Amidine- and Amidoxime-Substituted Heterocycles: Synthesis, Antiproliferative Evaluations and DNA Binding

The novel 1,2,3-triazolyl-appended N- and O-heterocycles containing amidine 4–11 and amidoxime 12–22 moiety were prepared and evaluated for their antiproliferative activities in vitro. Among the series of amidine-substituted heterocycles, aromatic diamidine 5 and coumarine amidine 11 had the most potent growth-inhibitory effect on cervical carcinoma (HeLa), hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (SW620), with IC50 values in the nM range. Although compound 5 was toxic to non-tumor HFF cells, compound 11 showed certain selectivity. From the amidoxime series, quinoline amidoximes 18 and 20 showed antiproliferative effects on lung adenocarcinoma (A549), HeLa and SW620 cells emphasizing compound 20 that exhibited no cytostatic effect on normal HFF fibroblasts. Results of CD titrations and thermal melting experiments indicated that compounds 5 and 10 most likely bind inside the minor groove of AT-DNA and intercalate into AU-RNA. Compounds 6, 9 and 11 bind to AT-DNA with mixed binding mode, most probably minor groove binding accompanied with aggregate binding along the DNA backbone.

Hybridization of coumarin moiety with other anticancer pharmacophores was found to provide novel anticancer candidates with low toxicity, high specificity and excellent efficacy against drug-susceptible and drug-resistant cancers [3,13,22]. It was observed that substituent, length and position of alkyl spacer had a profound effect on the anticancer potency. 1,2,3-Triazole-containing novobiocin analogues with triazole ring at C-3 of coumarin were investigated [23] and showed that coumarin-1,2,3-triazole-indole VII (Figure 2) exhibited potent antiproliferative activity against two breast cancer cell lines (SKBr-3 and MCF-7), which was directly related to inhibition of the Hsp90-mediated protein folding process. Artemisinin-coumarin compound VIII accumulated in cancer-cell mitochondria efficiently and induced the generation of intracellular ROS, triggered cancer cell apoptosis, and consequently caused cancer cell death [24]. In addition, chalcone-coumarin hybrids displayed cytotoxicity against lymphoblastic leukemia (MOLT-3) cells without affecting normal cells [25]. Among these, compound IX showed strong activity against human liver cancer (HepG2) cells and no toxicity to Vero cells. Further investigations revealed that coumarin-1,2,3-triazole-benzimidazole derivative X induced cell death, mainly due to early apoptosis, and its inhibition of 5-lipoxygenase activity and perturbation of sphingolipid signaling by interfering with intracellular acid ceramidase activity [26]. Another coumarin-1,2,3-triazole hybrid XI containing amide showed moderate to excellent activity against human breast cancer (MDA-MB-231) cells, revealing general enhancement of proliferationinhibiting activity under hypoxia, contrasted with normoxia [27]. 6-Hydroxycoumarin XII linked through triazole with ortho-substituted phenyl moiety showed to be selectively cytotoxic against lung cancer (A-549) cell line [28]. and MCF-7), which was directly related to inhibition of the Hsp90-mediated protein folding process. Artemisinin-coumarin compound VIII accumulated in cancer-cell mitochondria efficiently and induced the generation of intracellular ROS, triggered cancer cell apoptosis, and consequently caused cancer cell death [24]. In addition, chalcone-coumarin hybrids displayed cytotoxicity against lymphoblastic leukemia (MOLT-3) cells without affecting normal cells [25]. Among these, compound IX showed strong activity against human liver cancer (HepG2) cells and no toxicity to Vero cells. Further investigations revealed that coumarin-1,2,3-triazole-benzimidazole derivative X induced cell death, mainly due to early apoptosis, and its inhibition of 5-lipoxygenase activity and perturbation of sphingolipid signaling by interfering with intracellular acid ceramidase activity [26]. Another coumarin-1,2,3-triazole hybrid XI containing amide showed moderate to excellent activity against human breast cancer (MDA-MB-231) cells, revealing general enhancement of proliferation-inhibiting activity under hypoxia, contrasted with normoxia [27]. 6-Hydroxycoumarin XII linked through triazole with ortho-substituted phenyl moiety showed to be selectively cytotoxic against lung cancer (A-549) cell line [28]. Heterocyclic diamidines have been extensively studied as DNA minor groove binders [29]. The research on amidines has been successful in demonstrating a correlation between the structure and biological activity with their ability to bind in minor grooves [29,30].
To improve bioavailability and oral absorption of amidines, the strategy of amidoxime prodrugs was successfully applied [31,32].
Based on the above-mentioned data and in continuation of our research on 1,2,3-triazolyl-appended heterocycles [26,[33][34][35] and their amidino derivatives [8,36,37] as anticancer and antipathogenic agents, herein, we report the synthesis and antiproliferative evaluations of novel indole, quinoline and coumarin-based aromatic amidines 1-11 and amidoximes 12-22. Since nucleic acids are a major target for a large number of anticancer drugs, one of our aims was to investigate the binding of small molecules to DNA and RNA [29]. Assessing the binding strength and mode of binding of small molecule-DNA/RNA interactions was performed using UV-Vis, fluorescence and circular dichroism (CD) spectroscopy.

Chemistry
Novel hybrids of aromatic nitrile and heterocycle linked via 1,2,3-triazole scaffold 3a-3i were synthesized by regioselective Cu(I)-catalyzed azide-alkyne 1,3-dipolar Heterocyclic diamidines have been extensively studied as DNA minor groove binders [29]. The research on amidines has been successful in demonstrating a correlation between the structure and biological activity with their ability to bind in minor grooves [29,30].
To improve bioavailability and oral absorption of amidines, the strategy of amidoxime prodrugs was successfully applied [31,32].
Based on the above-mentioned data and in continuation of our research on 1,2,3triazolyl-appended heterocycles [26,[33][34][35] and their amidino derivatives [8,36,37] as anticancer and antipathogenic agents, herein, we report the synthesis and antiproliferative evaluations of novel indole, quinoline and coumarin-based aromatic amidines 1-11 and amidoximes 12-22. Since nucleic acids are a major target for a large number of anticancer drugs, one of our aims was to investigate the binding of small molecules to DNA and RNA [29]. Assessing the binding strength and mode of binding of small molecule-DNA/RNA interactions was performed using UV-Vis, fluorescence and circular dichroism (CD) spectroscopy.
Comparison of the activity of evaluated compounds and reference drug (5-FU) showed that from amidine series, compounds 5, 10 and 11 had better antiproliferative effect on HeLa and HepG2 cells, while from amidoximes, only compound 20 had activity comparable to that of 5-FU.

DNA Binding Study
Based upon the antiproliferative activity, several compounds from a series of novel amidine-and amidoxime-substituted heterocycles (5, 6, 811 and 20) were selected for the study with calf thymus DNA (ctDNA), alternating AT-DNA (poly (dAdT)2) and AU-RNA (poly A-poly U). Compounds 5, 6, 811 were soluble in water (c = 2  10 −3 mol dm −3 ). Due to the small aggregation of 20 in water, a stock solution was also prepared in DMSO (c = 2  10 −3 mol dm −3 ) to check the dependence of UV-Vis changes on concentration increase. The absorbencies of buffered aqueous solutions of studied compounds were proportional to their concentrations up to c = 2  10 −5 mol dm −3 (Supplementary information (SI), Figures S1-S8) indicating that studied compounds do not aggregate by intermolecular stacking at experimental conditions used. Absorption maxima and corresponding molar extinction coefficients (ε) are given in Table S1 (SI).
The addition of ctDNA, poly A-poly U and poly (dAdT)2 yielded a fluorescence decrease of 6, 9, 11 and 20 whereas an emission increase was noticed in the case of 5, 8 and 10 with all studied polynucleotides (Table 2, Figure 4). The fluorescence changes of studied compounds were not dependent on the structure of polynucleotide added. cells. Quinoline diamidoxime 20 was around 9-fold more active on HeLa and SW620 cells compared to its monoamidoxime 19. Both quinolines 19 and 20 with methyleneoxy spacer did not exhibit an inhibitory effect on non-tumor HFF cells and selectivity was more expressed by inhibition of compound 20 on HeLa and SW620 cells (SI > 14).
Comparison of the activity of evaluated compounds and reference drug (5-FU) showed that from amidine series, compounds 5, 10 and 11 had better antiproliferative effect on HeLa and HepG2 cells, while from amidoximes, only compound 20 had activity comparable to that of 5-FU.

DNA Binding Study
Based upon the antiproliferative activity, several compounds from a series of novel amidine-and amidoxime-substituted heterocycles (5, 6, 8-11 and 20) were selected for the study with calf thymus DNA (ctDNA), alternating AT-DNA (poly (dAdT) 2 ) and AU-RNA (poly A-poly U). Compounds 5, 6, 8-11 were soluble in water (c = 2 × 10 −3 mol dm −3 ). Due to the small aggregation of 20 in water, a stock solution was also prepared in DMSO (c = 2 × 10 −3 mol dm −3 ) to check the dependence of UV-Vis changes on concentration increase. The absorbencies of buffered aqueous solutions of studied compounds were proportional to their concentrations up to c = 2 × 10 −5 mol dm −3 (Supplementary information (SI), Figures S1-S8) indicating that studied compounds do not aggregate by intermolecular stacking at experimental conditions used. Absorption maxima and corresponding molar extinction coefficients (ε) are given in Table S1 (SI).
The addition of ctDNA, poly A-poly U and poly (dAdT) 2 yielded a fluorescence decrease of 6, 9, 11 and 20 whereas an emission increase was noticed in the case of 5, 8 and 10 with all studied polynucleotides (Table 2, Figure 4). The fluorescence changes of studied compounds were not dependent on the structure of polynucleotide added.    The binding constants Ks and ratios n (bound compound)/(DNA/RNA) obtained by processing of fluorometric titration data with Scatchard equation [40] are summarized in Table 2. Studied compounds showed similar affinity toward ds-DNA and ds-RNA. Still, some compounds such as 5, 6, 9 and 10 showed greater affinities towards p(dAdT) 2 compared to other compounds. In addition, 5 displayed the best affinity towards AU-RNA than other compounds. Fluorescence changes of 8 and 10 with ctDNA and poly A-poly U were too small for accurate calculation of binding constants. Compounds with cyclic amidines at both ends of heterocyclic core (5,6) or with cyclic amidine at one end and quinoline at the other (9) exhibited the preferential binding toward AT sequences. Interestingly, 9 exhibited greater affinities toward polynucleotides than 8, although the only difference was the linker between triazole and quinoline rings, O-alkyl vs. N-alkyl linker.
Compounds 9-11 and especially 5 and 6 showed moderate to big stabilization effect of AT-DNA (Table S2, Figure S41). In comparison to AT-DNA, stabilization of ctDNA by 5 and 6 was weaker as a consequence of the mixed base pair composition of ctDNA (58% AT, 42% GC). Other compounds showed either small or no stabilization effect of polynucleotides. Only a derivative possessing two imidazole moieties at the end of the heteroaromatic structure, 5 stabilized both DNA and RNA.
CD spectroscopy is a highly sensitive method for gaining insight into the conformational changes in the secondary structure of polynucleotides induced by small molecule binding [41]. In addition, it can provide information on the binding mode for small achiral molecules possessing UV-Vis spectra above 300 nm.
Generally, the addition of compounds caused either a small decrease or a small increase of CD intensity of DNA and RNA polynucleotides at their maximal values (ctDNA at 275 nm, AT-DNA and AU-RNA at 260 nm). Still, significantly induced CD (ICD) bands appeared in titrations of 5, 6 and 9-11 with AT-DNA ( Figure 5, SI). Addition of AT-DNA to 6, 9 and 11 induced positive ICD signals in the region from 270-303 nm at ratios, r < 0.2 which could be ascribed to minor groove binding [42]. However, positive and negative signals positioned at 320 and 380 nm appeared at ratios of r higher than 0.2, suggesting additional binding mode-formation and binding of dimers or higher-order aggregates along the polynucleotide backbone. In titrations with AT-DNA, compounds 5 and 10 caused strong positive ICD bands centered around 276 and 287 nm, respectively which is consistent with minor groove binding ( Figure 5, SI). In titrations with AU-RNA with 10 and 11, weak negative ICD signals visible around 300 nm are indicative of intercalative binding mode where the ligands are oriented "parallel" to the long axis of adjacent base pairs [42]. Compound 5 caused an appearance of similar ICD signals (strong positive ICD signals around 284 nm) with ctDNA as in titration with AT-DNA and additionally, positive and negative signals positioned at 320 and 390 nm were indicative of binding of aggregates along the polynucleotide backbone or most probably inside the groove.
Molecules 2021, 26, 7060 9 of 22 5 and 6 was weaker as a consequence of the mixed base pair composition of ctDNA (58% AT, 42% GC). Other compounds showed either small or no stabilization effect of polynucleotides. Only a derivative possessing two imidazole moieties at the end of the heteroaromatic structure, 5 stabilized both DNA and RNA. CD spectroscopy is a highly sensitive method for gaining insight into the conformational changes in the secondary structure of polynucleotides induced by small molecule binding [41]. In addition, it can provide information on the binding mode for small achiral molecules possessing UV-Vis spectra above 300 nm.
Generally, the addition of compounds caused either a small decrease or a small increase of CD intensity of DNA and RNA polynucleotides at their maximal values (ctDNA at 275 nm, AT-DNA and AU-RNA at 260 nm). Still, significantly induced CD (ICD) bands appeared in titrations of 5, 6 and 9-11 with AT-DNA ( Figure 5, SI). Addition of AT-DNA to 6, 9 and 11 induced positive ICD signals in the region from 270-303 nm at ratios, r < 0.2 which could be ascribed to minor groove binding [42]. However, positive and negative signals positioned at 320 and 380 nm appeared at ratios of r higher than 0.2, suggesting additional binding mode-formation and binding of dimers or higher-order aggregates along the polynucleotide backbone. In titrations with AT-DNA, compounds 5 and 10 caused strong positive ICD bands centered around 276 and 287 nm, respectively which is consistent with minor groove binding ( Figure 5, SI). In titrations with AU-RNA with 10 and 11, weak negative ICD signals visible around 300 nm are indicative of intercalative binding mode where the ligands are oriented "parallel" to the long axis of adjacent base pairs [42]. Compound 5 caused an appearance of similar ICD signals (strong positive ICD signals around 284 nm) with ctDNA as in titration with AT-DNA and additionally, positive and negative signals positioned at 320 and 390 nm were indicative of binding of aggregates along the polynucleotide backbone or most probably inside the groove.  In titrations of polynucleotides with 8 and 20, there were no significantly induced CD signals possibly due to the formation of aggregates such as in titration of 20 with ctDNA or a lack of any dominant orientation of 8 and 20 molecules with respect to the polynucleotide chiral axis.

Materials and Methods
All solvents were purified following recommended drying agents and/or distilled over 3 Å molecular sieves. For monitoring the progress of a reaction and for comparison purposes, thin layer chromatography (TLC) was performed on pre-coated Merck (Darmstadt, Germany) silica gel 60F-254 plates using an appropriate solvent system and the spots were detected under UV light (254 nm). For column chromatography silica gel (Fluka, Buchs, Switzerland, 0.063-0.2 mm) was employed, glass column was slurry-packed under gravity. Melting points (uncorrected) were determined with Kofler micro hot-stage (Reichert, Wien, Austria). The IR spectra were recorded on PerkinElmer Spectrum One spectrophotometer (Boston, MA, USA) with KBr disks. Elemental composition analyses of all novel compounds were within the 0.4% of the calculated values. 1 H and 13 C NMR spectra were acquired on a Bruker 300 and 600 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany). All data were recorded in DMSO-d 6 at 298 K. Chemical shifts were referenced to the residual solvent signal of DMSO-d 6 at δ 2.50 ppm for 1 H and δ 39.50 ppm for 13 C. Individual resonances were assigned on the basis of their chemical shifts, signal intensities, multiplicity of resonances and H-H coupling constants. 1 H and 13 C NMR spectra of compounds 4-22 are provided in Supporting Information (Figures S43-S61).

Synthetic Procedures
Compounds 4-azidobenzonitrile (2) [39] and alkynyl derivatives of benzonitrile (1a and 1b) [43,44], quinoline (1e and 1f) [45,46] and coumarin (1h and 1i) [47] were synthesized according to the known procedure. Suspension of corresponding cyano derivative 3a-3i in absolute EtOH was cooled in an ice-salt bath and was saturated with HCl gas. The flask was sealed, and the mixture was stirred at room temperature until the nitrile band disappeared (monitored by the IR analysis at 2200 cm −1 ). The reaction mixture was diluted with dry diethylether, and the crude imidate was filtered off and suspended in absolute ethanol. Ethylenediamine was added and the mixture was stirred at reflux for 24 h. The reaction mixture was diluted with dry diethylether, and the crude base was filtered off. Suspension of base in absolute EtOH was cooled in an ice-salt bath and saturated with HCl gas, sealed and stirred for 24 h. Mixture was diluted with dry diethyl ether and the crude product was then filtered off, washed with diethylether to give powdered product as hydrochloride salt.
T m values are the midpoints of the transition curves determined from the maximum of the first derivative and checked graphically by the tangent method. The ∆T m values were calculated by subtracting the T m of the free nucleic acid from the T m of the complex. Every ∆T m value reported here was the average of at least two measurements. The error in ∆T m is ±0.5 • C.

Fluorometric Measurements
Fluorescence spectra were recorded on a Varian Cary Eclipse spectrophotometer at 25 • C using appropriate 1cm path quartz cuvettes. Fluorometric experiments were performed at pH = 7 (I = 0.05 mol dm −3 , sodium cacodylate buffer) by adding portions of polynucleotide solution into the solution of the studied compound. In fluorometric experiments, an excitation wavelength of λ exc ≥ 300 nm was used to avoid the inner filter effect caused due to increasing absorbance of the polynucleotide. Emissions were determined in the range λ em = 300-600 nm. Values for K s obtained by processing titration data using the Scatchard equation [40], in most titrations have satisfactory correlation coefficients (≥0.98).

CD Measurements
CD spectra were recorded on a JASCO J815 spectrophotometer in 1cm path quartz cuvettes. CD parameters: range = 500-220 nm, data pitch = 2, standard sensitivity, scanning speed = 200 nm/min, accumulation = 3-5. CD experiments were performed at 25 • C and pH = 7 (I = 0.05 mol dm −3 , sodium cacodylate buffer) by adding portions of compound stock solution into the polynucleotide solution.

Conclusions
The novel 1,2,3-triazolyl-appended indole, quinoline and coumarin heterocycles containing aryl imidazoline 4-11 and amidoxime 12-22 moiety substituted at the various position of heterocycle or connected via nitrogen-and oxygen-containing linkers were synthesized with the aim to assess the influence of performed structural modification on antiproliferative activity.
Antiproliferative evaluations on hepatocellular carcinoma (HepG2), showed that asymmetrical aromatic diamidine 5 and coumarin amidine 11 were the most potent compounds, with IC 50 values in the nM range. However, 5 was also highly cytotoxic to normal HFF fibroblasts, while 11 exhibited less toxicity to non-tumor cells with a selectivity index (SI) of 11. Quinoline amidine 8 with -NCH 2 -spacer exhibited marked and selective effects against HepG2. Its analogue 9 with -OCH 2 -unit significantly inhibited HFF fibroblasts. From the amidoxime series, only quinoline amidoximes 18 and 20 showed a strong antiproliferative effect on A549, HeLa and SW620. Bis-amidoxime moiety in 20 contributed to its better activity compared to mono-amidoxime analogue 19. Based on the results of CD titrations and thermal melting experiments, it can be concluded that 5 and 10 most likely bind inside the minor groove of AT-DNA and intercalate into AU-RNA. 6, 9 and 11 bind to AT-DNA with mixed binding mode, most probably minor groove binding accompanied with aggregate binding along the DNA backbone.
Supplementary Materials: The following are available online. UV-VIS spectra, fluorescence spectra, 1 H and 13 C NMR spectra.