Сationic carboxamide derivatives of tricyclic heteroaromatic compounds: synthesis and preliminary evaluation of antiproliferative activity

This research was aimed at the synthesis and study of biological activity of the carboxamides of tricyclic heteroaromatic systems, acridone, phenazine, and thioxanthone, containing the aliphatic and aromatic cationic substituents at amide fragment. These heterocyclic cores are DNA intercalating agents, whereas the introduction of cationic groups provides additional ionic interactions of the ligands with their biological targets, such as DNA and enzymatic complexes of the system of nucleic acids biosynthesis. A convenient way of the introduction of such groups is a modification of heterocyclic carboxamides. A small library of new N-substituted cationic amide derivatives of acridone-4-, phenazine-1and thioxanthone-4-carboxylic acids was obtained. They were synthesized in 37-81% yield by mild and selective quaternization of the nitrogen atoms at N,N-dimethylaminoalkyl (alkyl = ethyl, propyl) and pyridylmethyl fragments of the neutral N-functionalized carboxamides with methyl iodide. Tricyclic heteroaromatic cores were not affected. Convenient protocol for the synthesis of thioxanthone-4-carboxylic acid (TCA) based on the reaction of 2-mercaptobenzoic and 2-iodobenzoic acids followed by cyclization of the intermediate was developed (yield 79%). A series of new N-functionalized neutral amides of TCA, the precursors of corresponding cationic carboxamide, were also obtained via the reaction of acyl chloride with amines. Preliminary in vitro testing of four compounds as potential antitumor agents in U87MG tumor cell culture (human malignant glioma) demonstrated their significant antiproliferative activity at low micromolar concentrations, with growth inhibition values GI50 in the range 1.7-11 μM. These results suggest that cationic carboxamides of tricyclic heteroaromatic systems are promising scaffolds for the design of new antitumor drugs.


Introduction
Condensed tricyclic heteroaromatic systems are privileged scaffolds for the design of therapeutic agents for the treatment of various diseases [1][2][3][4][5]. In particular, a broad variety of antitumor, antibacterial, and antiviral drugs belong to this class of compounds, including the derivatives of acridine, phenazine, and thioxanthone. In most cases, such compounds target the cellular enzymatic systems of nucleic acids biosynthesis. Small molecules based on condensed tricyclic heterocycles were reported as efficient Important factors influencing their biological activity are the structure of heterocycle, the nature of amide substituent, and the position of the carboxamide group in the core molecule [10,16].
Much less attention has been paid to the studies of bioactivity of compounds based on thioxanthone, a close acridone analogue. Among thioxanthone derivatives, antitumor agents have been reported [41][42][43], although we were unable to find have not found in the literature data on the activity of thioxanthone carboxamides.
We have previously obtained a series of carboxamides of acridone and phenazine whose amide groups were functionalized with N,N-dimethylaminoalkyl and pyridyl fragments [38]. The introduced basic functions can be protonated under physiological conditions to form cationic moieties. Structure design was based on the fact that the attachment of basic/cationic substituents to DNA-intercalating ligands could enhance their binding to DNA or enzymatic complexes formed by the enzymes of nucleic acids biosynthesis (topoisomerase, telomerase, DNA, and RNA polymerases, etc.) by additional interactions with either anionic DNA phosphates or acidic groups within the enzymes. At the same time, the aromatic pyridyl residues could also interact with nucleic acids bases or aromatic amino acids via the hydrophobic mechanism further enhancing the binding of ligands to their molecular targets. Phenazine and acridone derivatives containing aromatic pyridyl fragments were found to inhibit the topoisomerase I at 100 μM concentration, whereas their analogues with aliphatic basic substituents at carboxamide fragment were less efficient [38]. At the same time, the free core heterocycles and their non-substituted carboxamides are inactive against topoisomerase [38] and telomerase [24].
Thus, the introduction of protonable basic substituents significantly increased the biological activity of tricyclic carboxamides, and we could expect that the modification of core heterocycles with cationic fragments would result in even more efficient inhibitors with potential antitumor and/or antibacterial properties. In this work, we have significantly extended the range of N-substituted tricyclic carboxamides as potential antitumor agents. (Scheme 1). Their amide groups were functionalized with N,N-dimethylamino and isomeric pyridyl groups attached via the short (1-3 carbon atoms) alkyl linkers [38]. Taking into account the above considerations, we have decided to prepare cationic derivatives of tricyclic heterocycles. One of the possible ways to obtain compounds of this type would be a quaternization of nitrogen atoms of basic aliphatic and aromatic substituents present in the already prepared carboxamides 1,2a-e. We have also decided to extend the range of tricyclic heteroaromatic systems by adding their structural analogue, thioxanthone.

Results and discussion
Carboxamides 1,2a-e have been previously synthesized from carboxylic acids [38]. To obtain analogues based on thioxanthone, first of all, we have elaborated an efficient procedure for the synthesis of thioxanthone-4-carbocylic acid (TCA). This procedure is similar to that commonly used for the synthesis of acridone-4-carboxylic acid [44] and is based on intramolecular cyclization of bis-dicarboxyphenyl sulfide formed in the reaction of 2-mercaptobenzoic and 2-iodobenzoic acids.
We have modified a published protocol [45] using potassium carbonate as a base instead of NaOH, and 2-iodobenzoic acid in place of 2-chlorobenzoic acid. The condensation of two acids at 60 °С followed by the intermediate cyclization by heating in conc. sulfuric acid afforded TCA in 79% yield (Scheme 2). The use of less reactive bromobenzoic acid in the condensation required heating at a higher temperature (100 °С) and resulted in a significantly lower total yield of target tricyclic carboxylic acid (below 50%).
The synthesis of new N-substituted carboxamides of thioxanthone was based on our previous approach developed for phenazine and acridone series [38]. This convenient one-flask process consisted in the formation of acyl chloride followed by its reaction with amine. Target neutral amides 3a-e were obtained by the reaction of TCA chloride with corresponding amines in the presence of TEA (Scheme 1).
The neutral compounds 1-3a-e were found to easily react with methyl iodide, and the quaternization of nitrogen atoms in amide substituents allowed obtaining a series of novel cationic derivatives 1-3f-j. N-Alkylation reaction was carried out in polar solvent (methanol, acetonitrile) at room temperature or with some heating (up to 50 °C). Since salttype products precipitated from the reaction mixture, the use of crude amides instead of analytically pure samples did not significantly affect the total yield of iodides from starting carboxylic acids.
The primary centres of N-methylation are obviously tertiary aliphatic (AlkNMe2) and pyridine nitrogen atoms in carboxamide fragments. Only one additional methyl group signal appeared in 1 H NMR spectra of all cationic derivatives. It is known that the quaternization of phenazine under the applied conditions does not occur, but possible alkylation of endocyclic nitrogen or exocyclic oxygen atom of the acridone ring could not be excluded. However, NMR spectra of the obtained derivatives and their comparison with the spectral data of reference compounds confirmed that compounds 1f-j do not contain N-methyl group located at acridine ring, as one-proton low-field signals at δ ≥ 12 ppm characteristic of the 10-NH ring proton of neutral carboxamides of acridone carboxylic acid [44] are observed in the spectra. NMR spectra of cationic TCA carboxamides also contain the signals of methyl groups only from trimethylammonium or N-methylpyridinium residues.
Cationic aliphatic trimethylammonium group of compounds 1-3f-g is represented by singlets at 3.1-3.4 ppm, whereas the spectra of pyridinium derivatives 1h-j, 2i, and 3i contain the signals of cationic N-methyl group located at 4.2-4.4 ppm. Low-field shift of signals of the cationic fragments is observed in NMR spectra of the salts, as compared to corresponding neutral carboxamides. In general, the deshielding effect of cationic structures results in the shift of CONH and methylene protons (lowfield shift for 0.1-0.5 and 0.15-0.25 ppm, respectively) in comparison with neutral precursors.
Thus, we have prepared a series of 11 new compounds containing trimethylammonium group attached via the ethyl or propyl linker, and compounds with isomeric N-methylpyridinium fragments. This small library would allow analyzing the structure-activity relationship among the derivatives of three tricyclic systemsphenazine, acridone, and thioxanthone.

Investigation of antitumor activity of compounds in vitro
Preliminary evaluation of the antiproliferative activity of some new compounds was performed in vitro in the culture of U87MG tumor cells (human malignant glioma). To determine the effect of quaternization on biological activity, the representative pairs of the derivatives of two different heterocycles, acridone and phenazine, containing the same pyridyl and N-metyylpyridinium fragments (1d, i and 2d, i) were tested. The cells were cultured in 24-well plates and treated for 3 days by drugs added at concentrations ranging from 20 to 0.5 µM.
In vitro cytostatic activity of compounds towards cancer cell line was determined using the classic MTT assay [46]. MTT test is based on the transformation of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by the mitochondrial dehydrogenase of viable cells into the blue formazan which can then be measured spectrophotometrically. The optical density of the probe is proportional to the number of live cells. From the obtained data, the plots of the number of live cells in the probe as compared to a control (cell growth inhibition level) vs. drug concentration were built, from which the GI50 value was obtained for each tested carboxamide. GI50 was determined as a concentration of drug required for 50% of maximal inhibition of cell proliferation (decreasing cell vitality by 50%) as compared to the non-treated control.
Tested carboxamides demonstrated a significant dosedependent antiproliferative activity towards U87MG cells at low micromolar concentrations, with GI50 below 10 µM for three of four compounds (Table 1). It is interesting to note that the cationic acridone derivative 1i (GI50 7.2 µM) was more active than its neutral analogue 1d (GI50 11 µM), whereas for the pair of phenazine carboxamides the cationic compound 2i was three times less efficient as compared to its non-charged counterpart 2d (GI50 5.5 and 1.7 µM, respectively).
The opposite effects of quaternization on the biological activity of acridone and phenazine carboxamides may be due to different molecular targets of the studied compounds and/or different modes of inhibitor-target interaction. However, at this moment we have only limited information on biological properties of a small set of derivatives, and molecular mechanisms of bioactivity of new compounds require further investigation. In particular, it will include the studies on the inhibition of the enzymes of nucleic acid biosynthesis and ligand interactions with nucleic acids. It would be also interesting to access the antibacterial activity of these derivatives.

Conclusions
A convenient protocol was proposed for the synthesis of cationic N-functionalized carboxamide derivatives of acridone, phenazine, and thioxanthone, the tricyclic systems with DNA-intercalating properties. A series of compounds with aliphatic and aromatic cationic substituents were obtained. Their heteroaromatic cores contained the carboxamide functions modified with

General procedure for the synthesis of N-substituted amides of thioxanthone-4-carboxylic acid (3а-e).
0.5 Mmol (128 mg) of TCA was suspended in 3 ml of dry toluene, and 50 µl of thionyl chloride and 60 µl of dry pyridine (0.7 mmol each) were added with stirring and the mixture was heated at 90 °С for 1.5-2 h. After cooling to room temperature corresponding amine (1.25 mmol) and triethylamine (1.25 mmol) were added, and the mixture was stirred at ambient temperature until the reaction was complete (control TLC). The mixture was evaporated, the residue was treated with 10 ml of chloroform and washed with saturated NаНСО3 (3×5 ml). The organic phase was dried over Nа2SО4 and evaporated to dryness. The product was crystallized from the appropriate solvent.
Neutral heterocyclic N-substituted carboxamide 1-3a-e (0.1 mmol) and 150 µl of methyl iodide in 2 ml of methanol or acetonitrile were kept at room temperature or with weak heating (up to 50 °C) until the reaction was complete (control TLC). The precipitated product (1-3f-j) was collected by filtration and crystallized from the appropriate solvent.  The cells were grown in 24-well plastic plates (ТТР, Switzerland) in CO2-incubator at 37 °C, 5% CO2. The cells (2x10 3 per well) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma, USA) supplemented with 2.5% fetal bovine serum (Sigma, USA). In 24 h after cell seeding, tested compounds in DMSO were added to the culture at concentrations 20, 10, 5, 2, 1 and 0.5 µM (final drug concentration in the medium) using a serial dilution approach, and then cells were incubated for 72 h. In all cases, DMSO content in the medium was 0.2%. Preliminary experiments confirmed that DMSO at this concentration did not affect cell growth. The cells cultured in the presence of 0.2% DMSO without drugs were used as a control. After the incubation of cells with or without drugs, the number of viable cells in each well was determined using a standard MTT colorimetric assay [46]. After the treatment wit MTT reagent, optical density in the wells was measured at 570 nm using BioTek ELx800 plate reader (BioTek, USA). Using the absorbance measurements, the percent of growth inhibition as compared with a non-treated control was calculated for each drug concentration. Growth inhibition levels were plotted against inhibitor concentrations, and GI50 parameter (drug concentration giving a 50% growth inhibition in comparison with a control culture) was determined for each compound. Each experiment was performed in triplicate. The data are presented as the mean (M) ± standard deviation (SD).