the reaction proceeded smoothly and gave expected 6-substituted-3diethoxyphosphoryl-2-isopropyltetrahydropyran-4-ones 12k-o in good yields (Scheme 3, Table 3). The only drawback was poor stereoselectivity of these reactions. In each case, a complex mixture of four diastereoisomers and two enol forms were formed. Although the axial attack was still favored, the substantial amount of r-2-trans-3-cis-6-12k-o along with their epimers, r-2-cis-3-cis-6-12k-o, and cis-enols-12k-o, were formed.   Figure 3 shows all six possible isomers of 3-diethoxyphosphoryl-2,6-diisopropylpyran-4-one 12l and characteristic coupling constants which could be read from the NMR spectra. Despite many signals present in these spectra, we were able to assign signals and consequently relative configurations and conformations for five isomers in the mixture. The sixth isomer, r-2-cis-3-trans-6-12l, was present in too small an amount (1% according to 31 P NMR spectrum, Table 3) to find its characteristic signals in 1 H or 13 C NMR spectra. Small values of coupling constants J C1 -P for both r-2-trans-3-cis-6 and r-2-cis-3-cis-6-12l (3.5 Hz and 3.9 Hz, respectively) indicate a cis relationship between phosphoryl and isopropyl groups. In turn, small coupling constant J H2-P in r-2-trans-3-cis-6-12l (11.8 Hz) and big coupling constant J H2-P in r-2-cis-3-cis-6-12l (39.3 Hz) prove axial/equatorial and diaxial relationships between the H2 proton and phosphoryl group in these diastereoisomers, respectively. Similarities in the NMR spectra of 12l and remaining 6-substituted 2-isopropyl-3-diethoxyphosphoryltetrahydropyran-4-ones 12k-o allow us to propose the same stereochemical relationships in all pyranones 12k-o.

R 1 R 2 Yield a [%]
a Et  a Ratios of trans/enol/cis isomers were taken from 31 P NMR spectra of the crude products. b Yields of pure isolated products.
Relative configurations of the obtained compounds were assigned by the analysis of 1 H and 13 C NMR spectra, which showed that in all cases trans form dominated and the R 1 group was in equatorial position. Characteristic coupling constants for 14c are shown in  Characteristic coupling constants of all isomers of 5-diethoxyphosphoryl-2phenyltetrahydropyran-4-one 14c.

In Vitro Cytotoxicity
The cytotoxic activity of the obtained methylidenetetrahydropyran-4-ones was investigated in two cancer cell lines, human promyelocytic leukemia (HL-60) and breast cancer adenocarcinoma (MCF-7), and in healthy umbilical vein endothelial cells (HUVEC), for comparison. An anticancer drug, carboplatin, was used as a reference compound. Metabolic activity of the cells was assessed after 48 h incubation with various concentrations of the analogs ( Table 6). Purity of all final compounds was determined on the basis of their 1 H NMR spectra [24] (signal-to-noise ratio and presence of unidentified signals) and was at least 98%, in most cases even over 99%. Generally, the tested compounds were more cytotoxic for leukemic than for breast cancer cells. Analysis of the structure-activity relationship revealed that the cytotoxicity of methylidenetetrahydropyran-4-ones 13a-o strongly depends on the nature of the substituent in position 2 (R 2 ), and to a much lesser extent, on the structure of the substituent in position 6 (R 1 ). This observation can be explained by the fact that R 2 substituent is much closer to the methylidene group, which is believed to be the reaction site for various bionucleophiles, as mentioned in the Introduction. The best substituent in position 6 seems to be the isopropyl group; however, the differences in cytotoxic activity among other substituents in this position are not very significant. On the other hand, the presence and nature of the substituent next to the methylidene moiety is crucial for the activity. Compound 15d, with no substituent in this position, is practically inactive. Evidently, the isopropyl group is the best substituent for position 2 in both tested cell lines. Almost all methylidenetetrahydropyran-4-ones 13 (with the exception of 13a) bearing isopropyl in this position are highly cytotoxic, with IC 50 values below 10 µM. The n-butyl or phenyl groups contribute much less to the cytotoxic activity of the analogs.
The most cytotoxic analogs were 13g, 13j and 13m, with IC 50 values below 4 µM against the HL-60 cell line. However, these compounds showed low cancer/healthy cell IC 50 ratios, and therefore, poor selectivity for HL-60 cells compared to normal HUVEC cells. Analog 13d showed high cytotoxicity in HL-60 cells (IC 50 = 7.65 ± 0.05 µM) and was the most selective one, exhibiting over 3-fold higher inhibitory activity in HL-60 than in HUVEC cells.
Analogs 13d and 13g were selected for further assessment of their anticancer properties. The cytotoxicity of these two compounds in HL-60 cells was evaluated after 24 h incubation. The IC 50 values were 15.65 ± 0.61 µM ( Figure 5B) and 6.49 ± 0.31 µM ( Figure 6B), respectively, and they were used in the subsequent assays.  concentrations were stained with Anti-BrdU, Anti-Cleaved-PARP (Asp214) and Anti-H2AX (pS139) antibodies and DAPI solution and analyzed by flow cytometry. The error bars represent means of three replicates ± SEM. ns-statistically non-significant; * p < 0.05, ** p < 0.01, *** p < 0.001 were considered significant in comparison to untreated control.

Antiproliferative Activity
To examine the influences of the investigated compounds on HL-60 cell proliferation, cells were exposed to a thymidine analog, bromodeoxyuridine (BrdU), that incorporates into newly synthesized DNA during proliferation. The results indicate that both analogs sig-nificantly inhibited cell proliferation. Treatment with 13d at IC 50 and 2IC 50 concentrations decreased the BrdU-incorporating cell populations to 31.5 ± 1.65% and 26.4 ± 6.9%, respectively ( Figure 5F), whereas 13g at IC 50 and 2IC 50 concentrations decreased the populations to 35.3 ± 1.23% and 31.9 ± 6.02%, respectively ( Figure 6F).
The cell cycle has a critical role in regulation of cell growth, proliferation and division of cells after DNA damage. Therefore, it often serves as a target in cancer therapy development [25]. The cell cycle distribution was investigated using DAPI DNA staining and flow cytometry analysis. 13d at 2IC 50 concentration significantly raised the number of cells in the G2/M phase (24.1 ± 1.40% of cells compared to control 14.9 ± 1.37%), whereas the population of cells in G0/G1 phase was diminished ( Figure 5D,E). 13g caused a slight increase in the G2/M cell population; however, the change was not statistically significant ( Figure 6D,E).

Cell Morphology Changes
HL-60 cells grow in suspension and usually have a roughly spherical shape. Treatment with 13d or 13g for 24 h induced several morphological changes in the cells. Some became elongated and asymmetrical; others exhibited characteristics typical for apoptotic cells, including shrinkage, membrane blebbing and formation of apoptotic bodies ( Figures 5A and 6A).

Induction of Apoptosis
One of the typical features of apoptotic cell death is translocation of phosphatidylserine from the inside surface to the extracellular surface of the cell membrane [26], which can be detected with Annexin V [27]. Therefore, to examine the ability of the investigated compounds to induce apoptosis in HL-60 cells, we performed Annexin V and propidium iodide (PI) staining ( Figures 5C and 6C). In the control group, 90.3 ± 1.15% of HL-60 cells were healthy, 3.3 ± 0.36% were in early stage of apoptosis (Annexin V-positive and PInegative) and 5.5 ± 1.02% were in late-stage apoptosis (Annaxin V-positive and PI-positive). 13d did not induce a significant change in the number of apoptotic cells at IC 50 ; however, at 2IC 50 , the analog caused increases in both early and late apoptotic cell numbers to 13.0 ± 2.64% and 24.6 ± 2.34%, respectively. Similarly, 13g was not effective at IC 50 , but at 2IC 50 , early-stage apoptosis was detected in 8.7 ± 0.57% of cells, and 39.5 ± 1.16% of cells were in the late stage apoptosis.
Another hallmark of apoptosis is cleavage of poly(ADP-ribose) polymerase 1 (PARP1) into 24 kDa and 89 kDa fragments executed by caspases 3 and 7. PARP 1 fragmentation causes its inactivation, which in turn leads to inhibition of DNA repair and promotes caspase-mediated fragmentation of DNA during programmed cell death [28]. Therefore, detection of 89 kDa fragments of cleaved PARP with Anti-Cleaved PARP (Asp214) antibodies and flow cytometry was used to investigate apoptosis. Incubation of HL-60 cells with 13d only at 2IC 50 concentration led to significant increase in population of cells with cleaved PARP (49.0 ± 1.61%), in comparison to the control (0.7 ± 0.13%) ( Figure 5G). Treatment with 13g also caused a significant PARP cleavage in 13.5 ± 3.6% of the cell population only at 2IC 50 concentration ( Figure 6G).

Induction of DNA Damage
DNA damage can be the result of either single-or double-strand breaks (DSBs). DSB are considered the most dangerous, as it is assumed that in metazoan, just one DSB has the potential to trigger cell death [29]. Cancer cells are often more susceptible to DSBs than healthy cells. Therefore, drugs able to induce DSB formation are widely used in anticancer therapies [30]. One of the earliest signals for DSB detection include phosphorylation of the histone H2AX on the Ser139, generating γH2AX [31]. In our study, induction of DSB was investigated by flow cytometry of HL-60 cells stained with anti-γH2AX fluorescent antibody. Treatment of HL-60 cells with 13d at 2IC 50 led to significant γH2AX generation in 38.4 ± 5.89% of cells in comparison to the control (1.1 ± 0.17%) [ Figure 5H]. 13g was markedly less genotoxic, causing at 2IC 50 , H2AX phosphorylation in 9.6 ± 2.94% of the cell population ( Figure 6H).

Topoisomerase IIα Activity
Some of the most effective DNA-damaging anticancer drugs include inhibitors of topoisomerases [32]. DNA topoisomerases are enzymes capable of changing DNA topology by generating transient breaks to mitigate strain. There are two types of topoisomerases, namely, type I enzymes cleaving one of the DNA strands and type II enzymes creating DSB [33]. Type II topoisomerases separate into subfamilies α and β. Topoisomerase IIβ is evenly distributed in all cells, both normal and cancerous, whereas topoisomerase IIα is overexpressed in rapidly proliferating cells, what makes it an optimal target for anticancer therapy [34]. Therefore, the inhibitory activity of the investigated analogs on topoisomerase IIα was also explored.
The ability of the analogs to inhibit topoisomerase II activity was investigated by measuring decatenation of kinetoplast DNA (kDNA) by human topoisomerase IIα. Topoisomerase II has the ability to decatenate interlinked dsDNA minicircles. When the enzyme is inactive, the kDNA molecules remain in the well after gel electrophoresis, whereas when topoisomerase II is active, the minicircles are released and migrate into the gel. Both compounds inhibited topoisomerase II activity at 0.5 mM and 1 mM concentrations. 13g showed stronger inhibitory activity towards topoisomerase IIα than 13d (Figure 7).

Conclusions
In the present investigation, we showed that Horner-Wadsworth-Emmons methodology is a very useful tool for the efficient synthesis of 2,6-disubstituted 3-methylidenetetrahydropyran-4ones 13a-o and 2-(4-methoxyphenyl)-5-methylidenetetrahydropyran-4-one 15d. Addition of Gilman or Grignard reagents to intermediate diethoxyphosphoryltetrahydropyran-4ones 13a-e was fully or highly stereoselective, respectively, and finally gave access to diastereomerically pure trans-2,6-disubstituted 3-methylidenetetrahydropyran-4-ones 13ao with various substitution patterns. It is worth stressing that, in spite of very complex diastereoismeric mixtures of the obtained adducts, careful NMR analysis allowed us to establish their relative configurations and conformations. The in vitro assessment of the obtained analogs using the conventional MTT assay showed that in HL-60 and MCF-7 cells, analogs with isopropyl and phenyl substituents in position R2 were more cytotoxic than those with n-butyl substituents. Among the novel compounds, 13g was the most cytotoxic and 13d the most selective, as compared with normal HUVEC cells. Therefore, the anticancer potential of 13d and 13g in HL-60 cells was further investigated. Our findings showed that both analogs induced morphological changes characteristic of apoptosis in cancer cells, significantly inhibited proliferation and induced apoptotic cell death. Moreover, both compounds generated DNA damage, and analog 13d arrested the cell cycle of HL-60 cells in the G2/M phase. It has been reported that topoisomerase II inhibitory activity is associated with generation of DNA damage and induction of cell cycle arrest at the G2/M phase [35]. Both 13d and 13g were able to inhibit the activity of topoisomerase IIα; however, 13g was more potent. Based on these findings, the investigated analogs may be further optimized for the development of new and effective topoisomerase II inhibitors.