Xanthone-1,2,4-triazine and Acridone-1,2,4-triazine Conjugates: Synthesis and Anticancer Activity

A total of 21 novel xanthone and acridone derivatives were synthesized using the reactions of 1,2,4-triazine derivatives with 1-hydroxy-3-methoxy-10-methylacridone, 1,3-dimethoxy-, and 1,3-dihydroxanthone, followed by optional dihydrotiazine ring aromatization. The synthesized compounds were evaluated for their anticancer activity against colorectal cancer HCT116, glioblastoma A-172, breast cancer Hs578T, and human embryonic kidney HEK-293 tumor cell lines. Five compounds (7a, 7e, 9e, 14a, and 14b) displayed good in vitro antiproliferative activities against these cancer cell lines. Compounds 7a and 7e demonstrated low toxicity for normal human embryonic kidney (HEK-293) cells, which determines the possibility of further development of these compounds as anticancer agents. Annexin V assay demonstrated that compound 7e activates apoptotic mechanisms and inhibits proliferation in glioblastoma cells.


Introduction
Cancer, a malignant cell growth, is the second-leading cause of death in the world [1]. Annually, about 19 million people are diagnosed with cancer worldwide, leading to approximately 10 million deaths [1]. The constantly emerging incidence of cancer worldwide makes oncological diseases one of the most serious public health problems. Therefore, exploration and development of novel anticancer drugs are crucial tasks of medicinal chemistry.
A precursor in the acronicine biosynthesis is 3-Methoxy-1-hydroxy-9-methylacridone 2 [8] and it occurs in many plant species [9][10][11][12][13][14]. This compound demonstrates activity on HeLa [14] and colorectal cancer HCT116 [15] cells, human leukemic lymphoblasts CCRF-CEM, glioblastoma cell line U-87, and human alveolar basal epithelial adenocarcinoma A549 [16] cell lines. Symadex 3, also known as C-1311, is a synthetic antitumor agent that passed Phase II clinical trials [17]. Previously, it was shown experimentally that during the initial stage of its action, C-1311 forms stable intercalation complexes with DNA duplexes [17]. Thus, acridone and xanthone derivatives are privileged frameworks fo development of anticancer agents. However, anticancer agents currently development that contain these heterocycles have some drawbacks. They are not a sufficiently active and may have poor solubility [20]. At the same time, the synth modified analogues of natural acridones and xanthones is an effective method to op their antitumor properties [21]. Therefore, the synthesis of new acridone and xan derivatives is an urgent task in the search for new anticancer compounds.

Results and Discussion
In the continuation of our previous work on the synthesis of acridone analogue we have obtained new derivatives of xanthone and acridone and tested them fo activity against several cancer cell lines. Xanthone, a dibenzo-γ-pyrone, may be considered an oxa counterpart of acridone. Xanthone is another framework that is well known for its anti-cancer properties [18]. The anticancer activity of this heterocyclic system is mediated by multiple targets: caspase activation, RNA binding, DNA cross-linking, kinase, aromatase, and topoisomerase inhibition.
There are also many compounds with anticancer activity among xanthones. For example, it was demonstrated the anticancer activity of α-, β-, and γ-mangostin, and mangiferin, a natural product isolated from the fruit and Garcinia mangostana (Clusiaceae).
DMXAA is an example of a synthetic xanthone with antitumor activity [19] mediated by vascular collapse and tumor necrosis through tumor necrosis factor-α induction. DMXAA exhibits low toxicity to normal cells with suitable pharmacokinetic parameters. It successfully passed phase II clinical trials but was withdrawn from phase III clinical trials due to insufficient effectiveness in humans.
Thus, acridone and xanthone derivatives are privileged frameworks for the development of anticancer agents. However, anticancer agents currently under development that contain these heterocycles have some drawbacks. They are not always sufficiently active and may have poor solubility [20]. At the same time, the synthesis of modified analogues of natural acridones and xanthones is an effective method to optimize their antitumor properties [21]. Therefore, the synthesis of new acridone and xanthone derivatives is an urgent task in the search for new anticancer compounds.

Results and Discussion
In the continuation of our previous work on the synthesis of acridone analogues [22], we have obtained new derivatives of xanthone and acridone and tested them for their activity against several cancer cell lines. We chose 1-hydroxy-3-methoxyacridone 2 as a convenient starting material that has potential for solubility due to the presence of a hydroxyl group, has a low molecular weight, and was easily available through the condensation of anthranilic acid with phloroglucinol and subsequent N,O-dimethylation [23]. Thus, this acridone can be modified to obtain substances with the expected acceptable pharmacokinetic properties. The reaction of 1-hydroxy-3-methoxy-9-methylacridone 2 with the triazines 6a-f in acetic acid in the presence of methanesulfonic acid provided compounds 7a-f in good yields (Scheme 1).
The structure of compounds 7a-f is consistent with their NMR spectral data. The 13 C NMR spectra of compounds 7a-f comprise the characteristic resonances for the dihydrotriazine C5′ sp 3 carbon atom at 45 ppm, while the resonance of hydrogen at this sp 3 carbon is observed at 6.3-6.4 ppm in the 1 H NMR spectra. In order to exclude the alternative structure of the isomeric C4 substituted product 8, 2D 1 H-13 C HMBC spectra for compound 7f were recorded. In the HMBC spectra, the signals of the C1 atom at 161.6 has cross-peak with the C5′proton resonance at 6.3-6.4 ppm (compound 7, Scheme 1). In the case of an alternative substitution at the carbon atom in position 4 (compound 8, Scheme 1), this cross-peak cannot be observed since the dihydrotriazine fragment is too far from the carbon atom C1. We further modified the compounds 7a-e by oxidizing the dihydrotriazine ring to a fully aromatic triazine (Scheme 2). Oxidation was performed by boiling a dichloroethane solution of compound 7 with tetrachloro-p-benzoquinone (TCQ). During the oxidation, Scheme 1. Synthesis of dihydrotriazine-substituted acridones 7a-f.
The structure of compounds 7a-f is consistent with their NMR spectral data. The 13 C NMR spectra of compounds 7a-f comprise the characteristic resonances for the dihydrotriazine C5 sp 3 carbon atom at 45 ppm, while the resonance of hydrogen at this sp 3 carbon is observed at 6.3-6.4 ppm in the 1 H NMR spectra. In order to exclude the alternative structure of the isomeric C4 substituted product 8, 2D 1 H-13 C HMBC spectra for compound 7f were recorded. In the HMBC spectra, the signals of the C1 atom at 161.6 has cross-peak with the C5 proton resonance at 6.3-6.4 ppm (compound 7, Scheme 1). In the case of an alternative substitution at the carbon atom in position 4 (compound 8, Scheme 1), this cross-peak cannot be observed since the dihydrotriazine fragment is too far from the carbon atom C1.
We further modified the compounds 7a-e by oxidizing the dihydrotriazine ring to a fully aromatic triazine (Scheme 2). Oxidation was performed by boiling a dichloroethane solution of compound 7 with tetrachloro-p-benzoquinone (TCQ). During the oxidation, the sp 3 -carbon atom signal shifts downfield in the 13 C spectrum, while the hydrogen atom signal (δ = 6.3-6.4 ppm) at this carbon disappears in the 1 H NMR spectrum. The oxidation had no significant effect on the biological activity, as demonstrated using the MTT assay (Table 1, compound 9e), but the solubility of the aromatized triazines 9a-e in aqueous medium was significantly impaired by the oxidation. To further evaluate the role of the 1,2,4-triazine ring for anticancer activity, we transformed the 1,2,4-triazine moiety of compounds 9a-d into pyridine compounds 10a-d (Scheme 3). The reaction proceeds as [2+4] cycloaddition followed by nitrogen and cyclopentadiene extrusion (Boger reaction). We used previously described conditions suitable for this transformation [24,25]. The structure of compounds 10 is consistent with their NMR spectral data. The structure of the product 10b was proven using X-ray diffraction analysis ( Figure 2). To further evaluate the role of the 1,2,4-triazine ring for anticancer activity, we transformed the 1,2,4-triazine moiety of compounds 9a-d into pyridine compounds 10ad (Scheme 3). The reaction proceeds as [2+4] cycloaddition followed by nitrogen and cyclopentadiene extrusion (Boger reaction). We used previously described conditions suitable for this transformation [24,25]. The structure of compounds 10 is consistent with their NMR spectral data. The structure of the product 10b was proven using X-ray diffraction analysis ( Figure 2   transformed the 1,2,4-triazine moiety of compounds 9a-d into pyridine compounds 10ad (Scheme 3). The reaction proceeds as [2+4] cycloaddition followed by nitrogen and cyclopentadiene extrusion (Boger reaction). We used previously described conditions suitable for this transformation [24,25]. The structure of compounds 10 is consistent with their NMR spectral data. The structure of the product 10b was proven using X-ray diffraction analysis ( Figure 2

Synthesis of Xanthone Derivatives
Further, we modified compound 7a by replacing the NMe moiety with an oxygen atom in order to study the role and influence of the nitrogen atom in the tricyclic dibenzoannulated scaffold of the obtained compounds.
The reaction of 1,3-dihydroxyxanthone 11 with 3,5-diphenyl-and 3-methylthiotriazines 6a and 6b provides dihydrotriazines 12a and 12b, respectively (Scheme 4). To our surprise, the reaction proceeded at position 4 of 1,3-dihydroxyxanthone, not at position 2 as observed for acridones (Scheme 1). The position of addition of the triazine ring was supported by the HMBC 1 H-13 C spectrum of compound 12b, wherein cross-peaks between the proton at C2 (δ = 6.3 ppm) and C1 and C3 carbons are registered (Scheme 4). We can attribute the different methylthiotriazines 6a and 6b provides dihydrotriazines 12a and 12b, resp (Scheme 4). To our surprise, the reaction proceeded at position 4 dihydroxyxanthone, not at position 2 as observed for acridones (Scheme 1). The p of addition of the triazine ring was supported by the HMBC 1 H-13 C spectrum of com 12b, wherein cross-peaks between the proton at C2 (δ = 6.3 ppm) and C1 and C3 are registered (Scheme 4). We can attribute the different regiochemistry of the addition to the influence of the steric factor, which, in the presence of the N-methy directs the reaction to the sterically less difficult position 2. We also obtained xanthones in which both the hydroxy groups were replac methoxy groups (Scheme 5) via the reaction of xanthone 11 with triazine dihydrotriazines 14a-b obtained in this scheme were oxidized to triazines 15a similar manner as described earlier (Schemes 2 and 4). We also obtained xanthones in which both the hydroxy groups were replaced with methoxy groups (Scheme 5) via the reaction of xanthone 11 with triazine 6a. The dihydrotriazines 14a-b obtained in this scheme were oxidized to triazines 15a-b in a similar manner as described earlier (Schemes 2 and 4).

Synthesis of Xanthone Derivatives
Further, we modified compound 7a by replacing the NMe moiety with an oxygen atom in order to study the role and influence of the nitrogen atom in the tricyclic dibenzoannulated scaffold of the obtained compounds.
The reaction of 1,3-dihydroxyxanthone 11 with 3,5-diphenyl-and 3methylthiotriazines 6a and 6b provides dihydrotriazines 12a and 12b, respectively (Scheme 4). To our surprise, the reaction proceeded at position 4 of 1,3dihydroxyxanthone, not at position 2 as observed for acridones (Scheme 1). The position of addition of the triazine ring was supported by the HMBC 1 H-13 C spectrum of compound 12b, wherein cross-peaks between the proton at C2 (δ = 6.3 ppm) and C1 and C3 carbons are registered (Scheme 4). We can attribute the different regiochemistry of the triazine addition to the influence of the steric factor, which, in the presence of the N-methyl group, directs the reaction to the sterically less difficult position 2.
We also obtained xanthones in which both the hydroxy groups were replaced with methoxy groups (Scheme 5) via the reaction of xanthone 11 with triazine 6a. The dihydrotriazines 14a-b obtained in this scheme were oxidized to triazines 15a-b in a similar manner as described earlier (Schemes 2 and 4).

Anticancer Activity of the Obtained Acridone and Xanthone Compounds
Acridones 7a-e were tested for their antiproliferative activity by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [26,27]. The results of a single-concentration screening against human colorectal cancer cells (HCT116) are presented in Table 1. As evident from the screening results, compounds 7b and 7d exhibited no antiproliferative activity against the HCT116 cancer cell line. On the other hand, derivatives 7a, 7c, and 7e revealed profound antiproliferative action. We tested promising compounds 7a and 7e on other cancer lines, glioblastoma A-172 and breast ductal carcinoma Hs-578T, and found high activity of the tested compounds on these cell lines. In addition, an evaluation of the It is interesting to note that the introduction of the triazine ring significantly increases the anticancer activity of the acridone pharmacophore in some cases (Table 1, cf. compound 2 and compounds 7a, 7e, and 9e). However, in some cases, the activity does not change significantly (Table 1, compound 7b).
It is important to note the critical role of the triazine cycle for antitumor activity. Thus, the transformation of the triazine ring into pyridine results in the complete disappearance of activity (compounds 7a and 7c vs. 10a and 10c, Table 1).
Xanthone derivatives 14a-b showed moderate activity in the HCT116 cancer cell test, while the aromatic triazine 15b and dihydroxy counterparts 12a and 12b showed no activity (Table 1).

Investigation of the Anticancer Mechanism of Action of Acridone Compounds
A highly specific and sensitive method for detecting apoptosis is a method based on the treatment of cells with fluorescently-tagged Annexin V (FITC) proteins. Annexin V is able to bind to phosphatidylserines that appear on the cell surface during apoptosis. Additionally, propidium iodide (PI) dye was used to stain DNA inside dead cells or those with irreversibly damaged membranes (necrotic or late apoptotic cells).
Based on the results of cell staining with a mixture of Annexin V-FITC and PI, the number of A-172 and HEK-293 live, apoptotic, and dead cells was calculated for the compounds that showed the greatest effectiveness in suppressing the viability of malignant cells, 7a and 7e (Figure 3). ductal carcinoma Hs-578T, and found high activity of the tested compounds on these cell lines. In addition, an evaluation of the action of compounds 7a and 7e on normal human embryonic kidney (HEK-293) cells showed that these compounds demonstrated low toxicity and, as a result, good therapeutic indexes (IC50 (HEK-293)/IC50 (cancer) = 10-27) on the cancer cell lines studied.
It is interesting to note that the introduction of the triazine ring significantly increases the anticancer activity of the acridone pharmacophore in some cases (Table 1, cf.  compound 2 and compounds 7a, 7e, and 9e). However, in some cases, the activity does not change significantly (Table 1, compound 7b).
It is important to note the critical role of the triazine cycle for antitumor activity. Thus, the transformation of the triazine ring into pyridine results in the complete disappearance of activity (compounds 7a and 7c vs. 10a and 10c, Table 1).
Xanthone derivatives 14a-b showed moderate activity in the HCT116 cancer cell test, while the aromatic triazine 15b and dihydroxy counterparts 12a and 12b showed no activity (Table 1).

Investigation of the Anticancer Mechanism of Action of Acridone Compounds
A highly specific and sensitive method for detecting apoptosis is a method based on the treatment of cells with fluorescently-tagged Annexin V (FITC) proteins. Annexin V is able to bind to phosphatidylserines that appear on the cell surface during apoptosis. Additionally, propidium iodide (PI) dye was used to stain DNA inside dead cells or those with irreversibly damaged membranes (necrotic or late apoptotic cells).
Based on the results of cell staining with a mixture of Annexin V-FITC and PI, the number of A-172 and HEK-293 live, apoptotic, and dead cells was calculated for the compounds that showed the greatest effectiveness in suppressing the viability of malignant cells, 7a and 7e (Figure 3).  The data obtained confirm the marked effect of 7e against malignant cells (Figure 4), using the A-172 line as an example. In addition, the effect of the studied compounds was compared in the experiment with the widely used anticancer drug cisplatin at a similar concentration (6 µM) to compare the intensity of the cytotoxic effect. Compound 7e showed a more intense effect against tumor cells of the A-172 line than cisplatin. At the same time, there was no significant increase in the number of dead cells in the HEK-293 line (Figures 3 and 5). The data obtained confirm the marked effect of 7e against malignant cells (Figure 4), using the A-172 line as an example. In addition, the effect of the studied compounds was compared in the experiment with the widely used anticancer drug cisplatin at a similar concentration (6 μM) to compare the intensity of the cytotoxic effect. Compound 7e showed a more intense effect against tumor cells of the A-172 line than cisplatin. At the same time, there was no significant increase in the number of dead cells in the HEK-293 line (Figures 3 and 5).  Measurement of the cell's ability to synthesize DNA is one of the most important methods for evaluating cell proliferative activity, determining toxicity, and evaluating antitumor drugs. The EdU (5-ethyl-2'-deoxyuridine) assay is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. The detection is based on a copper-catalyzed covalent reaction between azide and alkyne, which are contained in the dye and EdU, respectively.
Assessing the effect of compounds 7a and 7e under study on the ability of cells to Measurement of the cell's ability to synthesize DNA is one of the most important methods for evaluating cell proliferative activity, determining toxicity, and evaluating antitumor drugs. The EdU (5-ethyl-2'-deoxyuridine) assay is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. The detection is based on a copper-catalyzed covalent reaction between azide and alkyne, which are contained in the dye and EdU, respectively.
Assessing the effect of compounds 7a and 7e under study on the ability of cells to synthesize DNA in the HEK-293 line (Figure 6), a minimal effect comparable to intact cells, which served as a negative control, can be noted. Some decrease in proliferative activity can be noted in cisplatin-treated cells. At the same time, a more pronounced effect of compounds 7a and 7e on DNA synthesis in cells is observed in line A-172 (Figure 7). It should also be noted that no signs of cell proliferation were detected in the experiment with compound 7e. In addition, Figures 4 and 7 show a significant decrease in the total number of cells in the experiment with compound 7e relative to the other groups.

Synthesis of Novel Acridone and Xanthone Compounds
All chemicals were purchased from commercial sources and used as received, unless otherwise specified. Column chromatography was performed on SiO2 (230-400 mesh). TLC plates coated with SiO2 60F254 were visualized under UV light.
1 H NMR and 13 C{1H} NMR spectra were recorded at room temperature on a BRUKER AVANCE 400 spectrometer ( 1 H, 400 MHz) or on a BRUKER NEO 600 spectrometer ( 1 H, 600 MHz) in DMSO-d6, DMF-d7, or CDCl3 as the deuterated solvent. Chemical shifts were reported in the δ scale relative to the residual DMSO-d6, DMF-d7, or CDCl3 solvent peaks (2.50 ppm and 39.52 ppm for 1 H and 13 C, respectively for DMSO-d6, 8.03, and 163.15 ppm for 1 H and 13 C, respectively for DMF-d7, and 7.26 and 77.17 for 1 H and 13 C, respectively for CDCl3). NMR spectra were registered at ambient temperature.

Synthesis of Novel Acridone and Xanthone Compounds
All chemicals were purchased from commercial sources and used as received, unless otherwise specified. Column chromatography was performed on SiO 2 (230-400 mesh Structural studies were performed using equipment available in the Collaborative Access Center "Testing Center of Nanotechnology and Advanced Materials" at the Mikheev Institute of Metal Physics, Ural Branch, Russian Academy of Sciences. The X-ray diffraction analysis was performed at room temperature on a Rigaku XtaLAB Synergy-S diffractometer. The calculations were performed using Olex 2 v. 171.41.120a 64-bit software. According to the published procedures, 1-hydroxy-3-methoxy-10-methylacridin-9(10H)-one 2 [23], 1,2,4-triazines 6 [27], and xanthones 11 and 13 [28] were prepared.

Viability Assessment
The compounds were dissolved in DMSO. The solutions were diluted with DMEM/F-12 culture medium with 10% fetal bovine serum to the studied concentrations: 1, 2, 4, 8, 16, 32, 64, 128, 256 µM. In all cases, the concentration of DMSO in the final solution did not exceed 1%.
Cells were seeded in 96-well plates at a concentration of 4 × 10 3 cells per well. After 24 h, test compounds were added to the wells in a given concentration range. Then the cells were incubated for 24 h, after which a solution of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was added to the cultures at 20 µL (5 mg/mL) to the well. After 2 h, the medium was removed from the wells, and 200 µL of a 1:1 mixture of DMSO and isopropanol was added. The optical density was measured on a plate spectrophotometer at a wavelength of 570 nm.

Statistical Analysis
Statistical data processing was carried out in the RStudio program (Version 1.4.1106 © 2009-2021 RStudio, PBC) using the R package (version 4.1.2). The cytotoxicity index (IC 50 ) was calculated by plotting dose-response curves using the "drc" package [32].
Stock solutions of the tested compounds of the concentration 0.0125 M were prepared by diluting the suspension in an appropriate volume of DMSO, and then diluting the obtained solution in the nutrient medium DMEM/F-12 (10% FBS) to obtain solutions of the tested concentration in the range from 1 to 128 µM.

Apoptosis
The cells of the lines A-172 and HEK-293 were incubated in 35 mm Petri dishes one day before adding the studied compounds and incubated under the conditions of 5% CO 2 , t = 37 • C, and 95% humidity. Then, solutions of the test compounds and cisplatin were added at a concentration of 6 µM. The cultures were returned to the incubator for 24 h, then the cells were stripped with 0.25% trypsin solution and centrifuged at 200× g for 5 min. The cell precipitate was resuspended in a 25 µL phosphate buffer. To the resulting suspension, 25 µL of freshly prepared dye mixture was added, pipetted, and incubated for 10 min in the dark. After that, 10 µL of the mixture was added to a cell counter slide (C100, RWD) for automatic counting.

DNA Synthesis
Cells were pre-dispersed into the wells of a 96-well plate at a seeding concentration of 4 × 10 3 cells per well the day before the addition of the test substances and incubated in a CO 2 incubator for 24 h. Subsequently, suspensions of the test compounds were added at a concentration of 6 µM. After 24 h, EdU reagent was added, and the cultures were incubated for 2 h. Then the cells were fixed with formaldehyde and permeabilized with Triton X-100. The cells were then incubated for 30 min at room temperature with the reaction mixture, washed, stained with DAPI, and microscopically examined (Optika, Italy).

Conclusions
Herein, we report the synthesis of acridone-triazine, acridone-pyridine, and xanthonetriazine conjugates. The synthetic strategy involves the coupling reaction between hydroxyand/or alkoxy derivatives of dibenzo-pyrones 11 and 13 or pyridones 2 with 1,2,4-triazines 6a-f, followed by optional aromatization of the dihydrotriazine ring leading to aromatic 1,2,4-triazines and the Boger reaction of the triazines yielding pyridines. The antitumor activity was evaluated in vitro on several cell lines. The conjugates of acridone with 1,2,4triazine, compounds 7a and 7c, were the most active among the compounds tested. These compounds show high activity on colorectal cancer (HCT116), glioblastoma (A-172), and breast ductal carcinoma (Hs578T) cell lines, combined with low toxicity on normal human embryonic kidney (HEK-293) cells, and can be used as a starting point in the development of new anticancer agents. It should be noted that aromatization of the dihydrotriazine ring appears to have little effect on activity but significantly reduces the solubility of the compounds. Pyridine derivatives of acridones demonstrated no anticancer activity on the tested cell lines. Conjugates of xanthones with 1,2,4-triazines showed moderate activity on colorectal cancer HCT116 cells. Preliminary mechanistic studies of anticancer activity using the Annexin V assay demonstrated that compound 7e activates apoptotic mechanisms and inhibits proliferation in glioblastoma cells.