Synthesis and In Vitro Anticancer Evaluation of Flavone—1,2,3-Triazole Hybrids

Hybrid compounds of flavones, namely chrysin and kaempferol, and substituted 1,2,3-triazole derivatives, were synthesized by click reaction of the intermediate O-propargyl derivatives. 4-Fluoro- and 4-nitrobenzyl-1,2,3-triazole-containing hybrid molecules were prepared. The mono- and bis-coupled hybrids were investigated on 60 cell lines of 9 common cancer types (NCI60) in vitro as antitumor agents. Some of them proved to have a significant antiproliferative effect.


Introduction
Cancer treatment is one of the most important medical challenges. Permanent research is in progress to produce more effective and less toxic derivatives. One of the exciting and promising directions of this research is the synthesis of antitumor hybrid molecules [1,2]. The concept of molecular hybridization is to incorporate two or more pharmacophores into one molecule with covalent bonds, increasing the chance of effectiveness and improving the drug kinetic properties of the resulting hybrid compared to the corresponding fixed-dose drug combination. It should be noted that rigid distance imposed by the structure of the compound between potentially active parts of the hybrid may prevent biological efficiency.
In this study, the above outlined results inspired us to develop synthetic possibilities for the preparation of flavone-1,2,3-triazole hybrids.

Coupling Components
Chrysin (3) is among the best-known flavones. It is abundant in nature and present in many edible plants and honey [21]. It has an anticancer effect through inducing apoptosis and autophagy [21,22]. Chrysin (3) seems to be suitable for use alone and/or in combination with other chemotherapeutic agents [21]. Kaempferol (4) and its derivatives are also found in many plants. They can prevent coronary heart disease and inflammatory problems, and they also show antiproliferative effects and may induce apoptosis [23].
It is known that 1,2,3-Triazole derivatives have been widely used as a pharmacophore in hybrids. In addition to the advantageous physico-chemical properties of this moiety, it is also known to exert various biological effects [24,25]. 1,2,3-Triazole derivatives are characterized by stability, the ability to form hydrogen bonds (increasing their water solubility), and weak basicity (they are not protonated at physiological pH). Moreover, 1,2,3-triazole derivatives have fungicidal, antibacterial, antituberculosis, and anticancer effects [26,27]. The well-known click reaction is used for the preparation of 1,2,3triazole derivatives, as one of the tools of modern organic synthetic methods based on structure-activity relationships, preferably the N 1 -(4-fluoro-and 4-nitrobenyzl)-1,2,3-triazole derivatives [24,28].

Coupling Components
Chrysin (3) is among the best-known flavones. It is abundant in nature and present in many edible plants and honey [21]. It has an anticancer effect through inducing apoptosis and autophagy [21,22]. Chrysin (3) seems to be suitable for use alone and/or in combination with other chemotherapeutic agents [21]. Kaempferol (4) and its derivatives are also found in many plants. They can prevent coronary heart disease and inflammatory problems, and they also show antiproliferative effects and may induce apoptosis [23].
It is known that 1,2,3-Triazole derivatives have been widely used as a pharmacophore in hybrids. In addition to the advantageous physico-chemical properties of this moiety, it is also known to exert various biological effects [24,25]. 1,2,3-Triazole derivatives are characterized by stability, the ability to form hydrogen bonds (increasing their water solubility), and weak basicity (they are not protonated at physiological pH). Moreover, 1,2,3-triazole derivatives have fungicidal, antibacterial, antituberculosis, and anticancer effects [26,27]. The well-known click reaction is used for the preparation of 1,2,3-triazole derivatives, as one of the tools of modern organic synthetic methods based on structure-activity relationships, preferably the N 1 -(4-fluoro-and 4-nitrobenyzl)-1,2,3-triazole derivatives [24,28].

Chemistry
Chrysin (3) reacted with an equimolar quantity of propargyl bromide (PPGBr) in dimethylformamide in the presence of cesium carbonate at room temperature (Scheme 1), resulting in the 7-substituted product (6) (known as an intermediate of antibacterial derivative prepared by a method different from ours [20]). The reason for the regioselectivity is that the proton of the 5-hydroxyl group forms an intramolecular H-bond with the neighboring oxo group. Certainly, with an excess of propargyl bromide (5 equivalent), exclusively the 5,7-disubstituted derivative (7) proved to be the product, as expected. Others also synthesized this compound using gold(I) complexes without reporting any preparative and characterization details [29].
The next reaction step was the click reaction (Scheme 2) using 4-fluoro-and 4-nitrobenzyl azide prepared in situ from the corresponding benzyl bromides with sodium azide in DMF at room temperature [30].
The reaction was carried out in the presence of copper(I) iodide, triphenylphosphine, and N,N-diisopropylethylamine, and resulted in known hybrids 8 and 9, respectively. These two hybrids were prepared previously with another method, however, only their antibacterial effect has been investigated [20]. Bis(propargyl) derivative 7 was also treated with the same reaction conditions and gave the bis-hybrids 10 and 11. Avoiding the difficult isolation from the triphenylphosphine oxide formed, the latter click reaction was successfully achieved also with further reagents, namely with copper sulfate pentahydrate and sodium L-ascorbate in a two-phase mixture. The next reaction step was the click reaction (Scheme 2) using 4-fluoro-and 4-nitrobenzyl azide prepared in situ from the corresponding benzyl bromides with sodium azide in DMF at room temperature [30].
The reaction was carried out in the presence of copper(I) iodide, triphenylphosphine, and N,N-diisopropylethylamine, and resulted in known hybrids 8 and 9, respectively. These two hybrids were prepared previously with another method, however, only their antibacterial effect has been investigated [20]. Bis(propargyl) derivative 7 was also treated with the same reaction conditions and gave the bis-hybrids 10 and 11. Avoiding the difficult isolation from the triphenylphosphine oxide formed, the latter click reaction was successfully achieved also with further reagents, namely with copper sulfate pentahydrate and sodium L-ascorbate in a two-phase mixture.  The next reaction step was the click reaction (Scheme 2) using 4-fluoro-and 4-nit benzyl azide prepared in situ from the corresponding benzyl bromides with sodium azi in DMF at room temperature [30].
The reaction was carried out in the presence of copper(I) iodide, triphenylphosphi and N,N-diisopropylethylamine, and resulted in known hybrids 8 and 9, respective These two hybrids were prepared previously with another method, however, only th antibacterial effect has been investigated [20]. Bis(propargyl) derivative 7 was also treat with the same reaction conditions and gave the bis-hybrids 10 and 11. Avoiding the di cult isolation from the triphenylphosphine oxide formed, the latter click reaction was su cessfully achieved also with further reagents, namely with copper sulfate pentahydr and sodium L-ascorbate in a two-phase mixture. Scheme 2. The synthesis of chrysin hybrids (8)(9)(10)(11) containing one or two 1,2,3-triazole units.
The second flavone building block, selected for the synthesis of hybrids, was kaempferol (4). The alkylation with propargyl bromide was investigated with different bases and in different solvents (Scheme 3). Using cesium carbonate or potassium carbonate as a base in dimethylformamide compounds 12 and 13 were isolated. However, in acetone solution compound 14 was obtained. bases and in different solvents (Scheme 3). Using cesium carbonate or potassium c bonate as a base in dimethylformamide compounds 12 and 13 were isolated. However acetone solution compound 14 was obtained.

Biological Evaluation
The in vitro antiproliferative activities of chrysin (3) and the synthesized compoun (8)(9)(10)(11)15) were examined against 60 human tumor cell lines according to the given pro cols of NCI (USA) [31][32][33][34][35]. The results are summarized in Table 1. The percentages growth show the amount of living cancer cells compared to a reference. The negat numbers indicate a significant decrease in the cell number. Since derivatives 8 and 10 h shown remarkable antiproliferative activity on several cancer cell lines during the on dose test, they were subjected to a five-dose screening. The GI50 (50% growth inhibitio values are also given in Table 1.

Scheme 3. The reaction between kaempferol (4) and propargyl bromide.
Derivative 13 was the compound isolated in the relatively largest quantity and was chosen for the click reaction (Scheme 4). Investigating both the reaction conditions resulted in the isolation of the bis-hybrid 15.
bases and in different solvents (Scheme 3). Using cesium carbonate or potassium c bonate as a base in dimethylformamide compounds 12 and 13 were isolated. However, acetone solution compound 14 was obtained.  (15) containing two 1,2,3-triazole units.

Biological Evaluation
The in vitro antiproliferative activities of chrysin (3) and the synthesized compoun (8)(9)(10)(11)15) were examined against 60 human tumor cell lines according to the given pro cols of NCI (USA) [31][32][33][34][35]. The results are summarized in Table 1. The percentages growth show the amount of living cancer cells compared to a reference. The negati numbers indicate a significant decrease in the cell number. Since derivatives 8 and 10 h shown remarkable antiproliferative activity on several cancer cell lines during the on dose test, they were subjected to a five-dose screening. The GI50 (50% growth inhibitio values are also given in Table 1.  (15) containing two 1,2,3-triazole units.

Biological Evaluation
The in vitro antiproliferative activities of chrysin (3) and the synthesized compounds (8)(9)(10)(11)15) were examined against 60 human tumor cell lines according to the given protocols of NCI (USA) [31][32][33][34][35]. The results are summarized in Table 1. The percentages of growth show the amount of living cancer cells compared to a reference. The negative numbers indicate a significant decrease in the cell number. Since derivatives 8 and 10 had shown remarkable antiproliferative activity on several cancer cell lines during the one-dose test, they were subjected to a five-dose screening. The GI 50 (50% growth inhibition) values are also given in Table 1. It can be seen from Table 1 that no antiproliferative effect was shown by chrysin (3) and compounds 9 and 11. Hybrids 8 and 10 cause cell death on several cell lines of different types of cancer and show inhibition effect also on some cases. Despite the relatively limited structural diversity of our compounds, the above results revealed some interesting structure-activity relationships. We found that (i) the bis-hybrid compounds Molecules 2023, 28, 626 7 of 16 also exert considerable antiproliferative effect and (ii) replacement of the fluorine atom by a nitro group reduces the bioactivity. The kaempferol-triazole hybrid (15) gave rather modest results.
The two promising compounds (8 and 10) were tested for their antiproliferative activity on further two human cervical cancer cell lines HeLa and SiHa (Table 2). Interestingly, the monohybrid derivative 8 was active only against HeLa cells, and SiHa cells were relatively resistant to it. The bis-hybrid derivative 10 was more potent and similarly active against both cell lines, with a sub-micromolar IC 50 value against HeLa. Both derivatives exhibited higher activity than the reference agent cisplatin against HeLa cells. The results obtained in this paper are encouraging for the future optimization of the derivatives. We want to emphasize that this study may be the starting point for more detailed synthetic and anticancer research.

General Materials and Methods
All chemicals were purchased from Sigma-Aldrich (Budapest, Hungary) and were used as received. Melting points were measured on a VEB Analytik Dresden PHMK-77/1328 apparatus (Dresden, Germany) and are uncorrected. IR spectra were recorded on Zeiss IR 75 and 80 instruments (Thornwood, NY, USA). NMR measurements were performed on a Bruker Avance III HDX 500 MHz NMR spectrometer equipped with a 1 H{ 13 C/ 15 N} 5 mm TCI CryoProbe (Bruker Corporation, Billerica, MA, USA). 1 H And 13 C chemical shifts are given on the delta scale as parts per million (ppm) relative to tetramethyl silane. One-dimensional 1 H, and 13 C spectra and two-dimensional 1 H-1 H COSY, 1 H-1 H NOESY, 1 H-13 C HSQC, and 1 H- 13 C HMBC spectra were acquired using pulse sequences included in the standard spectrometer software package (Bruker TopSpin 3.5, Bruker Corporation). ESI-HRMS and MS-MS analyses were performed on a Thermo Velos Pro Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) system. The ionization method was ESI, operated in positive ion mode. The protonated molecular ion peaks were fragmented by CID (collision-induced dissociation) at a normalized collision energy of 35-65%. For the CID experiment, helium was used as the collision gas. The samples were dissolved in methanol. EI-HRMS analyses were performed on a Thermo Q Exactive GC Orbitrap (Thermo Fisher Scientific, Bremen, Germany) system. The ionization method was EI and operated in positive ion mode. Electron energy was 70 eV and the source temperature was set at 250 • C. Data acquisition and analysis were accomplished with Xcalibur software version 4.0 (Thermo Fisher Scientific). TLC was carried out using DC-Alufolien Kieselgel 60 F 254 (Merck, Budapest, Hungary) plates. Preparative TLC analyses were performed on silica gel 60 PF 254+366 (Merck) glass plates. (6) Chrysin (3) (330 mg, 1.3 mmol) and cesium carbonate (426 g, 1.3 mmol) were dissolved in DMF (15 mL), the solution was stirred at 10 min, then propargyl bromide (0.142 mL, 1.3 mmol) was added in 80% toluene solution. After stirring at room temperature for 18.5 hrs, the reaction mixture was evaporated to dryness, and the residue was dissolved in dichloromethane (60 mL). Next, water (60 mL) was added, and the pH was adjusted to 1 with 2M hydrochloric acid solution. The water phase was extracted with dichloromethane (2 × 30 mL), then the combined organic phase was washed with water (60 mL) and saturated sodium chloride solution (60 mL). The organic phase after drying with magnesium sulfate was evaporated to dryness, and the crude product was purified with preparative TLC  (7) Chrysin (3) (500 mg, 1.97 mmol) and cesium carbonate (3.2 g, 9.84 mmol) were dissolved in dimethylformamide (20 mL), the solution was stirred at 10 min, then propargyl bromide (1.1 mL, 9.84 mmol) was added in 80% toluene solution. After stirring at room temperature for 45 min, the reaction mixture was evaporated to dryness, and the residue was dissolved in dichloromethane (40 mL). Next, water (40 mL) was added, and the pH was adjusted to 1 with 2M hydrochloric acid solution. The water phase was extracted with dichloromethane (3 × 20 mL), then the combined organic phase was washed with water (2 × 20 mL) and saturated sodium chloride solution (20 mL). The organic phase after drying with magnesium sulfate was evaporated to dryness and 620 mg (95%) pure product (7)

One-Dose Screen
All compounds were tested initially at a single high dose (10 −5 M) in the full NCI60 cell panel [31][32][33][34][35]. The number reported for the one-dose assay is growth relative to the no-drug control and relative to the time zero number of cells. This allowed the detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). For example, a value of 100 means no growth inhibition. A value of 30 would mean 70% growth inhibition. A value of 0 means no net growth over the course of the experiment. A value of −30 would mean 30% lethality. A value of −100 means all cells are dead.

Five-Dose Screen
Compounds that exhibited significant growth inhibition in the one-dose screen were evaluated against the 60-cell panel at five concentration levels. The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM l-glutamine. Typically, cells were inoculated in 96-well microtiter plates in 100 µL at plating densities ranging from 5000 to 40,000 cells/well, depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 °C, 5% CO2, 95% air, and 100% relative humidity for 24 h prior to the addition of experimental drugs. After 24 h, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA), to represent a measurement of the cell population for each cell line at the time of drug addition (tz). Experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 µg ml −1 gentamicin. Additional four, 10-fold or ½ log serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 µL of these different drug dilutions were added to the appropriate microtiter wells already containing 100 µL of medium, resulting in the required final drug concentrations.
Following drug addition, the plates were incubated at 37 °C, 5% CO2, 95% air, and 100% relative humidity for an additional 48 h. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the addition of 50 µL of cold 50% (w/v) TCA, and incubated at 4 °C for 60 min. The supernatant was discarded, and the plates were washed with water (5×) and dried in air. Sulforhodamine B (SRB) solution (100 µL) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated at room temperature for 10 min. After staining, the unbound dye was removed by washing five times with 1% acetic acid, and the plates were dried in the air. The bound stain is

One-Dose Screen
All compounds were tested initially at a single high dose (10 −5 M) in the full NCI60 cell panel [31][32][33][34][35]. The number reported for the one-dose assay is growth relative to the no-drug control and relative to the time zero number of cells. This allowed the detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). For example, a value of 100 means no growth inhibition. A value of 30 would mean 70% growth inhibition. A value of 0 means no net growth over the course of the experiment. A value of −30 would mean 30% lethality. A value of −100 means all cells are dead.

Five-Dose Screen
Compounds that exhibited significant growth inhibition in the one-dose screen were evaluated against the 60-cell panel at five concentration levels. The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM l-glutamine. Typically, cells were inoculated in 96-well microtiter plates in 100 µL at plating densities ranging from 5000 to 40,000 cells/well, depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 • C, 5% CO 2 , 95% air, and 100% relative humidity for 24 h prior to the addition of experimental drugs. After 24 h, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA), to represent a measurement of the cell population for each cell line at the time of drug addition (t z ). Experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 µg ml −1 gentamicin. Additional four, 10-fold or 1 2 log serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 µL of these different drug dilutions were added to the appropriate microtiter wells already containing 100 µL of medium, resulting in the required final drug concentrations.
Following drug addition, the plates were incubated at 37 • C, 5% CO 2 , 95% air, and 100% relative humidity for an additional 48 h. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the addition of 50 µL of cold 50% (w/v) TCA, and incubated at 4 • C for 60 min. The supernatant was discarded, and the plates were washed with water (5×) and dried in air. Sulforhodamine B (SRB) solution (100 µL) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated at room temperature for 10 min. After staining, the unbound dye was removed by washing five times with 1% acetic acid, and the plates were dried in the air. The bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at λ = 515 nm. Using the seven absorbance measurements [time zero (t z ), control growth (c), and test growth in the presence of drug at the five concentration levels (t i )], the percentage growth was calculated at each of the drug concentration levels. Growth inhibition (%) was calculated as: [(t i − t z )/(t z )] × 100, for concentrations where t i < t z .
Three dose-response parameters were calculated as follows. GI 50 (growth inhibition of 50%) was calculated from Equation (3), which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Equation (4), where t i = t z . The LC 50 indicating a 50% net loss of cells following treatment was calculated from Equation (5): [(t i − t z )/(c − t z )] × 100 = 0 (4) [(t i − t z )/(t z )] × 100 = −50.

Antiproliferative Assay on HeLa and SiHa Cells
Cervical adenocarcinoma (HeLa) and cervical carcinoma (SiHa) cells were obtained from the European Collection of Cell Cultures (Salisbury, UK) and the American Type Tissue Culture Collection (Manassas, VA, USA), respectively. The cells were cultured in Minimum Essential Medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin at 37 • C in a humidified atmosphere. Media and supplements were purchased from Lonza Group Ltd. (Basel, Switzerland). Cell viability was assessed by the MTT assay as published before [36]. Briefly, the cells were seeded in 96 well plates at 5000 cells/well density. After 24 h, 100 µL of new media containing the test samples was added. After incubation for 72 h, an aliquot of 44 µL of MTT solution (5 mg/mL) was added. After incubation for a further 4 h, the medium was removed by aspiration, the precipitated formazan crystals were dissolved by adding 100 µL of DMSO to each well, and the plates were shaken at 37 • C for 1 h. The absorbance was measured at 545 nm with a microplate reader. IC 50 values were calculated by fitting sigmoidal dose-response curves by the nonlinear regression model log (inhibitor) vs. normalized response and variable slope fit of GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). Clinically utilized anticancer agent cisplatin (Ebewe GmbH, Unterach, Austria) was included as a reference molecule.

Conclusions
As a result of the current study, hybrid compounds containing chrysin coupled with substituted 1,2,3-triazole pharmacophores showed significant in vitro anticancer activities on several cell lines of different types of cancer. Moreover, the activity of the bis-conjugated derivatives of chrysin was also considerable. Therefore, it may be a reasonable strategy to prepare further hybrid molecules of flavones with more complex structures to obtain potentially valuable new antitumor leads.