Biological Activity of Amidino-Substituted Imidazo [4,5-b]pyridines

A series of cyano- and amidino-substituted imidazo[4,5-b]pyridines were synthesized using standard methods of organic synthesis, and their biological activity was evaluated. Biological evaluation included in vitro assessment of antiproliferative effects on a diverse selection of human cancer cell lines, antibacterial activity against chosen Gram-positive and Gram-negative bacterial strains, and antiviral activity on a broad panel of DNA and RNA viruses. The most pronounced antiproliferative activity was observed for compound 10, which contained an unsubstituted amidino group, and compound 14, which contained a 2-imidazolinyl amidino group; both displayed selective and strong activity in sub-micromolar inhibitory concentration range against colon carcinoma (IC50 0.4 and 0.7 μM, respectively). All tested compounds lacked antibacterial activity, with the exception of compound 14, which showed moderate activity against E. coli (MIC 32 μM). Bromo-substituted derivative 7, which contained an unsubstituted phenyl ring (EC50 21 μM), and para-cyano-substituted derivative 17 (EC50 58 μM) showed selective but moderate activity against respiratory syncytial virus (RSV).


Introduction
Heterocycles are important building blocks in the rational design of novel biologically active molecules in medicinal and pharmaceutical chemistry due to their structural and chemical diversity and the fact that they are utilized throughout many biochemical processes [1,2]. One of the great benefits of heterocyclic chemistry is the fact that there are numerous ways to manipulate such structures in order to change the type and number of heteroatoms, the size of the ring or the incorporation of functional groups as substituents or as part of the ring itself [3,4]. Thus, the structure of heterocyclic derivatives can be optimized towards more biologically active, selective and stable molecules with potential medicinal applications [5,6].
Among all known heterocycles, nitrogen-containing heterocycles are one of the most important subclasses, with benzimidazole, benzothiazole, benzoxazole and imidazopyridine being the most important ones [7,8]. Consequently, nitrogen heterocycles have been the subject of intensive studies in organic and medicinal chemistry [8,9]. Due to their presence in many natural and synthetic molecules and the fact that they possess a wide range of different biological features, nitrogen heterocycles have been selected as building blocks for the synthesis of various therapeutic small molecules in drug design [10,11]. Imidazopyridines are nitrogen heterocycles containing a pyridine and an imidazole ring in their structure. They can exist as several isomer forms as presented in Figure 1, with to their presence in many natural and synthetic molecules and the fact that they possess a wide range of different biological features, nitrogen heterocycles have been selected as building blocks for the synthesis of various therapeutic small molecules in drug design [10,11]. Imidazopyridines are nitrogen heterocycles containing a pyridine and an imidazole ring in their structure. They can exist as several isomer forms as presented in Figure  1, with imidazo [4,5-b]pyridine, imidazo [4,5-c]pyridine and imidazo [1,2-a]pyridine being the most studied derivatives [12]. Imidazopyridine derivatives have great therapeutic potential, and they play an important role in the treatment of numerous diseases due to the wide range of their biological activities [13][14][15]. Because the structure of the abovementioned isomers is similar to naturally occurring purines, imidazopyridine derivatives can also easily interact with essential biomolecules of living systems.
Among the most important biological properties displayed by imidazopyridine derivatives are antitumor [16][17][18], antibacterial [19,20], antiviral [21] and anti-inflammatory [22] activity. Recently, we have published several studies involving the synthesis, biological evaluation and spectroscopic properties [23] of various imidazo [4,5-b]pyridine derivatives. We confirmed the antioxidative potential of novel unsubstituted and N-substituted imidazo [4,5-b]pyridine derived acrylonitriles, with some derivatives displaying significantly improved activity compared with reference standard BHT [24]. Additionally, imidazo [4,5-b]pyridine-derived acrylonitriles were explored for their in vitro antiproliferative effects on a diverse panel of human cancer cell lines, which yielded three compounds with potent sub-micromolar activity (IC50 0.2-0.6 μM). Immunofluorescence staining and in vitro tubulin polymerization assays confirmed that tubulin was the main target. The most active lead compound potently inhibited cancer cell proliferation and migration while not affecting the viability of normal cells even at the highest concentration tested, making it a very promising anticancer agent [25]. Additionally, we have synthesized substituted imidazo [4,5-b] derivatives and their tetracyclic triaza-benzo[c]fluorenes, which demonstrated prominent antitumoral effects, with the amidino-substituted derivatives being the most active ( Figure 2).  Imidazopyridine derivatives have great therapeutic potential, and they play an important role in the treatment of numerous diseases due to the wide range of their biological activities [13][14][15]. Because the structure of the abovementioned isomers is similar to naturally occurring purines, imidazopyridine derivatives can also easily interact with essential biomolecules of living systems.
Among the most important biological properties displayed by imidazopyridine derivatives are antitumor [16][17][18], antibacterial [19,20], antiviral [21] and anti-inflammatory [22] activity. Recently, we have published several studies involving the synthesis, biological evaluation and spectroscopic properties [23] of various imidazo [4,5-b]pyridine derivatives. We confirmed the antioxidative potential of novel unsubstituted and N-substituted imidazo [4,5-b]pyridine derived acrylonitriles, with some derivatives displaying significantly improved activity compared with reference standard BHT [24]. Additionally, imidazo [4,5-b]pyridine-derived acrylonitriles were explored for their in vitro antiproliferative effects on a diverse panel of human cancer cell lines, which yielded three compounds with potent sub-micromolar activity (IC 50 0.2-0.6 µM). Immunofluorescence staining and in vitro tubulin polymerization assays confirmed that tubulin was the main target. The most active lead compound potently inhibited cancer cell proliferation and migration while not affecting the viability of normal cells even at the highest concentration tested, making it a very promising anticancer agent [25]. Additionally, we have synthesized substituted imidazo [4,5-b] derivatives and their tetracyclic triaza-benzo[c]fluorenes, which demonstrated prominent antitumoral effects, with the amidino-substituted derivatives being the most active ( Figure 2). wide range of different biological features, nitrogen heterocycles have been selected as building blocks for the synthesis of various therapeutic small molecules in drug design [10,11]. Imidazopyridines are nitrogen heterocycles containing a pyridine and an imid azole ring in their structure. They can exist as several isomer forms as presented in Figure  1, with imidazo [4,5-b]pyridine, imidazo [4,5-c]pyridine and imidazo[1,2-a]pyridine being the most studied derivatives [12]. Imidazopyridine derivatives have great therapeutic potential, and they play an important role in the treatment of numerous diseases due to the wide range of their bio logical activities [13][14][15]. Because the structure of the abovementioned isomers is simila to naturally occurring purines, imidazopyridine derivatives can also easily interact with essential biomolecules of living systems.
Among the most important biological properties displayed by imidazopyridine de rivatives are antitumor [16][17][18], antibacterial [19,20], antiviral [21] and anti-inflammatory [22] activity. Recently, we have published several studies involving the synthesis, bio logical evaluation and spectroscopic properties [23] of various imidazo [4,5-b]pyridine derivatives. We confirmed the antioxidative potential of novel unsubstituted and N-substituted imidazo [4,5-b]pyridine derived acrylonitriles, with some derivatives dis playing significantly improved activity compared with reference standard BHT [24] Additionally, imidazo [4,5-b]pyridine-derived acrylonitriles were explored for their in vitro antiproliferative effects on a diverse panel of human cancer cell lines, which yielded three compounds with potent sub-micromolar activity (IC50 0.2-0.6 μM). Immunofluo rescence staining and in vitro tubulin polymerization assays confirmed that tubulin was the main target. The most active lead compound potently inhibited cancer cell prolifera tion and migration while not affecting the viability of normal cells even at the highes concentration tested, making it a very promising anticancer agent [25]. Additionally, we have synthesized substituted imidazo [4,5-b] derivatives and their tetracyclic tria za-benzo[c]fluorenes, which demonstrated prominent antitumoral effects, with the amidino-substituted derivatives being the most active ( Figure 2).  The strongest broad-spectrum antiproliferative effect was observed for triaza-benzo[c]fluorenes bearing a 2-imidazolinyl moiety, which exert their activity by intercalating into dsDNA [26]. Following up on our previously published work on the biological activity of imidazo[4,5-b]pyridines and the fact that the amidino-substituted imidazo [4,5-b]pyridines are underexplored but promising pharmaceuticals, we have now designed and prepared a novel series of imidazo [4,5-b]pyridine derivatives bearing amidino substituents to explore their antiproliferative, antibacterial and antiviral activity.
The strongest broad-spectrum antiproliferative effect was observed for triaza-benzo[c]fluorenes bearing a 2-imidazolinyl moiety, which exert their activity by intercalating into dsDNA [26]. Following up on our previously published work on the biological activity of imidazo [4,5-b]pyridines and the fact that the amidino-substituted imidazo [4,5-b]pyridines are underexplored but promising pharmaceuticals, we have now designed and prepared a novel series of imidazo [4,5-b]pyridine derivatives bearing amidino substituents to explore their antiproliferative, antibacterial and antiviral activity.

Chemistry
Novel amidino-substituted 2-phenyl-substituted imidazo [4,5- Precursors 5-8 were prepared from corresponding 2,3-diaminopyridines 1-2 and benzaldehydes 3-4 in moderate to high yields (58-94%) using DMSO-mediated cyclization in the presence of Na2S2O5. Targeted compounds 9 and 10 bearing an unsubstituted amidino group were synthesized by a one-pot procedure. The previously described method [27] included protection of the NH proton using N,O-bis(trimethylsilyl)acetamide (BSA), nucleophilic addition of lithium hexamethyldisilazane (LiHMDS) and HCl work-up that resulted in deprotected amidines isolated as hydrochloric salts in low to moderate yields (18 and 40%). The reaction was monitored by the appearance of two singlets related to the amino protons in the 9.61-9.37 ppm range. Substituted amidino compounds 11-16 were prepared using a two-step Pinner reaction in 2-methoxyethanol. The progress of the first reaction step was monitored by IR spectroscopy in order to determine the conversion of cyano-substituted analogues 6 and 8 to imino ester. The targeted amidines were synthesized in the second reaction step from imino ester and an excess of corresponding amines, which gave moderate yields (42-70%). The appearance of peaks in the 10.91-10.31 ppm range and in the aliphatic region of the 1 H NMR spectrum related to the protons of the amino group confirmed the formation of the substituted amidino compounds. All amidino-substituted compounds were prepared as hydrochloride salts in order to achieve better solubility. All structures were confirmed by 1 H and 13 C NMR spectroscopy, mass spectrometry and elemental analysis. Precursors 5-8 were prepared from corresponding 2,3-diaminopyridines 1-2 and benzaldehydes 3-4 in moderate to high yields (58-94%) using DMSO-mediated cyclization in the presence of Na 2 S 2 O 5 . Targeted compounds 9 and 10 bearing an unsubstituted amidino group were synthesized by a one-pot procedure. The previously described method [27] included protection of the NH proton using N,O-bis(trimethylsilyl)acetamide (BSA), nucleophilic addition of lithium hexamethyldisilazane (LiHMDS) and HCl work-up that resulted in deprotected amidines isolated as hydrochloric salts in low to moderate yields (18 and 40%). The reaction was monitored by the appearance of two singlets related to the amino protons in the 9.61-9.37 ppm range. Substituted amidino compounds 11-16 were prepared using a two-step Pinner reaction in 2-methoxyethanol. The progress of the first reaction step was monitored by IR spectroscopy in order to determine the conversion of cyanosubstituted analogues 6 and 8 to imino ester. The targeted amidines were synthesized in the second reaction step from imino ester and an excess of corresponding amines, which gave moderate yields (42-70%). The appearance of peaks in the 10.91-10.31 ppm range and in the aliphatic region of the 1 H NMR spectrum related to the protons of the amino group confirmed the formation of the substituted amidino compounds. All amidino-substituted compounds were prepared as hydrochloride salts in order to achieve better solubility. All structures were confirmed by 1 H and 13 C NMR spectroscopy, mass spectrometry and elemental analysis. N-methyl-substituted derivatives 17-19 presented in Scheme 2 were prepared in low yields (12-16%), so the desired Pinner reaction to prepare the corresponding amidino-substituted analogues was not conducted. The alkylation reaction on the imidazo[4,5-b]pyridine core is not selective and often results in different monoalkylated and polyalkylated products [28,29]. Successful alkylation using methyl-iodide in DMF was confirmed by the appearance of signals in the 3.97-3.95 ppm range related to the protons of the methyl group.
Additionally, since some of our previously synthesized benzazole derivatives substituted with different types of amidino substituents showed promising and selective activity against colon adenocarcinoma SW620 and endocervical adenocarcinoma HeLa cells [23,30,31], and since some pentamidine analogues were reported to strongly inhibit the proliferation of prostate cancer [32], all synthesized compounds were tested against SW620, HeLa and PC3 cancer cell lines ( Table 2).
The results were compared to etoposide and doxorubicine as standard antitumor drugs. All derivatives showed moderate to strong antiproliferative activity against at least one of these cell lines, with the exception of compounds 9, 12, 13 and 17. Compounds 5, 6 and 18 displayed selectivity towards HeLa cells, while compounds 10 and 11 showed activity against both HeLa and SW620 cells. The bromo-substituted imidazo[4,5-b]pyridine 8 bearing a 4-cyanophenyl group at position 2 potently inhibited proliferation of all three cell lines (IC 50 1.8-3.2 µM). Generally, we can conclude that the substitution of the pyridine nuclei with bromine markedly increased the antiproliferative activity of the tested imidazo [4,5-b] pyridines (Figure 3). The most promising activity was observed for compound 10, a bromo-substituted derivative bearing an unsubstituted amidino group at the phenyl ring, and (once again) compound 14, the bromo-substituted derivative bearing a 2-imidazolinyl group at the phenyl ring. Both derivatives showed inhibitory activity at sub-micromolar concentrations against colon carcinoma (SW620), with IC 50 values of 0.4 and 0.7 µM, respectively. The N-methyl-substituted derivative 18 showed decreased antiproliferative activity in comparison with the N-unsubstituted analogues 6-8.
The results were compared to etoposide and doxorubicine as standard antitumor drugs. All derivatives showed moderate to strong antiproliferative activity against at least one of these cell lines, with the exception of compounds 9, 12, 13 and 17. Compounds 5, 6 and 18 displayed selectivity towards HeLa cells, while compounds 10 and 11 showed activity against both HeLa and SW620 cells. The bromo-substituted imidazo[4,5-b]pyridine 8 bearing a 4-cyanophenyl group at position 2 potently inhibited proliferation of all three cell lines (IC50 1.8-3.2 μM). Generally, we can conclude that the substitution of the pyridine nuclei with bromine markedly increased the antiproliferative activity of the tested imidazo[4,5-b]pyridines ( Figure 3). The most promising activity was observed for compound 10, a bromo-substituted derivative bearing an unsubstituted amidino group at the phenyl ring, and (once again) compound 14, the bromo-substituted derivative bearing a 2-imidazolinyl group at the phenyl ring. Both derivatives showed inhibitory activity at sub-micromolar concentrations against colon carcinoma (SW620), with IC50 values of 0.4 and 0.7 μM, respectively. The N-methyl-substituted derivative 18 showed decreased antiproliferative activity in comparison with the N-unsubstituted analogues 6-8.

Antibacterial Activity In Vitro
The bactericidal activity of all synthesized derivatives was evaluated against four different bacterial strains ( Table 3).
The Gram-positive bacterial strains comprised S. aureus and S. pneumoniae, while the panel of Gram-negative bacteria included two different strains of E. coli. As reference drugs, four antibiotics (ampicillin, ceftazidime, ciprofloxacin and meropenem) were included. Based on the obtained results, we can conclude that all tested imidazo[4,5-b]pyridine derivatives were devoid of antibacterial activity, with MIC values > 64 μM (Table 3). Derivative 14 substituted with bromine at the pyridine nuclei and bearing a 2-imidazolinyl group at the phenyl ring showed moderate activity against E. coli (MIC 32 μM).

Cytotoxicity and Antiviral Activity in Vitro
All prepared imidazo[4,5-b]pyridines 5-19 were also tested for their cytotoxicity on three virus host cell lines (HEL 299, Huh7 and MDCK) as well as their antiviral activity against several viruses (human coronavirus 229E, NL63 and OC43; influenzavirus A and B; respiratory syncytial virus; herpes simplex virus-1; yellow fever virus; Zika virus and sindbis virus), as depicted in Tables 4 and 5. The results are expressed as CC50 (50% cytotoxic concentration) and EC50 (50% effective concentration) values, respectively, and all inactive derivatives were excluded from Table 5. For comparison of the results, five standard antiviral drugs were included. To assess the possible antiviral activity of the synthesized derivatives, their cytotoxic effect on the virus host cell lines was first determined using the colorimetric MTS assay. No negative impact on the cell viability was measured for the majority of the tested compounds, as can be seen in Table 4. Derivatives 5, 11, 13 and 15 were mildly cytotoxic, with CC50 values above 30 μM on either one or two of the tested host cell lines.

Antibacterial Activity In Vitro
The bactericidal activity of all synthesized derivatives was evaluated against four different bacterial strains (Table 3). The Gram-positive bacterial strains comprised S. aureus and S. pneumoniae, while the panel of Gram-negative bacteria included two different strains of E. coli. As reference drugs, four antibiotics (ampicillin, ceftazidime, ciprofloxacin and meropenem) were included. Based on the obtained results, we can conclude that all tested imidazo [4,5-b]pyridine derivatives were devoid of antibacterial activity, with MIC values > 64 µM (Table 3). Derivative 14 substituted with bromine at the pyridine nuclei and bearing a 2-imidazolinyl group at the phenyl ring showed moderate activity against E. coli (MIC 32 µM).

Cytotoxicity and Antiviral Activity In Vitro
All prepared imidazo [4,5-b]pyridines 5-19 were also tested for their cytotoxicity on three virus host cell lines (HEL 299, Huh7 and MDCK) as well as their antiviral activity against several viruses (human coronavirus 229E, NL63 and OC43; influenzavirus A and B; respiratory syncytial virus; herpes simplex virus-1; yellow fever virus; Zika virus and sindbis virus), as depicted in Tables 4 and 5. The results are expressed as CC 50 (50% cytotoxic concentration) and EC 50 (50% effective concentration) values, respectively, and all inactive derivatives were excluded from Table 5. For comparison of the results, five standard antiviral drugs were included. To assess the possible antiviral activity of the synthesized derivatives, their cytotoxic effect on the virus host cell lines was first determined using the colorimetric MTS assay. No negative impact on the cell viability was measured for the majority of the tested compounds, as can be seen in Table 4. Derivatives 5, 11, 13 and 15 were mildly cytotoxic, with CC 50 values above 30 µM on either one or two of the tested host cell lines.
The most pronounced cytotoxicity was seen for the bromo-substituted compound 14 bearing a 2-imidazolinyl amidino group, which had a relatively high CC 50 on the MDCK cell line (33.9 µM) but CC 50 values of 9.3 µM and even below 0.8 µM on HEL 299 and Huh7 Molecules 2023, 28, 34 8 of 16 cells, respectively. Similar to the antiproliferation results, among amidino-and bromosubstituted imidazo [4,5-b]pyridines, compound 10 bearing an unsubstituted amidino group did not show any cytotoxicity at all, while isopropyl-amidino-substituted derivative 16 showed mild toxicity on MDCK (CC 50 71.2 µM) and a more pronounced cytotoxic effect on Huh7 cells (CC 50 3.0 µM). The whole set of newly synthesized compounds was tested in vitro against a panel of DNA and RNA viruses. Reference antiviral drugs remdesivir, ribavirin, zanamivir, rimantadine and brivudine (BVDU) were included in the CPE reduction assays as positive controls. Overall, no significant broad-spectrum antiviral activity was detected for the imidazo[4,5-b]pyridine derivatives. Compound 8, a bromo-substituted imidazo [4,5-b]pyridine substituted with a 4-cyanophenyl group at position two, as well as compound 10, a bromo-substituted derivative bearing an unsubstituted amidino group at the phenyl ring, both showed weak but broad anti-influenza virus activity, inhibiting all tested subtypes (H1N1, H3N2 and B). The most promising antiviral activity was seen for bromo-substituted derivative 7 with an unsubstituted phenyl ring showing selective activity against RSV (EC 50 21.0 µM) as well as for para-cyano-substituted derivative 17 (EC 50 79.0 µM).

Conclusions
In this study, we describe the synthesis, structural characterization and biological evaluation of imidazo [4,5-b]pyridine derivatives substituted either with bromo, cyano or acyclic and cyclic amidino groups at the phenyl ring. Unsubstituted derivatives were prepared to study the influence of the type of substituent on the biological activity. We used previously published and well-optimized procedures for the synthesis of amidino derivatives from cyano precursors via a Pinner reaction or through reaction with lithium hexamethyldisilazane (LiHMDS).
All synthesized imidazo [4,5-b]pyridines were evaluated for their in vitro biological activity including antiproliferative activity, antibacterial activity and antiviral activity.
Antiproliferative activity was evaluated on a diverse panel of human cancer cell lines and on HeLa, PC3 and SW620 cell lines, which were included because earlier publications pointed out that amidino-substituted derivatives could be active against these three cell lines. Overall, the in vitro evaluation of antiproliferative activity revealed that the most significant impact on cancer cell proliferation was detected for the compound in which bromine was placed at the pyridine nuclei as well as for the cyclic amidino group, namely the 2-imidazolinyl group placed at the para position on the phenyl ring. Furthermore, compounds 10 and 14 substituted with either an unsubstituted amidino or a 2-imidazolinyl group, proved to be the most promising derivatives for pronounced and selective activity against SW620 cells (IC 50 0.4 and 0.7 µM, respectively). These two compounds have been chosen as lead compounds for further optimization of the presented skeleton. The prepared imidazo[4,5-b]pyridines 5-19 showed little or no antibacterial activity against four chosen bacterial strains. Additionally, all compounds were evaluated for their antiviral activity against several viruses. Four compounds showed antiviral activity, with derivatives 7 and 17 showing selective antiviral activity against respiratory syncytial virus (RSV).
In conclusion, we have shown that imidazo [4,5-b]pyridines bearing either a bromine at the pyridine nuclei or an amidine group at the phenyl ring have promising biological potential with pronounced antiproliferative activity. All presented results point out that this chemical class harbors promising candidates for further design and optimization in order to developed potent antiproliferative agents.

General Methods
Melting points were determined using an Original Kofler Mikroheitztisch apparatus (Reichert, Wien, Austria). The 1 H NMR and the 13 C NMR spectra were recorded with the Bruker Avance DPX-300 or Bruker AV-600 using TMS as an internal standard. Chemical shifts were reported in parts per million (ppm) relative to TMS. Elemental analyses for carbon, Molecules 2023, 28, 34 9 of 16 hydrogen and nitrogen were performed on PerkinElmer 2400 elemental analyzer. Analyses were indicated as symbols of elements, and analytical results obtained were within 0.4% of the theoretical value. All compounds were routinely checked by TLC using Merck silica gel 60F-254 glass plates. All NMR spectra are given in Supplementary Materilas.

General Procedure for Preparation of Compounds 5-8
Compounds 5-8 were prepared by heating equimolar amounts of 2,3-pyridines 1-2 and benzaldehyde derivatives 3-4 in the presence 0.55 equivalents of Na 2 S 2 O 5 in DMSO at 165 • C in an oil bath.

General Method for Preparation of Compounds 9-10
Equivalents of BSA were added to the suspension of 6 or 8 in anhydrous THF 1.1, and the reaction mixture was stirred under argon for 30 min. The reaction mixture was cooled to 0 • C, and 15 equivalents of LiHMDS solution (1M in THF) were added dropwise. After completion of addition, the reaction mixture was stirred for 6 h at room temperature, cooled to 0 • C and quenched with 4 M HCl in ethanol. The solvent was removed under pressure, and solid residue was treated with ethanol and 4 M HCl in ethanol until the mixture became acidic. Additional ethanol-HCl was added, and the mixture was stirred for 2 h. After removing the solvent under reduced pressure, diethyl ether was added, and the solid was separated by filtration. The solid was suspended in water and treated with 20% NaOH to pH 9. The mixture was filtered, and the air-dried solid was suspended in ethanol and treated with HCl. Diethyl ether was added and the solid was separated by filtration, which gave amidine HCl salt.

General Method for Preparation of Compounds 11-16
A nitrile compound was suspended in anhydrous 2-methoxyethanol and cooled to 0 • C. Dry HCl gas was bubbled into a cooled suspension for 4 h. The suspension was monitored with IR spectroscopy and stirred at room temperature until the -CN band was undetectable. Anhydrous diethyl ether was added to the suspension and the solid was filtered off and dried in vacuo. The dry product was suspended in anhydrous ethanol, and corresponding amine was added. The crude product was filtered off and washed with diethyl ether to give powder products, which were suspended in anhydrous ethanol and concentrated HCl was added. For the synthesis of imidazolinyl-substituted amidines 11 and 14, 3.5 equivalents of ethylendiamine were added to a suspension of imidate ester, and the reaction was heated for 24 h. For the synthesis of 1,4,5,6-tetrahydropyrimidinesubstituted amidines 12 and 15, two equivalents of 1,3-propylenediamine were added to a suspension of imidate ester, and the reaction was heated for 24 h. For the synthesis of isopropyl-substituted amidines 13 and 16, five equivalents of isopropyl amine were added to a suspension of imidate ester, and the reaction was heated for 24 h.
Leibniz-Institut, Germany). Culture media were purchased from Gibco Life Technologies, USA, and supplemented with 10% fetal bovine serum (HyClone, GE Healthcare Life Sciences, Chicago, IL, USA), except for the media for SW620, PC3 and HeLa cells, which were purchased from Sigma.

Proliferation Assays
Adherent cell lines LN-229, HCT-116, NCI-H460 and Capan-1 cells were seeded at a density between 500 and 1500 cells per well in 384-well tissue culture plates (Greiner). After overnight incubation, cells were treated with seven different concentrations of the test compounds, ranging from 100 to 0.006 µM. Suspension cell lines HL-60, K-562, Z-138 and DND-41 were seeded at densities ranging from 2500 to 5500 cells per well in 384well culture plates containing the test compounds at the same concentration points. Cells were incubated for 72 h with compounds and were then analyzed using the CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) reagent (Promega) according to the manufacturer's instructions. Absorbance of the samples was measured at 490 nm using a SpectraMax Plus 384 (Molecular Devices), and OD values were used to calculate the 50% inhibitory concentration (IC 50 ). Compounds were tested in two independent experiments. SW620, PC3 and HeLa cells were seeded in 96-well microtiter plates at 1 × 10 4 to 3 × 10 4 cells/mL, depending on the doubling times of the specific cell line. Test agents were then added in five 10-fold dilutions (10 −8 to 10 −4 M). Working dilutions were freshly prepared on the day of testing. After 72 h of incubation, the cell growth rate was evaluated using the MTT assay as described previously [33]. Absorbance of the samples was measured at 570 nm, and OD values were used to calculate the 50% inhibitory concentration (IC 50 ). Each test was performed in quadruplicate in at least two individual experiments.

Antibacterial Activity In Vitro Materials
In addition to the synthesized compounds, the standard antibiotics ampicillin, ceftazidime, ciprofloxacin and meropenem from USP were tested. Selected bacterial strains were Gram-negative Escherichia coli (ATCC 25922) and Gram-positive Staphylococcus aureus (ATCC 29213) and S. Pneumoniae (ATCC 49619). Saccharomyces cerevisiae ATCC 7752 strain was tested as a eukaryotic model organism. Synthesized compounds were prepared as 10 mM DMSO solutions and tested in a final concentration range of 100-0.2 µM [34]. Standard antibiotics were prepared as 5 mg/mL DMSO solutions and tested in a final concentration range of 64-0.125 µg/mL.

Methods
Broth microdilution testing was performed according to CLSI (Clinical Laboratory Standards Institute) guidelines. MIC (minimal inhibitory concentration) values were defined as the last tested concentration of the compound at which there was no visible growth of bacteria. Inoculums for each microorganism were prepared using the direct colony suspension method, where broth solutions that achieved turbidity equivalent to 0.5 McFarland standard were additionally diluted 100× with Ca-adjusted MH media (Becton Dickinson). All test plates were incubated for 16-24 h at 37 • C. MIC values for reference antibiotics against quality control strains were used to confirm the validity of the screen according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically were obtained from M07, 11th edition, 2018. and Clinical and Laboratory Standards Institute (CLSI) guidelines. Performance standards for antimicrobial susceptibility testing were obtained from M100, 28th edition, 2018.