Synthesis and Cytotoxic Evaluation of 3-Methylidenechroman-4-ones

In the search for new anticancer agents, a library of variously substituted 3-methylidenechroman-4-ones was synthesized using Horner–Wadsworth–Emmons methodology. Acylation of diethyl methylphosphonate with selected ethyl salicylates furnished 3-diethoxyphosphorylchromen-4-ones which were next used as Michael acceptors in the reaction with various Grignard reagents. The adducts were obtained as the mixtures of trans and cis diastereoisomers along with a small amount of enol forms. Their relative configuration and preferred conformation were established by NMR analysis. The adducts turned up to be effective Horner–Wadsworth–Emmons reagents giving 2-substituted 3-methylidenechroman-4-ones, which were then tested for their possible cytotoxic activity against two leukemia cell lines, HL-60 and NALM-6, and against MCF-7 breast cancer cell line. All new compounds (14a–o) were highly cytotoxic for the leukemic cells and showed a moderate or weak effect on MCF-7 cells. Analog 14d exhibited the highest growth inhibitory activity and was more potent than carboplatin against HL-60 (IC50 = 1.46 ± 0.16 µM) and NALM-6 (IC50 = 0.50 ± 0.05 µM) cells. Further tests showed that 14d induced apoptosis in NALM-6 cells, which was mediated mostly through the extrinsic pathway.


Introduction
Chroman-4-one skeleton 1 is a core structure for a large group of plant metabolites called flavonoids, which possess many desirable biological activities including anticancer, antibacterial, and antioxidant properties [1,2]. One relatively small subgroup of flavonoids are homoisoflavonoids 2-4 which are characterized by the presence of arylmethyl or arylidene group in position 3 ( Figure 1). A special place within this group belongs to 3-arylidenechroman-4-ones 4 which were found in many plants. For example, Bonducellin 5 was isolated from Caesalpina bonducella [3] and Eucomin 6 from Eucomis bicolor BAK (Liliaceae) [4].
Both natural and synthetic 3-arylidenechroman-4-ones 4 display valuable biological activities. They are potent and selective monoamine oxidase-B (MAO-B) inhibitors [5,6] and possess anti-cholinesterase activity [7,8], what makes them good candidates for the treatment of various neurological diseases such as Alzheimer's or Parkinson's disease. Furthermore, they show significant cytotoxicity for several cancer cell lines [9,10] and are cytochrome P450 aromatase inhibitors [11] being used for the treatment of Both natural and synthetic 3-arylidenechroman-4-ones 4 display valuable biological activities. They are potent and selective monoamine oxidase-B (MAO-B) inhibitors [5,6] and possess anticholinesterase activity [7,8], what makes them good candidates for the treatment of various neurological diseases such as Alzheimer's or Parkinson's disease. Furthermore, they show significant cytotoxicity for several cancer cell lines [9,10] and are cytochrome P450 aromatase inhibitors [11] being used for the treatment of advanced breast cancer. Also, their antifungal [12,13], antioxidant [14,15], and anti-inflammatory [16] activity was reported.
In contrast to 3-arylidenechroman-4-ones 4, biological activity of 3-methylidenechroman-4-ones 7 is poorly recognized. There is only one report describing their significant bacteriostatic activity against Gram-positive microorganisms [17]. However, an exo-cyclic methylidene bond conjugated with a carbonyl group present in 3-methylidenechroman-4-ones 7 is a structural motif found also in a large number of natural products, such as α-methylidene-γ-and δ-lactones [25,26] which react by the Michael-type addition with various bionucleophiles, disrupting key biological processes and are considered promising anticancer agents [27,28]. Consequently, we reasoned that also 3methylidenechroman-4-ones 7 might have considerable cytotoxic activity.
In contrast to 3-arylidenechroman-4-ones 4, biological activity of 3-methylidenechroman-4-ones 7 is poorly recognized. There is only one report describing their significant bacteriostatic activity against Gram-positive microorganisms [17]. However, an exo-cyclic methylidene bond conjugated with a carbonyl group present in 3-methylidenechroman-4-ones 7 is a structural motif found also in a large number of natural products, such as α-methylidene-γand δ-lactones [25,26] which react by the Michael-type addition with various bionucleophiles, disrupting key biological processes and are considered promising anticancer agents [27,28]. Consequently, we reasoned that also 3-methylidenechroman-4-ones 7 might have considerable cytotoxic activity.
In this report, we present a new, general synthetic method for obtaining variously substituted 3-methylidenechroman-4-ones 14, based on the (well-recognized in our laboratory) Horner-Wadsworth-Emmons approach for the construction of exo-methylidene bond [29][30][31]. All obtained 3-methylidenechroman-4-ones 14 were evaluated in terms of their cytotoxic activity against three human cancer cell lines: promyelocytic leukemia HL-60, NALM-6, and breast adenocarcinoma cell line MCF-7. The most cytotoxic compound, 14d was selected for further experiments and its effect on the induction of apoptosis was investigated.

Chemistry
The first step was the synthesis of 3-diethoxyphosphorylchromen-4-ones 12a-c, which are crucial intermediates in our methodology. Literature search revealed that there is no efficient method for obtaining 2-unsubstituted 3-phosphorylchromen-4-ones. In the only report we have found, 3-diethoxyphosphorylchromen-4-one was formed in 5% yield, as a side product in free radical phosphorylation of chromen-4-one [32]. Therefore, we worked out a two-step procedure, which starts with the reaction of commercially available ethyl salicylates 8a-c with diethyl methylphosphonate 9 in the presence of three equivalents of LDA (Scheme 1). Using this stoichiometry, protection of the hydroxyl group is avoided and we believe this is the major improvement over the reported acylation of dimethyl methylphosphonate using benzyl protected methyl salicylate [33]. The standard work-up and column chromatography purification gave 2-(2-hydroxyphenyl)-2-oxoethylphoshonates 10a-c in good yields (Table 1). In the next step, reaction between phophonates 10a-c and dimethylformamide dimethyl acetal 11 gave, after purification by column chromatography, 3-diethoxyphosphorylchromen-4-ones 12a-c in high yields (

Chemistry
The first step was the synthesis of 3-diethoxyphosphorylchromen-4-ones 12a-c, which are crucial intermediates in our methodology. Literature search revealed that there is no efficient method for obtaining 2-unsubstituted 3-phosphorylchromen-4-ones. In the only report we have found, 3diethoxyphosphorylchromen-4-one was formed in 5% yield, as a side product in free radical phosphorylation of chromen-4-one [32]. Therefore, we worked out a two-step procedure, which starts with the reaction of commercially available ethyl salicylates 8a-c with diethyl methylphosphonate 9 in the presence of three equivalents of LDA (Scheme 1). Using this stoichiometry, protection of the hydroxyl group is avoided and we believe this is the major improvement over the reported acylation of dimethyl methylphosphonate using benzyl protected methyl salicylate [33]. The standard workup and column chromatography purification gave 2-(2-hydroxyphenyl)-2-oxoethylphoshonates 10ac in good yields (Table 1). In the next step, reaction between phophonates 10a-c and dimethylformamide dimethyl acetal 11 gave, after purification by column chromatography, 3diethoxyphosphorylchromen-4-ones 12a-c in high yields (Table 1).
Scheme 1. Synthesis of 3-diethoxyphosphorylchromen-4-ones 12a-c. With 3-diethoxyphosphorylchromen-4-ones 12a-c in hand, we performed their reactions with various Grignard reagents (Scheme 2). In all cases, after standard work-up, we received adducts 13ao, which were purified by column chromatography with yields given in Table 2. Interestingly, examination of the 1 H, 13 C and 31 P NMR spectra revealed that all adducts 13 were formed as mixtures of trans and cis diastereoisomers (trans-or cis-13a-o), along with small amount of enol form (enol-13a-o), with trans diastereoisomers strongly predominating. Ratios, determined from the 31 P NMR spectra of the crude reaction mixtures, are given in Table 2.  With 3-diethoxyphosphorylchromen-4-ones 12a-c in hand, we performed their reactions with various Grignard reagents (Scheme 2). In all cases, after standard work-up, we received adducts 13a-o, which were purified by column chromatography with yields given in Table 2. Interestingly, examination of the 1 H, 13 C and 31 P NMR spectra revealed that all adducts 13 were formed as mixtures of trans and cis diastereoisomers (trans-or cis-13a-o), along with small amount of enol form (enol-13a-o), with trans diastereoisomers strongly predominating. Ratios, determined from the 31 P NMR spectra of the crude reaction mixtures, are given in Table 2.  Careful analysis of the NMR spectra showed also that both trans-and cis-13a-o exist in the halfchair conformation and diethoxyphosphoryl group occupies the axial position ( Figure 2). Simple application of Karplus correlation between corresponding dihedral angles and coupling constants 3 JH2-H3, 3 JH2-P and 3 JC(R3)-P, determined from the 1 H and 13 C NMR spectra of trans-and cis-13a-o shows full agreement with the proposed configurations and conformations. Corresponding coupling constants are given in Figure 2. Similar half-chair conformation with diethoxyphosphoryl group in axial position was reported for 3-diethoxyphosphorylchroman-2-ones [34,35].   Careful analysis of the NMR spectra showed also that both transand cis-13a-o exist in the half-chair conformation and diethoxyphosphoryl group occupies the axial position ( Figure 2). Simple application of Karplus correlation between corresponding dihedral angles and coupling constants 3 J H2-H3 , 3 J H2-P and 3 J C(R3)-P , determined from the 1 H and 13 C NMR spectra of transand cis-13a-o shows full agreement with the proposed configurations and conformations. Corresponding coupling constants are given in Figure 2. Similar half-chair conformation with diethoxyphosphoryl group in axial position was reported for 3-diethoxyphosphorylchroman-2-ones [34,35].  Careful analysis of the NMR spectra showed also that both trans-and cis-13a-o exist in the halfchair conformation and diethoxyphosphoryl group occupies the axial position ( Figure 2). Simple application of Karplus correlation between corresponding dihedral angles and coupling constants 3 JH2-H3, 3 JH2-P and 3 JC(R3)-P, determined from the 1 H and 13 C NMR spectra of trans-and cis-13a-o shows full agreement with the proposed configurations and conformations. Corresponding coupling constants are given in Figure 2. Similar half-chair conformation with diethoxyphosphoryl group in axial position was reported for 3-diethoxyphosphorylchroman-2-ones [34,35].  On the other hand, 1 H NMR spectra of enols-13a-o revealed very characteristic doublets with coupling constant 4 J P-H~1 Hz and chemical shift in the range of 11-12 ppm, which can be assigned to the proton of the hydroxyl group. Coupling between this proton and phosphorus indicates the presence of resonance-assisted hydrogen bond (RAHB) [36] and can be visualized by resonance structures shown in Figure 3. Recently, we have reported on the existence of RAHB in 3-(dimenthoxyphosphoryl)-2-phenyl-1,2-dihydroquinolin-4-ol [37], what was the first example of this phenomenon in organophosphorus compounds. Now, we can confirm the existence of RAHB also in 3-diethoxyphosphoryl-2H-chromen-4-oles.

In Vitro Cytotoxicity of New Analogs Against Three Cancer Cell Lines
All obtained 3-methylidenechroman-4-ones 14a-o were evaluated for their possible cytotoxic activity against three human cancer cell lines: leukemia HL-60 and NALM-6 and breast adenocarcinoma MCF-7 using the MTT assay (after 48 h incubation) ( Table 3). Carboplatin served as a reference compound.
Analysis of the structure-activity relationship revealed that chromanones 14a-j were, in general, more potent than benzochromanones 14k-o, containing additional benzene ring ortho-fused with a chromanone skeleton. All compounds (14a-o) were much more cytotoxic for leukemia cells than for the solid tumor MCF-7 cells. In both series of chromanones, 14a-e (R 2 = H) and 14f-j (R 2 = Me), the most potent compounds against leukemic HL-60 and NALM-6 cells were these containing an i-propyl substituent in position 2, i.e., 14d and 14i, respectively. For HL-60 cells only 14d was more cytotoxic than the reference carboplatin. The highest cytotoxicity was observed against NALM-6 cells, with three analogs 14b, 14d, and 14i exhibiting lower half maximal inhibitory concentration values (IC50) than carboplatin. Analog 14d was the most cytotoxic chromanone for NALM-6 cells (IC50 of 0.5 ± 0.05 µM) and was selected for further investigation of its potential antineoplastic properties. Finally, all adducts 13a-o were transformed into 3-methylidenechroman-4-ones 14a-o performing Horner-Wadsworth-Emmons olefination of formaldehyde. The best results were obtained with K 2 CO 3 used as a base and formalin as a source of formaldehyde (Scheme 3). Standard work-up and purification by column chromatography furnished 3-methylidenechroman-4-ones 14a-o in moderate to good yields ( Table 2). presence of resonance-assisted hydrogen bond (RAHB) [36] and can be visualized by resonance structures shown in Figure 3. Recently, we have reported on the existence of RAHB in 3-(dimenthoxyphosphoryl)-2-phenyl-1,2-dihydroquinolin-4-ol [37], what was the first example of this phenomenon in organophosphorus compounds. Now, we can confirm the existence of RAHB also in 3-diethoxyphosphoryl-2H-chromen-4-oles.  (Table 2).

In Vitro Cytotoxicity of New Analogs Against Three Cancer Cell Lines
All obtained 3-methylidenechroman-4-ones 14a-o were evaluated for their possible cytotoxic activity against three human cancer cell lines: leukemia HL-60 and NALM-6 and breast adenocarcinoma MCF-7 using the MTT assay (after 48 h incubation) ( Table 3). Carboplatin served as a reference compound.
Analysis of the structure-activity relationship revealed that chromanones 14a-j were, in general, more potent than benzochromanones 14k-o, containing additional benzene ring ortho-fused with a chromanone skeleton. All compounds (14a-o) were much more cytotoxic for leukemia cells than for the solid tumor MCF-7 cells. In both series of chromanones, 14a-e (R 2 = H) and 14f-j (R 2 = Me), the most potent compounds against leukemic HL-60 and NALM-6 cells were these containing an i-propyl substituent in position 2, i.e., 14d and 14i, respectively. For HL-60 cells only 14d was more cytotoxic than the reference carboplatin. The highest cytotoxicity was observed against NALM-6 cells, with three analogs 14b, 14d, and 14i exhibiting lower half maximal inhibitory concentration values (IC50) than carboplatin. Analog 14d was the most cytotoxic chromanone for NALM-6 cells (IC50 of 0.5 ± 0.05 µM) and was selected for further investigation of its potential antineoplastic properties.

In Vitro Cytotoxicity of New Analogs Against Three Cancer Cell Lines
All obtained 3-methylidenechroman-4-ones 14a-o were evaluated for their possible cytotoxic activity against three human cancer cell lines: leukemia HL-60 and NALM-6 and breast adenocarcinoma MCF-7 using the MTT assay (after 48 h incubation) ( Table 3). Carboplatin served as a reference compound.
Analysis of the structure-activity relationship revealed that chromanones 14a-j were, in general, more potent than benzochromanones 14k-o, containing additional benzene ring ortho-fused with a chromanone skeleton. All compounds (14a-o) were much more cytotoxic for leukemia cells than for the solid tumor MCF-7 cells. In both series of chromanones, 14a-e (R 2 = H) and 14f-j (R 2 = Me), the most potent compounds against leukemic HL-60 and NALM-6 cells were these containing an i-propyl substituent in position 2, i.e., 14d and 14i, respectively. For HL-60 cells only 14d was more cytotoxic than the reference carboplatin. The highest cytotoxicity was observed against NALM-6 cells, with three analogs 14b, 14d, and 14i exhibiting lower half maximal inhibitory concentration values (IC 50 ) than carboplatin. Analog 14d was the most cytotoxic chromanone for NALM-6 cells (IC 50 of 0.5 ± 0.05 µM) and was selected for further investigation of its potential antineoplastic properties.

Apoptotic Cell Death Determination
It is now well documented that most anticancer drugs induce apoptosis. One of the main characteristics of apoptosis, phosphatidylserine (PS) translocation to the outer surface of the cellular membrane, was investigated by double-staining with Annexin V and propidium iodide (PI). Annexin V is a protein exerting high affinity to PS exposed on the outer surface of the plasma membrane, enabling detection of even an early stage apoptosis. The late stage of apoptosis is characterized by loss of membrane integrity allowing permeation of a dye such as PI into the cells [38,39]. Treatment of NALM-6 cells for 24 h ( Figure 4A) with the selected analog 14d at 1.25 µ M (IC50) and 2.5 µ M (2 IC50) concentrations led to the increase of Annexin V and PI-positive cells from 2.2% to 29.4% and 91.4%, respectively ( Figure 4B,C), showing that 14d induced the late stage of apoptosis in NALM-6 cells.

Apoptotic Cell Death Determination
It is now well documented that most anticancer drugs induce apoptosis. One of the main characteristics of apoptosis, phosphatidylserine (PS) translocation to the outer surface of the cellular membrane, was investigated by double-staining with Annexin V and propidium iodide (PI). Annexin V is a protein exerting high affinity to PS exposed on the outer surface of the plasma membrane, enabling detection of even an early stage apoptosis. The late stage of apoptosis is characterized by loss of membrane integrity allowing permeation of a dye such as PI into the cells [38,39]. Treatment of NALM-6 cells for 24 h ( Figure 4A) with the selected analog 14d at 1.25 µM (IC 50 ) and 2.5 µM (2 IC 50 ) concentrations led to the increase of Annexin V and PI-positive cells from 2.2% to 29.4% and 91.4%, respectively ( Figure 4B,C), showing that 14d induced the late stage of apoptosis in NALM-6 cells. Apoptosis occurs when caspases, which have proteolytic activity, cleave specific substrates, causing cell death. Depending on the initiator caspase involved in this process, the apoptosis may be mediated by the intrinsic (caspase 9) or extrinsic (caspase 8) pathway [40]. To investigate which caspases were involved in the apoptosis inducted by analog 14d in NALM-6 cells, the cells were treated with 14d at 1.25 µ M and 2.5 µ M concentrations for 6 h. Then, the activity of executioner caspase 3 and initiator caspases 8 and 9 was quantified using fluorogenic indicators. Results presented in Figure 5 indicate that the levels of caspase 3, 8 and 9 were significantly increased: 2.7and 3.7-fold for caspase 3, 4.9-and 11.3-fold for caspase 8, and 1.25-and 5.7-fold for caspase 9 after treatment of the cells with 1.25 µ M and 2.5 µ M concentration of 14d, respectively. Activation of the extrinsic pathway was more prominent than the intrinsic pathway.
Presented results indicate that compound 14d is a potent cytotoxic agent that significantly inhibits metabolic activity of NALM-6 cells with IC50 value as low as 0.5 µ M. Analog 14d also promotes apoptosis in the investigated cell line, which is mediated by the extrinsic and in a much lesser extend intrinsic pathway. Apoptosis occurs when caspases, which have proteolytic activity, cleave specific substrates, causing cell death. Depending on the initiator caspase involved in this process, the apoptosis may be mediated by the intrinsic (caspase 9) or extrinsic (caspase 8) pathway [40]. To investigate which caspases were involved in the apoptosis inducted by analog 14d in NALM-6 cells, the cells were treated with 14d at 1.25 µM and 2.5 µM concentrations for 6 h. Then, the activity of executioner caspase 3 and initiator caspases 8 and 9 was quantified using fluorogenic indicators. Results presented in Figure 5 indicate that the levels of caspase 3, 8 and 9 were significantly increased: 2.7-and 3.7-fold for caspase 3, 4.9-and 11.3-fold for caspase 8, and 1.25-and 5.7-fold for caspase 9 after treatment of the cells with 1.25 µM and 2.5 µM concentration of 14d, respectively. Activation of the extrinsic pathway was more prominent than the intrinsic pathway.
Presented results indicate that compound 14d is a potent cytotoxic agent that significantly inhibits metabolic activity of NALM-6 cells with IC 50 value as low as 0.5 µM. Analog 14d also promotes apoptosis in the investigated cell line, which is mediated by the extrinsic and in a much lesser extend intrinsic pathway.

General Information
NMR spectra were recorded on a Bruker DPX 250 or Bruker Avance II instrument at 250.13 MHz or 700 MHz for 1 H, 62.9 MHz or 176 MHz for 13 C, and 101.3 MHz for 31 P NMR with tetramethylsilane used as an internal and 85% H3PO4 as an external standard. 31 P NMR spectra were recorded using broadband proton decoupling. IR spectra were recorded on a Bruker Alpha ATR spectrophotometer. Melting points were determined in open capillaries and are uncorrected. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. The [α]D values are given in deg·cm2·g −1 and concentration c in g·(100 mL) −1 . Column chromatography was performed on silica gel 60 (230-400 mesh) (Aldrich, Steinheim, Germany). Thin-layer chromatography was performed on the pre-coated TLC sheets of silica gel 60 F254 (Aldrich, Steinheim, Germany). The purity of the synthesized compounds was confirmed by the combustion elemental analyses (CHN, elemental analyzer EuroVector 3018, Elementar Analysensysteme GmbH (Langenselbold, Germany). MS spectra were recorded on Waters 2695-Waters ZQ 2000 LC/MS apparatus (Waters Corporation, Milford, MA, USA). 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.

General Information
NMR spectra were recorded on a Bruker DPX 250 or Bruker Avance II instrument at 250.13 MHz or 700 MHz for 1 H, 62.9 MHz or 176 MHz for 13 C, and 101.3 MHz for 31 P NMR with tetramethylsilane used as an internal and 85% H 3 PO 4 as an external standard. 31 P NMR spectra were recorded using broadband proton decoupling. IR spectra were recorded on a Bruker Alpha ATR spectrophotometer. Melting points were determined in open capillaries and are uncorrected. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. The [α] D values are given in deg·cm2·g −1 and concentration c in g·(100 mL) −1 . Column chromatography was performed on silica gel 60 (230-400 mesh) (Aldrich, Steinheim, Germany). Thin-layer chromatography was performed on the pre-coated TLC sheets of silica gel 60 F254 (Aldrich, Steinheim, Germany). The purity of the synthesized compounds was confirmed by the combustion elemental analyses (CHN, elemental analyzer EuroVector 3018, Elementar Analysensysteme GmbH (Langenselbold, Germany). MS spectra were recorded on Waters 2695-Waters ZQ 2000 LC/MS apparatus (Waters Corporation, Milford, MA, USA). 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.

General Procedure for the Synthesis of 2-Substituted 3-Metylidenechroman-4-ones 14a-o.
To the vigorously stirred solution of 2-substituted 3-diethoxychroman-4-on 13a-o (0.15 mmol) in THF (1.5 mL), formaldehyde (36-38% solution in water, 0.125 mL, ca. 1.50 mmol) was added at 0 • C, followed by addition of K 2 CO 3 (41 mg, 0.30 mmol) in water (0.4 mL). The resulting mixture was stirred vigorously at 0 • C for 3 h. Next Et 2 O (5 mL) was added and layers were separated. The water fraction was washed with Et 2 O (5 mL). Organic fractions were combined, washed with brine (5 mL) and dried over MgSO 4 . The solvents were evaporated under reduced pressure and the resulting crude product was then purified by column chromatography (eluent CH 2 Cl 2 ). all measurements and the fold change of fluorescent intensity between control and the treated cells was calculated.
Furthermore, relative configuration of the crucial intermediates for this methodology, trans-or cis-2-substituted 3-diethoxyphosphorylchroman-4-ones 13a-o, was elaborated using NMR analysis. The obtained library of 3-methylidenechroman-4-ones 14a-o was evaluated for the anti-proliferative activity against leukemia and breast cancer cell lines. Several members of this library were determined to be capable of killing leukemia cells with improved activity compared to carboplatin used as a reference compound, exhibiting IC 50 values in the low micromolar range. The most potent 2-isopropyl-3-methylidenechroman-4-one 14d was then evaluated for its possible apoptotic activity against NALM-6 cells. Compound 14d promoted apoptosis mediated by caspase 3/8 and in a much lesser extend by caspase 3/9 induction, which indicated that mostly the extrinsic pathway was engaged in the programmed cell death caused by this analog. These results show that compound 14d is a good lead in a search for new anticancer agents.