Synthesis of Morpholine-, Piperidine-, and N-Substituted Piperazine-Coupled 2-(Benzimidazol-2-yl)-3-arylquinoxalines as Novel Potent Antitumor Agents

Abstract

A novel series of 2-(benzimidazol-2-yl)quinoxalines with three types of pharmacophore groups, namely, piperazine, piperidine, and morpholine moieties, which are part of known antitumor drugs, was designed and synthesized. The compounds have been characterized by NMR and IR spectroscopy, high- and low-resolution mass spectrometry, and X-ray crystallography. 2-(Benzimidazol-2-yl)quinoxalines with N-methylpiperazine substituents showed promising activity against a wide range of cancer lines, without causing hemolysis and showing little cytotoxicity against normal human Wi-38 cells (human fetal lung). A mixture of regioisomers 2-(benzimidazol-2-yl)-3-(4-fluorophenyl)-6(and 7)-(4-methylpiperazin-1-yl)quinoxalines (mriBIQ 13da/14da) showed a highly selective cytotoxic effect against human lung adenocarcinoma (cell line A549) with a half-maximal inhibitory concentration at the level of doxorubicin with a selectivity index of 12. The data obtained by flow cytometry, fluorescence microscopy, and multiparametric fluorescence analysis suggested that the mechanism of the cytotoxic effect of the mriBIQ 13da/14da on A549 cells may be associated with the stopping of the cell cycle in phase S and inhibition of DNA synthesis as well as with the induction of mithochondrial apoptosis. Thus, mriBIQ 13da/14da can be considered as a leading compound deserving further study, optimization, and development as a new anticancer agent.

Cancer has become the leading cause of death worldwide, especially in developing countries. According to the International Agency for Research on Cancer (IARC), there were 19.3 million new cancer cases and nearly 10.0 million cancer deaths that occurred in 2020 worldwide, and furthermore, the global cancer burden is estimated to increase to 28.4 million cases in 2040 by a 41% rise from 2020.¹ As for Russia in 2020, there were 591 371 newly diagnosed cases of cancer, and 312 122 deaths were recorded this year. At the same time, lung cancer was the most common cause of death from oncological diseases (there were 1.8 million deaths) in 2020 worldwide.² Thus, the investigation and identification of more effective agents for the treatment of cancer are urgently needed.

DNA is one of the key targets for cytotoxic antiproliferative drugs. As a rule, these antitumor agents damage DNA or block DNA synthesis; thus, they are responsible for inhibiting the biosynthesis of nucleic acid precursors or disrupt the hormonal stimulation of cell growth.³ Thus, it is necessary to develop a more effective, less toxic, and target-specific noncovalent DNA-binding anticancer drug. Currently, significant efforts are focused on the development of new anticancer drugs based on a combination of two active pharmacophores, in particular, bicyclic fragments, which are effective for DNA binding and cleavage⁴ under physiological conditions. The structures of benzimidazole and quinoxaline promote strong binding of the compound to DNA and therefore affect critical metabolic pathways. A large number of derivatives based on these pillars have been developed and evaluated for antitumor activity. Among these compounds, Nocodazole,^5,6 Bendamustine,^5,7 Mebendazole,^8,9 and Veliparib^10,11 are used in the clinic, while Carbendazim^12,13 is currently undergoing clinical trials (Figure 1). Simple quinoxaline heterocycles also display a broad spectrum of promising biological activities.¹⁴⁻¹⁷ Potent anticancer quinoxalines include XK469 (NSC 697887)^18,19 and CQS (NSC 339004),¹⁹⁻²¹ as depicted in Figure 1. The anticancer activity of benzimidazoles and quinoxalines is apparently due to their ability to form strong complexes with nucleic acids and, thus, cause DNA damage and have concomitant effects, such as topoisomerase poisoning, telomerase inhibition, and gene transcription inhibition.²²⁻²⁵ Consequently, extensive studies of the method and mechanism of interaction of benzimidazoles for binding to DNA have also been reported in various laboratories.²⁶⁻²⁸ To regulate the properties of the interaction of drugs with the benzimidazole backbone, various substituents as well as new cycles such as quinoxaline (1),²⁹ pyrimidine (2),³⁰ pyrrole (3),³¹ pyrazole (4),³² quinoline (5, Dovitinib),³³ etc. were introduced at position 2 (Figure 2).

Figure 1.

Examples of benzimidazole-containing drugs and promising structures of quinoxaline derivatives.

Figure 2.

Structures of 2-heteroarylbenzimidazole derivative as potent antitumor agents.

With these derivatives, pharmacological data showed that the maximum cytotoxicity was obtained with compounds substituted at positions 2 or 6 of benzimidazole and, in particular, with 2-quinoxalinone, 2-pyrimidine, 2-pyrrole, and 2-quinoline ring systems. Additionally, it turned out that some of these compounds that were evaluated against cell lines and DNA showed positive activity against the tumor and strong binding interactions with DNA. We expected that the titled molecules with 2-substituted benzimidazole containing morpholine, piperidine, and N-substituted piperazine moieties at position 6 would also bind to DNA and promote interaction with nucleophilic sites of nucleic acid bases since both morpholine and piperidine and N-substituted piperazine cycles are part of many anticancer drugs. Elucidation of the DNA-binding activity of these analogues may provide new opportunities for the search for other pharmacological activities.

The most promising compounds for oncological practice inhibit tumor growth, which is associated with their ability to bind DNA, and structures such as substituted bicyclic systems with piperazine, piperidine, and morpholine fragments on the one hand and various heterocyclic fragments on the other with a common C–N bond (Figure 3, structures of type A)^16,34−37 carbo- and hetarylquinoxalines (Figure 3, structures of type B and C),³⁸⁻⁴¹ and heteroarylbenzimidazoles (Figure 3, structures of type D)⁴²⁻⁴⁴ with a common C–C bond, including benzimidazolylquinoxalines^29,38 with various substituents, exhibit pronounced antitumor activity. From all the above-mentioned thought this prompted us to obtain new regioisomeric derivatives of 2-(benzimidazol-2-yl)-3-arylquinoxalines with piperazine, piperidine, and morpholine fragments at positions 6 and 7 (Figure 3, structures 13/14: X = NMe, NPh, CH₂, O) for a detailed study of their interaction with DNA and antitumor activity on a panel of cancer cell lines.

Figure 3.

Design strategy using a concept of molecular hybridization in a novel series of compounds as potent antitumor agents: target compounds 13/14.

To check these binding interactions and antitumor activity of a novel series of regioisomeric 2-(benzimidazol-2-yl)-3-arylquinoxalines designed using a concept of molecular hybridization with the mentioned substituents in positions 6 and 7 of the benzene ring of the quinoxaline system, we studied the reactivity of 3-aroylquinoxalinones with respect to specially synthesized benzene-1,2-diamines with N-methyl, N-phenylpiperazine, piperidine, and morpholine substituents in position 4 of the benzene ring under Mamedov rearrangement⁴⁵⁻⁵¹ conditions.

Results and Discussion

Chemistry

The synthesis of three types of new regioisomeric derivatives of 2-(benzimidazol-2-yl)quinoxaline 13 and 14 containing, in position 6 or 7, 4-methylpiperazine, 4-phenylpiperazine (Table 1, entries 1–5 and 6) (type I), piperidine (Table 1, entries 7 and 8) (type II), and morpholine (Table 1, entries 9 and 10) (type III) fragments, was carried out via Mamedov rearrangement⁴⁵⁻⁵¹ by the interaction of 3-aroylquinoxalinones 9a–9f with benzene-1,2-diamines 12a–12d at reflux in acetic acid for 4 h. Along with the target products 13ea, 14ea (Table 1, entry 5) and 13ed, 14ed (Table 1, entry 10), byproducts 15ea, 16ea and 15ed, 16ed are formed, respectively (Figure 4), as a result of nucleophilic substitution of the fluorine atom of the aryl ring with the nitrogen atom of the benzimidazole system;^57,58 mixtures of regioisomers 15ea/16ea and 15ed/16ed were isolated by column chromatography. The structures of compounds 13da and 14da were confirmed by the single-crystal X-ray analysis (Figure 5), and the phase state of a powder mixture of these regiosomers was analyzed by powder X-ray diffraction (see Supporting Information). 3-Aroylquinoxalinones 9 have been prepared by a procedure described by us^52,53 (Scheme 1). Diamines 12 were obtained by reduction of the corresponding ortho-nitroanilines 11(54,55) according to a literature procedure⁵⁶ (Scheme 2).

Table 1. Synthesis of Target Compounds 13 and 14.

	substrates					product
entry	9	12	R	Ar	X	13 + 14	yield, %,a ratio of 13/14b	yield of 13, %a	yield of 14, %a
1	9a	12a	H	Ph	NMe	13aa+14aa	73 (50:50/52:48)	n/i	n/i
2	9b	12a	H	4-ClC6H4	NMe	13ba+14ba	73 (50:50/50:50)	n/i	n/i
3	9c	12a	H	2,4-Cl2C6H3	NMe	13ca+14ca	79 (75:25/90:10)	n/i	n/i
4	9d	12a	H	4-FC6H4	NMe	13da+14da	75 (50:50/52:48)	13dad	14dad
5	9e	12a	H	2-FC6H4	NMe	13ea+14ead	63 (60:40/48:52)	13eae	n/i
6	9d	12b	H	4-FC6H4	NPh	13db+14db	71 (50:50/48:52)	n/i	n/i
7	9d	12c	H	4-FC6H4	CH2	13dc+14dc	54 (50:50/48:52)	13dc (12)	14dc (8)
8	9f	12c	5/6-Cl	4-FC6H4	CH2	13fc+14fcc	75 (50:50/52:48)	n/i	n/i
9	9d	12d	H	4-FC6H4	O	13dd+14dd	73 (50:50/48:52)	n/i	n/i
10	9e	12d	H	2-FC6H4	O	13ed+14edd	48 (60:40/48:52)	13ed (12)	n/i

^aIsolated yields of purified products. n/i = not isolated.

^bThe ratio was determined by ¹H NMR; the ratio of isomers in evaporated mass/analytically pure samples is indicated in parentheses.

^cEach of the regioisomers 13 fc and 14 fc in DMSO-d₆ solution exists in two tautomeric forms in a 1:1 ratio (see Materials and Methods).

^dIndividual compounds 13da and 14da were obtained by recrystallization of the mixture of regioisomers 13da/14da.

^eThe compound 13ea was isolated from the mixture of regioisomers 13ea/14ea by silica gel column chromatography.

Figure 4.

Structures of compounds 15 and 16.

Figure 5.

Molecular structures of 13da (A) and 14da (B) obtained by X-ray crystallography. The triclinic unit cell of 13da contained two independent molecules with different conformations; the monoclinic unit cell of 14da contained four independent different conformers.

Scheme 1. Synthesis of Starting Compounds 9.

Reagents and conditions: (a) benzene-1,2-diamine, AcOH, rt. (b) DMF, NaN₃, rt. (c) aq. AcOH, reflux.

Scheme 2. Synthesis of Starting Compounds 12.

Reagents and conditions: (a) 1-methyl(phenyl)piperazine, piperidine or morpholine, K₂CO₃, DMF, 120 °C. (b) H₂, 10% Pd/C, EtOAc-MeOH (4:1).

Cytotoxicity Assay

All types of synthesized 2-benzimidazolylquinoxaline derivatives with piperazine, piperidine, and morpholine fragments were tested for cytotoxic activity against seven cancer cell lines and the Normal Human Fetal Lung Fibroblast line (WI38). Cytotoxicity on human cancer and normal cell lines was studied at concentrations of 1–100 μM. Table 2 shows the half-maximal inhibitory concentration (IC₅₀) data for the compounds under study. In the series of 2-(benzimidazol-2-yl)-3-arylquinoxalines containing an N-methylpiperazine fragment (entries 1–8), a significant increase in the cytotoxic effect against cancer lines M-HeLa, MCF-7, HuTu 80, and A549 was observed in the case of mixtures of regioisomers with a 2-fluorophenyl substituent (13ea/14ea) and against all cancer lines used in the experiment—in the case of mixtures of regioisomers with a 4-fluorophenyl substituent (13da/14da). Among compounds with an N-methylpiperazine fragment, an mriBIQ 13da/14da should be distinguished. The IC₅₀ values of this mixture on lung adenocarcinoma (A549) and cervical carcinoma (M-HeLa) cancer cell lines were at the level of the comparison drug doxorubicin (entries 4 and 16). It is important to note that, relative to the normal cell line (Wi38), the mriBIQ 13da/14da was less toxic than doxorubicin. Despite the fact that the mriBIQ 13da/14da is inferior in activity to doxorubicin against other cancer cell lines, its cytotoxic effect is much higher than that of other mixtures of 13/14 regioisomers in the tested series. An important step in the presented studies was the isolation of individual 13da and 14da isomers and the study of their cytotoxic properties. It was shown that the 13da and 14da isomers are significantly inferior in activity to the mixture 13da/14da (entries 4–6). Next, an mriBIQ 13db/14db containing a 4-fluorophenyl substituent in position 3 and an N-phenylpiperazine fragment in position 7 or 6 was obtained. The study of the cytotoxic activity of the mriBIQ 13db/14db showed that the introduction of the N-phenylpiperazine fragment instead of the N-methylpiperazine fragment into the molecule led to a complete loss of cytotoxic properties compared to the mriBIQ 13da/14da (entries 6 and 9). The replacement of the N-substituted piperazine fragment in the structure of isomers 13da/14da with the piperidine (13dc/14dc) or morpholine (13dd/14dd) fragment resulted in a significant decrease or complete loss of cytotoxic activity (entries 10 and 14). Regioisomers 13dc and 14dc were isolated individually. The study of their cytotoxic activity showed that they exhibit greater activity against most of the cancer lines used in the experiment compared to mriBIQ 13dc/14dc (entries 10–12). We note that the regioisomer 13dc shows a selective cytotoxic effect against lung adenocarcinoma (A549) with an IC₅₀ value of 26.3 μM (entry 11). Modification of the structure of compounds 13dc/14dc by introducing a Cl atom (13 fc/14 fc) into the benzene ring led to the appearance of antitumor activity against the pancreatic cancer line (PANC-1) with an IC₅₀ value of 29.7 (entry 13). Replacement of the N-methylpiperazine fragment with a morpholine fragment in 2-benzimidazolylquinoxalines containing a 2-fluorophenyl substituent in position 3 leads to a decrease in cytotoxic activity against all cancer lines, with the exception of HuTu 80 and A549, which was shown in the study of the cytotoxic activity of 13ea isomers (entry 8) and 13ed (entry 15).

Table 2. Cytotoxic Effects of Compounds 13 and/or 14 on Cancer and Normal Human Cell Lines.

^aM-HeLa is a human cervix epitheloid carcinoma.

^bMCF-7 is a human breast adenocarcinoma (pleural fluid).

^cHuTu-80 is a duodenal adenocarcinoma.

^dPANC-1 is a human pancreatic cancer cell line.

^eA549 is a adenocarcinomic human alveolar basal epithelial cell line.

^fPC3 is a human prostate adenocarcinoma.

^gT98G is a glioblastoma cell line.

^hWi38 is a human embryonic lung. The experiments were repeated three times.

Thus, in the tested series of three types of 2-benzimidazolylquinoxalines, the mriBIQ 13da/14da is the most promising for further study as a new agent against lung cancer.

We do not have an unambiguous explanation of the reason that individual regioisomers 13da, 14da are not active in cytotoxicity assay; however, mriBIQ 13da/14da is active, but we tried to explain this by structural differences found as a result of crystallographic studies of regioisomers 13da and 14da when they are in an individual form and mixed state. So, comparison of the experimentally obtained diffraction pattern of the polycrystalline mixture of regioisomers 13da and 14da with the diffraction patterns theoretically calculated from single-crystal data for crystals of individual compounds 13da and 14da (Figure S5) convincingly indicates the presence of an additional unknown crystalline phase in the mixture. Upon careful examination of the powdered product of the mixture of regioisomers, very small crystals were isolated. A preliminary X-ray diffraction analysis of the crystals led to obtaining triclinic unit cell parameters that differ from those previously established. The calculation for a given crystalline form of the theoretical diffraction pattern (Figure S7, brown curve) and comparison with the experimental diffraction pattern of a mixture of regioisomers convincingly indicates the presence of this particular crystalline form as an additional phase. Thus, a polycrystalline sample of a mixture of regioisomers contains three crystalline phases: individual regioisomers 13da and 14da and a crystalline form that is possibly a cocrystal of two regioisomeric forms 13da and 14da. Taking into account the lability of the molecules of these compounds, as evidenced by the presence in their crystals of several independent molecules in different conformations, it can be assumed that it is quite easy to form their various polymorphic forms or the cocrystals of regioisomers in various stoichiometric ratios. Thus, it is possible that the latter is responsible for the cytotoxicity of regioisomeric mixtures. The analysis of the obtained experimental data continues.

The selectivity of compounds in relation to cancer cells is an important criterion for evaluating the cytotoxic effect. For this purpose, the selectivity index (SI) was calculated as the ratio between the IC₅₀ value for normal embryonic lung cells (Wi38) and the IC₅₀ value for cancer cells. The SI values for the mriBIQ 13da/14da are given in Table 3. Compounds with SI ≥ 10 are considered highly selective.⁵⁹ According to these data, the mriBIQ 13da/14da exhibits high selectivity for the A549 lung adenocarcinoma cell line. The SI value for this line was 12. At the same time, the reference drug doxorubicin was significantly inferior to the leading compound in selectivity (SI < 1).

Table 3. In Vitro Cytotoxic Effects (μM) and Selectivity Index Values (SI) of mriBIQ 13da/14da.

cancer cell lines	IC50 (μM)	SI
M-HeLa	5.1	6.6
MCF-7	16.6	2.0
HuTu 80	26.6	1.3
PANC1	9.2	3.7
A549	2.8	12.0
PC3	31.0	1.1
T98G	14.5	2.3

Hemolytic Activity

Hemolysis of erythrocytes by molecules of tested compounds is of great importance in the development of new drugs.⁶⁰ The hemolytic activity of the compounds can be used as a means for their toxicological evaluation. In this regard, for 2-(benzimidazol-2-yl)quinoxalines 13 and/or 14, the concentration (HC₅₀) causing hemolysis of 50% of erythrocytes was determined. The test compounds were shown to have no hemolytic activity in the concentration range tested (values of HC₅₀ > 100 μM).

Real-Time Monitoring of A549 Cell Proliferation

Figure 6 shows the data of real-time monitoring of A549 cell proliferation after the addition of the mriBIQ 13da/14da. Cell index (CI) is a dimensionless parameter that is calculated as a relative change in electrical impedance in a well and reflects the biological state of the cells. The CI value characterizes the change in cell proliferation in the population. The higher the CI value, the higher the level of cell proliferation. It can be seen that, when mriBIQ 13da/14da is added at high concentrations of 100 and 50 μM (curves 2 and 3), the CI sharply decreases to 0 (curves 1 and 2). These data indicate the complete cessation of proliferative processes in the A549 cell population. When the concentration of mriBIQ 13da/14da was reduced to 25 μM, a slight decrease in the CI of the cells was observed compared to the control. However, they did not lose the ability to proliferate and continued to actively divide, albeit with a lower cell index (curve 3). At concentrations in the range of IC values of mriBIQ 13da/14da, proliferative processes remained at the control level for 7.5 h (curves 5 and 6). Then their CI sharply decreased, which characterized a decrease in the activity of proliferative processes in A549 cells. The cell growth rate was calculated from the slope of the line between the values at certain time points. Figure 7A,B shows the change in cell growth rate over the time period from adding samples to the end of the experiment. The results obtained are consistent with the data on the evaluation of the cellular index (Figure 6). Figure 7A shows the period of cell growth from the point of application to the moment of a sharp decrease in CI of cells (period from 25 to 35 h) treated with mriBIQ 13da/14da at concentrations of 1 and 5 μM. When the lead compound was added at concentrations of 100 and 50 μM, the cell growth rate was dramatically reduced (columns 2 and 3). At a concentration of 25 μM (column 4), the cell growth rate decreased slightly, and when treated with the lowest concentrations of mriBIQ 13da/14da 1 and 5 μM (columns 5 and 6), it appeared at the control level (column 1). Figure 7B illustrates the cell growth period of A549 from 35 to 40 h. It can be seen that, at high concentrations, the cell growth rate does not change over time (columns 2 and 3); that is, the cells stop dividing. The addition of mriBIQ 13da/14da at a concentration of 25 μM (column 4) leads to some increase in the rate of cell division, which indicates the response of A549 cells to stress conditions. They begin to actively divide to preserve the population under adverse conditions. The presence of mriBIQ 13da/14da at concentrations close to the IC₅₀ values (columns 5 and 6) leads to a sharp decrease in the rate of cell division, which may be associated with cell cycle disruption and induction of apoptosis in A549 cells.

Figure 6.

Real-time monitoring of A549 cell proliferation. (1) Control. (2) 100 μM mriBIQ 13da/14da. (3) 50 μM mriBIQ 13da/14da. (4) 25 μM mriBIQ 13da/14da. (5) 5 μM mriBIQ 13da/14da. (6) 1 μM mriBIQ 13da/14da.

Figure 7.

Cell growth rate calculated by the slope of the line between the values at time points. (A) Time point (25–35) h. (B) Time point (35–40) h. (1) Control. (2) 100 μM mriBIQ 13da/14da. (3) 50 μM mriBIQ 13da/14da. (4) 25 μM mriBIQ 13da/14da. (5) 5 μM mriBIQ 13da/14da. (6) 1 μM mriBIQ 13da/14da.

Cell Cycle Analysis

As practice shows, the effect of cytotoxic agents on cells may be associated with a violation of the cell cycle passage by cells and a slowdown in their proliferation. Cell cycle analysis by quantifying the DNA content of a cell is a reliable method for assessing at what phase the cell cycle has been stopped. We have evaluated the effect of mriBIQ 13da/14da on the cell cycle of A549 cells using the fluorescent dye propidium iodide. The classic cell cycle analysis on a flow cytometer is based on measuring the amount of DNA in a cell to detect it at different stages of the cell cycle at the level of each cell, and the resulting histogram shows the number of cells in each phase. The results of the cell cycle analysis after treatment of the cell line A549 mriBIQ 13da/14da are shown in Figure 8.

Figure 8.

Effect of mriBIQ 13da/14da on A549 cell cycle arrest. (1) mriBIQ 13da/14da at concentration 1 μM. (2) mriBIQ 13da/14da at concentration 2.5 μM. (3) mriBIQ 13da/14da at concentration 5 μM. (A) Cell distribution histograms. (B) Percentage of cells in the G0/G1, S, and G2/M phases (data are presented as mean ± standard deviation of three independent experiments). *Values indicate P < 0.05.

Based on the data of real-time monitoring of cell proliferation, the cell cycle was assessed 12 h after the addition of the compound at concentrations of 1, 2.5, and 5 μM, since during this period of time, at the given concentrations, mriBIQ 13da/14da begins to exhibit a cytotoxic effect. Figure 8 shows that stable retention of A549 cells is observed in the S-phase of the cell cycle. The number of cells in the S-phase after treatment with mriBIQ 13da/14da at concentrations of 1, 2.5, and 5 μM increased and stand at 49.0%, 66.3%, and 68.0%, respectively, while 8.7% of cells were observed in the control. The main event of the S-phase of the cell cycle is DNA synthesis (replication). It is known that some antitumor drugs (e.g., methotrexate, cytarabine, hydroxyurea) are effective precisely in the S-phase of the cell cycle, being replication inhibitors. Based on data from Cell cycle analysis, we hypothesized that the compound may exhibit a phase-specific cytotoxic effect and possibly be an inhibitor of DNA synthesis.

Multiplex Analysis of Markers DNA Damage/Genotoxicity

Traditional genotoxicity analyses detect mutations associated with gain or loss of function but provide little or no data on possible molecular mechanisms of DNA damage. Therefore, to assess the possible genotoxic properties of mriBIQ 13da/14da, we used multiplex analysis and the MILLIPLEX MAP 7-plex DNA Damage/Genotoxicity Magnetic Bead Kit, which allows us to detect the expression and phosphorylation of a number of proteins involved in the detection and repair of DNA damage. This method provides a faster and more accurate assessment of the condition of cells for researchers exposing them to potentially genotoxic compounds. The 7-plex DNA Damage/Genotoxicity Magnetic Bead Kit is designed to detect changes in phosphorylated Chk1 (Ser345), Chk2 (Thr68), H2AX (Ser139), and p53 (Ser15) as well as total ATR, MDM2, and p21 protein levels in cell lysates with using the Luminex system.

DNA damage in living cells is often caused by exposure to various genotoxic agents. The cell tries to repair this damage in order to maintain its viability. There are many proteins involved in the detection and repair of DNA damage. These proteins are sensors, mediators, converters, and effectors. Sensory proteins such as Rad9, Rad1, and Hus1 accumulate at the site of DNA damage and promote phosphorylation of checkpoint proteins; this phosphorylation is influenced by intranuclear kinases ataxia-telangiectasia (ATR) and ataxia-telangiectasia mutated (ATM). Activation of mediator proteins such as H2AX, BRCA1, and SMC1 results in persistent protein–protein interactions that facilitate the transmission of signals to intranuclear ATR and ATM kinases, which in turn activate checkpoint kinases Chk1 and Chk2. Checkpoint kinases are required to stop the cell cycle before mitosis in response to DNA damage. Various mediators, such as Mre11 and MDC1, receive post-translational modifications that are created using detector proteins. These modified mediator proteins then amplify the DNA damage signal and transduce the signals to downstream effectors such as MDM2 and p53. The p53 effector is a tumor suppressor protein that plays a critical role in the decision of a cell to undergo cell cycle arrest or apoptosis after various stresses, including chemical DNA damage. The main targets of p53 are the genes for the p21 protein (a cyclin-dependent kinase inhibitor), which causes cell cycle arrest at the G1 stage. The coordinated functioning of these and a number of other proteins causes the cell cycle to stop, which makes it possible to repair genetic damage, and if it is impossible to carry out repair processes, it induces p53-dependent apoptosis.⁶¹

Figure 9 shows the data of the multiplex analysis performed using the 7-plex DNA Damage/Genotoxicity Magnetic Bead Kit. The results obtained characterize the effect of mriBIQ 13da/14da on the DNA of A549 cells. The studies were carried out at concentrations of 2.5 and 5 μM, leading to the most effective delay of the cell cycle in the S-phase, in which DNA damage is recognized. It can be seen that the values of the average fluorescence intensity of all the presented markers of DNA damage and genotoxicity are much higher than in the control. Moreover, a dose-dependent effect is observed. The results obtained characterize a significant degree of ATR kinase activation, which is the most important marker of DNA damage. The fluorescence intensity values of checkpoint kinases Chk1 and Chk2, mediator protein H2AX, and effectors DM2, p53, and p21 were also higher than in the control. The not very high content of the p21 protein, which characterizes the arrest of the cell cycle in the G1 phase, testifies in favor of the data on the possible S-phase-specific action of mriBIQ 13da/14da.

Figure 9.

Multiplex analysis of markers DNA Damage/genotoxicity in A549 cells treated mriBIQ 13da/14da. (1) mriBIQ 13da/14da at concentration 2.5 μM. (2) mriBIQ 13da/14da at concentration 5 μM. A549 cells untreated with the test substance (control).

Currently, apoptosis is one of the main mechanisms used in the creation of new anticancer drugs. The identification of markers of the mitochondrial or caspase-independent apoptotic pathway (proteins p53 and p21) using multiplex analysis suggests that mriBIQ 13da/14da may have an apoptosis-inducing effect.

The caspase-independent pathway of apoptosis induction is accompanied by damage to the mitochondrial membrane and a decrease in its potential.⁶² The possibility of induction of mitochondrial apoptosis as a result of treatment of the A549 cell line mriBIQ 13da/14da was assessed by flow cytometry using the fluorescent dye JC-10 from the Mitochondria Membrane Potential Kit. In normal cells with a high mitochondrial membrane potential, JC-10 forms aggregates (J-aggregate) with red fluorescence. At the same time, in apoptotic cells, where the membrane potential decreases, JC-10 transforms into a monomeric form (J-monomer) and emits green fluorescence, which is recorded by a flow cytometer. After treatment of A549 cells with mriBIQ 13da/14da at concentrations of 1, 2.5, and 5 μM, a decrease in the mitochondrial membrane potential was observed, which increased with increasing concentration. From the results in Figure 10, it can be seen that the intensity of the red fluorescence decreases as the concentration of the test compound increases. The results obtained suggest that mriBIQ 13da/14da induces mitochondrial apoptosis in A549 cells.

Figure 10.

Flow cytometry analysis of A549 cells treated with mriBIQ 13da/14da. (1) mriBIQ 13da/14da at concentration 1 μM. (2) mriBIQ 13da/14da at concentration 2.5 μM. (3) mriBIQ 13da/14da at concentration 5 μM. (A) Cell distribution histograms. (B) Quantitative determination of % cells with red aggregates.

The ability of compounds to increase the production of reactive oxygen species (ROS) also characterizes the development of mitochondrial apoptosis. It is known that mitochondria are both a potential source and target of ROS, which leads to loss of mitochondrial functions and cell death. In this regard, we studied how treatment of cells A549 with mriBIQ 13da/14da at concentrations of 1, 2.5, and 5 μM affects ROS production using fluorescence microscopy and 2′,7′-dichlorofluorescein (2′,7′-dichloro-3′,6′-dihydroxy-3H-spiro[[2]benzofuran-1,9′-xanthen]-3-one (DCFH-DA)) fluorescent dye. The DNA intercalating dye 4′,6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei (blue emission).

The data presented in Figure 11 demonstrate a significant increase in the intensity of DCFH-DA fluorescence as the concentration of mriBIQ 13da/14da increases compared to the control, which indicates an increase in ROS generation.

Figure 11.

Induction of the production of intracellular ROS in A549 cells incubated with mriBIQ 13da/14da. (1) mriBIQ 13da/14da at concentration 1 μM. (2) mriBIQ 13da/14da at concentration 2.5 μM. (3) mriBIQ 13da/14da at concentration 5 μM.

With the method of fluorescence microscopy, the morphology of A549 cells was analyzed after treatment with the mriBIQ 13da/14da at concentrations of 1, 2.5, and 5 μM (Figure 11). The use of DAPI as a fluorescent dye made it possible to observe dose-dependent nuclear damage compared to controls. As the concentration increased, a significant decrease in the size of the nucleus was observed. Normal oblong-shaped cells contracted and acquired a rounded shape. The nature of chromatin distribution has changed (condensation into irregular lumps, compaction). Micrographs showed a bright blue glow of fragmented DNA, which is characteristic of apoptosis.

Conclusion

In summary, based on a combination of pharmacologically significant structures, a series of new 2-(benzimidazol-2-yl)quinoxaline derivatives containing piperazine, piperidine, and morpholine fragments from 3-aroylquinoxalinones and corresponding benzene-1,2-diamines according to the Mamedov rearrangement was designed and synthesized with good and high final yields. A study of the cytotoxicity of synthesized compounds in relation to normal and tumor human cell lines revealed a selective cytotoxic effect of a mixture of regioisomers 2-(benzimidazol-2-yl)-3-(4-fluorophenyl)-6(and 7)-(4-methylpiperazin-1-yl)quinoxaline (mriBIQ 13da/14da) against the lung adenocarcinoma line human A549 with IC₅₀ at the doxorubicin level with a selectivity index of 12. Flow cytometry, laser confocal microscopy, and multiplex analysis have demonstrated that the mechanism of cytotoxic action of mriBIQ 13da/14da on A549 cells can be associated with both cell cycle arrest in phase S and inhibition of DNA synthesis and with induction of mitochondrial apoptosis. All tested compounds do not cause hemolysis and exhibit little cytotoxicity in relation to normal human cells Wi38 (human embryo lung). Thus, mriBIQ 13da/14da can be considered as a promising basis for the creation of new effective antitumor agents.

Materials and Methods

General Procedures

Melting points were determined on a Boetius hot-stage apparatus (Dresden, Germany) and are uncorrected. NMR spectra were obtained on a Bruker AVANCE 500 MHz spectrometer (Bruker company, Germany) with DMSO-d₆ as solvents at ambient temperature. IR spectra were recorded on a Bruker Vector-22 spectrometer (Karlsruhe, Germany). Mass spectrometry (MS) spectra were performed using an Ultraflex III TOF/TOF mass spectrometer (Bruker Daltonic GmbH, Germany). 4-Nitroaniline was used as the matrix. PEG-300, PEG-400, and Triton X-100 were used for calibration of accurate masses. Column chromatography was performed on a Kieselgel silica gel (0.060–200 mm, 40 A). The purity of all tested compounds was at least 95% as determined by absolute qNMR.

General Procedure for the Synthesis of Compounds 13 and 14 (with the Exception of 13ea/14ea and 13ed/14ed)

A mixture of the corresponding quinoxalin-2(1H)-one 9 (1.8 mmol) and benzene-1,2-diamine 12 (2.16 mmol) in AcOH (15 mL) was heated at reflux for 4 h. The solvent was evaporated under reduced pressure. The residue was chromatographed on silica gel using the mixture of various solvents as eluent. The mixtures of regioisomers 13/14 (Table 1, entries 1–10) and individual regioisomers 13, 14 (Table 1, entry 7) were obtained. The isolated products 13 and/or 14 were dried at 120 °C for 2 h.

3-(Benzimidazol-2-yl)-6-(4-methylpiperazin-1-yl)-2-phenylquinoxaline (13aa) and 2-(Benzimidazol-2-yl)-6-(4-methylpiperazin-1-yl)-3-phenylquinoxaline (14aa)

A mixture of regioisomers 13aa/14aa was obtained from quinoxalin-2(1H)-one 9a (0.45 g, 1.80 mmol) and benzene-1,2-diamine 12a (0.45 g, 2.16 mmol). Eluent: CHCl₃–MeOH (25/1). Yield: 0.55 g (73%), bright yellow solid, mp 230–231 °C. 13aa (52%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.26 (s, 3H, CH₃–Pz), 2.51–2.53 (m, 4H, H3/H5-Pz), 3.42–3.45 (m, 4H, H2/H6-Pz), 7.16 (brdd, J = 8.1, 8.1 Hz, 1H, H6–Bi), 7.24 (brdd, J = 8.1, 8.1 Hz, 1H, H5–Bi), 7.30 (d, J = 2.8 Hz, 1H, H5-Q), 7.35 (dd, J = 7.1, 7.0 Hz, 2H, H3/H5–Ar), 7.35–7.39 (m, 1H, H4–Ar), 7.48 (brd, J = 8.1 Hz, 1H, H7–Bi), 7.511 (d, J = 8.1 Hz, 1H, H4–Bi), 7.55 (d, J = 7.1 Hz, 2H, H2/H6–Ar), 7.82 (dd, J = 9.4, 2.8 Hz, 1H, H7-Q), 7.98 (d, J = 9.4 Hz, 1H, H8-Q), 13.04 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.6 (CH₃–Pz), 47.3 (C2/C6-Pz), 54.2 (C3/C5-Pz), 107.3 (C5-Q), 111.7 (C4–Bi), 119.5 (C7–Bi), 121.7 (C6–Bi), 122.7 (C7-Q), 123.2 (C5–Bi), 127.6 (C3/C5–Ar), 128.5 (C4–Ar), 129.0 (C8-Q), 129.1 (C2/C6–Ar), 134.3 (C3a-Bi), 136.0 (C8a-Q), 138.8 (C1–Ar), 141.5 (C4a-Q), 142.9 (C3-Q), 143.4 (C7a-Bi), 149.8 (C2–Bi), 152.0 (C6-Q), 153.1 (C2-Q). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 35.9 (N4-Pz), 73.8 (N1-Pz), 322.5 (N4-Q), 329.6 (N1-Q). The signals of N1–Bi, N3–Bi have not been observed. 14aa (48%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.26 (s, 3H, CH₃–Pz), 2.51–2.53 (m, 4H, H3/H5-Pz), 3.46–3.49 (m, 4H, H2/H6-Pz), 7.15 (brdd, J = 8.1 Hz, J = 8.1 Hz, 1H, H6–Bi), 7.30 (brdd, J = 8.1, 8.1 Hz, 1H, H5–Bi), 7.32 (d, J = 2.8 Hz, 1H, H5-Q), 7.36 (dd, J = 7.1, 7.0 Hz, 2H, H3/H5–Ar), 7.36–7.40 (m, 1H, H4–Ar), 7.50 (brd, J = 8.1 Hz, 1H, H7–Bi), 7.513 (d, J = 8.1 Hz, 1H, H4–Bi), 7.53 (d, J = 7.1 Hz, 2H, H2/H6–Ar), 7.80 (dd, J = 9.4, 2.8 Hz, 1H, H7-Q), 7.99 (d, J = 9.4 Hz, 1H, H8-Q), 12.96 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.6 (CH₃–Pz), 47.1 (C2/C6-Pz), 54.2 (C3/C5-Pz), 107.5 (C5-Q), 111.6 (C4–Bi), 119.3 (C7–Bi), 121.5 (C6–Bi), 122.9 (C5–Bi), 123.5 (C7-Q), 127.6 (C3/C5–Ar), 128.2 (C4–Ar), 129.1 (C8-Q), 129.2 (C2/C6–Ar), 134.3 (C3a-Bi), 134.5 (C8a-Q), 138.9 (C1–Ar), 139.4 (C2-Q), 143.0 (C4a-Q), 143.4 (C7a-Bi), 149.3 (C3-Q), 150.0 (C2–Bi), 152.4 (C6-Q). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 36.1 (N4-Pz), 75.9 (N1-Pz), 323.9 (N4-Q), 325.8 (N1-Q). The signals of N1–Bi, N3–Bi have not been observed. IR (KBr, ν, cm^–1): 3428, 3054, 2794, 1614, 1486, 1448, 1336, 1219, 744, 659. Low-resolution mass spectrometry (LRMS) (matrix-assisted laser desorption time-of-flight (MALDI-TOF)) m/z: [M + H]⁺ 421.2.

3-(Benzimidazol-2-yl)-2-(4-chlorophenyl)-6-(4-methylpiperazin-1-yl)quinoxaline (13ba) and 2-(Benzimidazol-2-yl)-3-(4-chlorophenyl)-6-(4-methylpiperazin-1-yl)quinoxaline (14ba)

A mixture of regioisomers 13ba/14ba was obtained from quinoxalin-2(1H)-one 9b (0.51 g, 1.80 mmol) and benzene-1,2-diamine 12a (0.45 g, 2.16 mmol). Eluent: CHCl₃–MeOH (50/1.5). Yield: 0.59 g (73%), orange solid, mp 250–251 °C. 13ba (50%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.30 (s, 3H, CH₃–Pz), 2.56–2.58 (m, 4H, H3/H5-Pz), 3.46–3.48 (m, 4H, H2/H6-Pz), 7.16 (brdd, J = 8.1, 8.1 Hz, 1H, H6–Bi), 7.26 (brdd, J = 8.1, 8.1 Hz, 1H, H5–Bi), 7.31 (d, J = 2.7 Hz, 1H, H5-Q), 7.42 (d, J = 8.6 Hz, 2H, H3/H5–Ar), 7.53 (d, J = 8.1 Hz, 1H, H7–Bi), 7.562 (d, J = 8.6 Hz, 2H, H2/H6–Ar), 7.57 (d, J = 8.1 Hz, 1H, H4–Bi), 7.82 (dd, J = 9.4, 2.7 Hz, 1H, H7-Q), 7.98 (d, J = 9.4 Hz, 1H, H8-Q), 13.08 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.5 (CH₃–Pz), 47.1 (C2/C6-Pz), 54.1 (C3/C5-Pz), 107.2 (C5-Q), 111.8 (C4–Bi), 119.6 (C7–Bi), 121.8 (C6–Bi), 123.4 (C5–Bi), 123.6 (C7-Q), 127.6 (C3/C5–Ar), 129.3 (C8-Q), 131.2 (C2/C6–Ar), 133.1 (C4–Ar), 134.3 (C3a-Bi), 135.9 (C8a-Q), 137.8 (C1–Ar), 141.7 (C4a-Q), 143.0 (C3-Q), 143.4 (C7a-Bi), 148.1 (C2-Q), 149.6 (C2–Bi), 152.1 (C6-Q). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 36.9 (N4-Pz), 74.1 (N1-Pz), 321.6 (N4-Q), 329.2 (N1-Q). The signals of N1–Bi, N3–Bi have not been observed. 14ba (50%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.30 (s, 3H, CH₃–Pz), 2.56–2.58 (m, 4H, H3/H5-Pz), 3.49–3.51 (m, 4H, H2/H6-Pz), 7.15 (brdd, J = 8.1, 8.1 Hz, 1H, H6–Bi), 7.25 (brdd, J = 8.1, 8.1 Hz, 1H, H5–Bi), 7.32 (d, J = 2.7 Hz, 1H, H5-Q), 7.44 (d, J = 8.6 Hz, 2H, H3/H5–Ar), 7.50 (d, J = 8.1 Hz, 1H, H7–Bi), 7.558 (d, J = 8.1 Hz, 1H, H4–Bi), 7.58 (d, J = 8.6 Hz, 2H, H2/H6–Ar), 7.84 (dd, J = 9.4, 2.7 Hz, 1H, H7-Q), 8.00 (d, J = 9.4 Hz, 1H, H8-Q), 12.99 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.5 (CH₃–Pz), 47.0 (C2/C6-Pz), 54.1 (C3/C5-Pz), 107.5 (C5-Q), 111.7 (C4–Bi), 119.4 (C7–Bi), 121.6 (C6–Bi), 122.9 (C7-Q), 123.1 (C5–Bi), 127.6 (C3/C5–Ar), 129.0 (C8-Q), 131.1 (C2/C6–Ar), 133.4 (C4–Ar), 134.3 (C3a-Bi), 134.7 (C8a-Q), 137.9 (C1–Ar), 139.1 (C2-Q), 142.9 (C4a-Q), 143.5 (C7a-Bi), 149.8 (C2–Bi), 152.0 (C3-Q), 152.4 (C6-Q). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 36.8 (N4-Pz), 74.0 (N1-Pz), 324.2 (N4-Q), 325.2 (N1-Q). The signals of N1–Bi, N3–Bi have not been observed. IR (KBr, ν, cm^–1): 3431, 3052, 2938, 2798, 1615, 1486, 1452, 1333, 1222, 1008, 741, 539. High-resolution mass spectrometry (HRMS) (MALDI-TOF) m/z: [M + Cs]⁺ calcd for C₂₆H₂₃N₆ClCs 587.0722, found 587.0724.

3-(Benzimidazol-2-yl)-2-(2,4-dichlorophenyl)-6-(4-methylpiperazin-1-yl)quinoxaline (13ca) and 2-(Benzimidazol-2-yl)-3-(2,4-dichlorophenyl)-6-(4-methylpiperazin-1-yl)quinoxaline (14ca)

A mixture of regioisomers 13ca/14ca was obtained from quinoxalin-2(1H)-one 9c (0.57 g, 1.80 mmol) and benzene-1,2-diamine 12a (0.45 g, 2.16 mmol). Eluent: CHCl₃–MeOH (50/1). Yield: 0.69 g (79%), yellow solid, mp 247–248 °C. 13ca (90%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.84–2.86 (m, 4H, H3/H5-Pz), 3.56–3.60 (m, 4H, H2/H6-Pz), 7.13 (ddd, J = 7.8, 7.8, 1.1 Hz, 1H, H6–Bi), 7.25 (ddd, J = 7.8, 7.8, 1.1 Hz, 1H, H5–Bi), 7.382 (d, J = 2.7 Hz, 1H, H5-Q), 7.404 (d, J = 7.8 Hz, 1H, H7–Bi), 7.568 (d, J = 7.8 Hz, 1H, H4–Bi), 7.58 (dd, J = 8.2, 2.0 Hz, 1H, H5–Ar), 7.63 (d, J = 2.0 Hz, 1H, H3–Ar), 7.645 (d, J = 8.2 Hz, 1H, H6–Ar), 7.87 (dd, J = 9.4, 2.7 Hz, 1H, H7-Q), 8.00 (d, J = 9.4 Hz, 1H, H8-Q), 13.12 (s, 1H, NH-Bi). The signal of CH₃-Pz has not been observed. ¹³C NMR (126 MHz, DMSO-d₆): δ 46.5 (C2/C6-Pz), 53.4 (C3/C5-Pz), 107.4 (C5-Q), 111.9 (C4–Bi), 119.9 (C7–Bi), 121.8 (C6–Bi), 123.5 (C7-Q), 123.7 (C5–Bi), 127.2 (C5–Ar), 128.1 (C3–Ar), 129.4 (C8-Q), 132.6 (C6–Ar), 133.4 (C4–Ar), 133.4 (C2–Ar), 134.4 (C3a-Bi), 135.8 (C8a-Q), 138.2 (C1–Ar), 138.4 (C3-Q), 142.9 (C4a-Q), 142.9 (C2-Q), 143.8 (C7a-Bi), 149.1 (C2–Bi), 152.1 (C6-Q). The signal of CH₃–Pz has not been observed. 14ca (10%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.84–2.86 (m, 4H, H3/H5-Pz), 3.56–3.60 (m, 4H, H2/H6-Pz), 7.11 (ddd, J = 7.8, 7.8, 1.1 Hz, 1H, H6–Bi), 7.23 (ddd, J = 7.8, 7.8, 1.1 Hz, 1H, H5–Bi), 7.377 (d, J = 2.7 Hz, 1H, H5-Q), 7.403 (d, J = 8.1 Hz, 1H, H7–Bi), 7.567 (d, J = 8.1 Hz, 1H, H4–Bi), 7.57 (dd, J = 8.2, 2.0 Hz, 1H, H5–Ar), 7.62 (d, J = 8.2 Hz, 1H, H6–Ar), 7.649 (d, J = 2.0 Hz, 1H, H3–Ar), 7.88 (dd, J = 9.4, 2.7 Hz, 1H, H7-Q), 8.07 (d, J = 9.4 Hz, 1H, H8-Q), 13.03 (s, 1H, NH-Bi). The signal of CH₃-Pz has not been observed. ¹³C NMR (126 MHz, DMSO-d₆): δ 47.1 (C2/C6-Pz), 54.2 (C3/C5-Pz), 107.3 (C5-Q), 111.7 (C4–Bi), 119.4 (C7–Bi), 121.6 (C6–Bi), 122.8 (C7-Q), 123.1 (C5–Bi), 127.6 (C3–Ar), 129.0 (C8-Q), 131.2 (C2–Ar), 133.4 (C4–Ar), 134.3 (C3a-Bi), 134.6 (C8a-Q), 137.9 (C1–Ar), 139.0 (C2-Q), 142.9 (C4a-Q), 143.5 (C7a-Bi), 149.8 (C2–Bi), 151.9 (C3-Q), 152.4 (C6-Q). The signals of CH₃–Pz, C5–Ar, C6–Ar have not been observed. IR (KBr, ν, cm^–1): 2934, 2081, 1613, 1489, 1452, 1400, 1372, 1333, 1210, 1104, 1006, 740. LRMS (MALDI-TOF) m/z: [M + H]⁺ 489.1.

3-(Benzimidazol-2-yl)-2-(4-fluorophenyl)-6-(4-methylpiperazin-1-yl)quinoxaline (13da) and 2-(Benzimidazol-2-yl)-3-(4-fluorophenyl)-6-(4-methylpiperazin-1-yl)quinoxaline (14da)

A mixture of regioisomers 13da/14da was obtained from quinoxalin-2(1H)-one 9d (0.48 g, 1.80 mmol) and benzene-1,2-diamine 12a (0.45 g, 2.16 mmol). Eluent: EtOAc–c-hexane (6/1). Yield: 0.59 g (75%), bright yellow solid, mp 219–221 °C. 13da (52%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.30 (s, 3H, CH₃–Pz), 2.57–2.59 (m, 4H, H3/H5-Pz), 3.48–3.50 (m, 4H, H2/H6-Pz), 7.14–7.18 (m, 1H, H6–Bi), 7.197 (dd, J_HF = 9.0 Hz, J_HH = 8.5 Hz, 2H, H3/H5–Ar), 7.250 (brdd, J = 8.1, 8.1 Hz, 1H, H5–Bi), 7.315 (d, J = 2.8 Hz, 1H, H5-Q), 7.54 (d, J = 8.1 Hz, 1H, H7–Bi), 7.56–7.62 (m, 3H, H4–Bi, H2/H6–Ar), 7.82 (dd, J = 9.5, 2.8 Hz, 1H, H7-Q), 7.99 (d, J = 9.5 Hz, 1H, H8-Q), 13.07 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.5 (CH₃–Pz), 47.2 (C2/C6-Pz), 54.2 (C3/C5-Pz), 107.5 (C5-Q), 111.9 (C4–Bi), 114.6 (d, J_CF = 21.7 Hz, C3/C5–Ar), 119.7 (C7–Bi), 121.9 (C6–Bi), 123.4 (C5–Bi), 123.7 (C7-Q), 129.3 (C8-Q), 131.6 (d, J_CF = 8.5 Hz, C2/C6–Ar), 134.4 (C3a-Bi), 135.4 (d, J_CF = 2.2 Hz, C1–Ar), 136.0 (C8a-Q), 141.7 (C3-Q), 143.2 (C4a-Q), 143.5 (C7a-Bi), 149.8 (C2–Bi), 152.1 (C6-Q), 152.3 (C2-Q), 162.4 (d, J_CF = 268.2 Hz, C4–Ar).¹⁵N NMR (51 MHz, DMSO-d₆): δ 36.5 (N4-Pz), 72.8 (N1-Pz), 322.2 (N4-Q), 328.6 (N1-Q). The signals of N1–Bi, N3–Bi have not been observed. 14da (48%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.30 (s, 3H, CH₃–Pz), 2.57–2.59 (m, 4H, H3/H5-Pz), 3.48–3.50 (m, 4H, H2/H6-Pz), 7.14–7.18 (dd, J = 8.1, 8.1 Hz, 1H, H6–Bi), 7.201 (dd, J_HF = 9.0 Hz, J_HH = 8.5 Hz, 2H, H3/H5–Ar), 7.246 (brdd, J = 8.1, 8.1 Hz, 1H, H5–Bi), 7.319 (d, J = 2.8 Hz, 1H, H5-Q), 7.51 (d, J = 8.1 Hz, 1H, H7–Bi), 7.56–7.62 (m, 3H, H4–Bi, H2/H6–Ar), 7.83 (dd, J = 9.5, 2.8 Hz, 1H, H7-Q), 8.00 (d, J = 9.5 Hz, 1H, H8-Q), 12.99 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.5 (CH₃–Pz), 47.0 (C2/C6-Pz), 54.2 (C3/C5-Pz), 107.4 (C5-Q), 111.8 (C4–Bi), 114.5 (d, J_CF = 21.7 Hz, C3/C5–Ar), 119.7 (C7–Bi), 121.7 (C6–Bi), 123.3 (C5–Bi), 123.7 (C7-Q), 129.2 (C8-Q), 131.5 (d, J_CF = 8.5 Hz, C2/C6–Ar), 134.4 (C3a-Bi), 134.7 (C8a-Q), 135.5 (d, J_CF = 2.2 Hz, C1–Ar), 139.2 (C2-Q), 143.0 (C4a-Q), 143.7 (C7a-Bi), 148.5 (C3-Q), 150.0 (C2–Bi), 152.4 (C6-Q), 162.6 (d, J_CF = 268.2 Hz, C4–Ar). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 36.5 (N4-Pz), 72.8 (N1-Pz), 322.0 (N4-Q), 326.6 (N1-Q). The signals of N1–Bi, N3–Bi have not been observed. IR (KBr, ν, cm^–1): 3432, 3063, 2939, 2798, 1614, 1492, 1335, 1222, 1158, 1142, 841, 744, 544. HRMS (MALDI-TOF) m/z: [M + Cs]⁺ calcd for C₂₆H₂₃N₆FCs 571.1017, found 571.0992. The purified mixture of regioisomers 13da/14da (0.50 g) was heated at reflux in i-PrOH; the undissolved precipitate was filtered off and dried in air to afford an analytically pure isomer 13da (0.19 g, 38%). The filtrate was evaporated. The residue after evaporation was boiled in MeCN; the part was not dissolved during boiling was filtered off. An additional amount of the isomer 13da (70.0 mg, 14%) was obtained. The precipitate that fell out of the filtrate was filtered out to afford an analytically pure isomer 14da (0.20 g, 39%). 13da: yellow brown solid, mp 165–167 °C. ¹H NMR (500 MHz, DMSO-d₆): δ 2.41 (s, 3H, CH₃–Pz), 2.73–2.75 (m, 4H, H3/H5-Pz), 3.46–3.49 (m, 4H, H2/H6-Pz), 7.17 (dd, J_HF = 9.9 Hz, J_HH = 8.6 Hz, 2H, H3/H5–Ar), 7.23–7.27 (m, 2H, H6–Bi, H5–Bi), 7.34 (d, J = 2.2 Hz, 1H, H5-Q), 7.51–7.59 (m, 2H, H7–Bi, H4–Bi), 7.56 (dd, J_HH = 8.6 Hz, J_HF = 5.6 Hz, 2H, H2/H6–Ar), 7.83 (dd, J = 9.4, 2.2 Hz, 1H, H7-Q), 7.99 (d, J = 9.4 Hz, 1H, H8-Q), 13.06 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.0 (CH₃–Pz), 47.0 (C2/C6-Pz), 53.9 (C3/C5-Pz), 107.7 (C5-Q), 112.0 (C4–Bi), 114.7 (d, J_CF = 21.6 Hz, C3/C5–Ar), 119.7 (C7–Bi), 122.0 (C6–Bi), 123.6 (C7-Q), 123.8 (C5–Bi), 129.4 (C8-Q), 131.5 (d, J_CF = 8.4 Hz, C2/C6–Ar), 134.5 (C3a-Bi), 135.4 (d, J_CF = 1.8 Hz, C1–Ar), 136.2 (C8a-Q), 141.8 (C4a-Q), 143.3 (C7a-Bi), 143.5 (C2-Q), 148.6 (C3-Q), 149.9 (C2–Bi), 152.0 (C6-Q), 162.4 (d, J_CF = 245.2 Hz, C4–Ar). IR (KBr, ν, cm^–1): 3412, 3058, 2797, 1615, 1511, 1491, 1451, 1333, 1221, 1142, 1006, 823, 744. LRMS (MALDI-TOF) m/z: [M + H]⁺ 439.2. 14da: yellow green solid, mp 292–293 °C. ¹H NMR (500 MHz, DMSO-d₆): δ 2.27 (s, 3H, CH₃–Pz), 2.58–2.60 (m, 4H, H3/H5-Pz), 3.48–3.50 (m, 4H, H2/H6-Pz), 7.15 (dd, J = 8.0, 8.0 Hz, 1H, H6–Bi), 7.19 (dd, J_HF = 9.0 Hz, J_HH = 8.9 Hz, 2H, H3/H5–Ar), 7.24 (dd, J = 8.0, 8.0 Hz, 1H, H5–Bi), 7.30 (d, J = 2.7 Hz, 1H, H5-Q), 7.49 (d, J = 8.0 Hz, 1H, H7–Bi), 7.56 (d, J = 8.0 Hz, 1H, H4–Bi), 7.60 (dd, J_HH = 8.9 Hz, J_HF = 5.5 Hz, 2H, H2/H6–Ar), 7.81 (dd, J = 9.5 Hz, J = 2.7 Hz, 1H, H7-Q), 8.00 (d, J = 9.5 Hz, 1H, H8-Q), 12.98 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.6 (CH₃–Pz), 47.1 (C2/C6-Pz), 54.3 (C3/C5-Pz), 107.5 (C5-Q), 111.8 (C4–Bi), 114.6 (d, J_CF = 21.6 Hz, C3/C5–Ar), 119.5 (C7–Bi), 121.7 (C6–Bi), 122.8 (C7-Q), 123.2 (C5–Bi), 129.1 (C8-Q), 131.6 (d, J_CF = 8.4 Hz, C2/C6–Ar), 134.4 (C3a-Bi), 134.7 (C8a-Q), 135.5 (d, J_CF = 2.2 Hz, C1–Ar), 139.2 (C3-Q), 143.0 (C4a-Q), 143.5 (C7a-Bi), 150.0 (C2–Bi), 152.2 (C2-Q), 152.5 (C6-Q), 162.5 (d, J_CF = 245.5 Hz, C4–Ar). IR (KBr, ν, cm^–1): 3432, 3057, 2943, 2841, 2795, 1618, 1496, 1338, 1225, 1158, 840, 750, 548. LRMS (MALDI-TOF) m/z: [M + H]⁺ 439.2.

3-(Benzimidazol-2-yl)-2-(4-fluorophenyl)-6-(4-phenylpiperazin-1-yl)quinoxaline (13db) and 2-(Benzimidazol-2-yl)-3-(4-fluorophenyl)-6-(4-phenylpiperazin-1-yl)quinoxaline (14db)

A mixture of regioisomers 13db/14db was obtained from quinoxalin-2(1H)-one 9d (0.48 g, 1.80 mmol) and benzene-1,2-diamine 12b (0.58 g, 2.16 mmol). Eluent: CHCl₃–MeOH (50/0.75). Yield: 0.64 g (71%), yellow-brown solid, mp 235–236 °C. 13db (48%): ¹H NMR (500 MHz, DMSO-d₆): δ 3.36–3.39 (m, 4H, H3/H5-Pz), 3.60–3.62 (m, 4H, H2/H6-Pz), 6.828 (dd, J = 7.8, 7.8 Hz, 1H, H4-Ph), 7.027 (d, J = 7.8 Hz, 2H, H2/H6-Ph), 7.20 (dd, J_HF = 9.0 Hz, J_HH = 8.7, 2H, H3/H5–Ar), 7.18–7.24 (br, 2H, H5/H6–Bi), 7.260 (dd, J = 7.8 Hz, 2H, H3/H5-Ph), 7.38 (d, J = 2.8 Hz, 1H, H5-Q), 7.51–7.57 (brm, 2H, H4/H7–Bi), 7.58 (dd, J_HH = 8.7 Hz, J_HF = 5.9 Hz, 2H, H2/H6–Ar), 7.90 (dd, J = 9.7, 2.8 Hz, 1H, H7-Q), 8.04 (d, J = 9.7 Hz, 1H, H8-Q). The signal of NH-Bi has not been observed. ¹³C NMR (126 MHz, DMSO-d₆): δ 47.4 (C2/C6-Pz), 48.1 (C3/C5-Pz), 107.5 (C5-Q), 114.6 (d, J_CF = 21.7 Hz, C3/C5–Ar), 115.7 (C2/C6-Ph), 119.3 (C4-Ph), 122.6 (C5/C6–Bi), 122.6 (C7-Q), 129.1 (C3/C5-Ph), 129.2 (C8-Q), 131.5 (d, J_CF = 8.5 Hz, C2/C6–Ar), 134.5 (C3a-Bi), 134.8 (C8a-Q), 135.4 (d, J_CF = 2.2 Hz, C1–Ar), 139.1 (C2-Q), 143.0 (C4a-Q), 148.5 (C3-Q), 149.8 (C2–Bi), 150.8 (C1-Ph), 152.4 (C6-Q), 162.3 (d, J_CF = 268.2 Hz, C4–Ar). The signals of C4/C7–Bi, C7a-Bi have not been observed. 14db (52%): ¹H NMR (500 MHz, DMSO-d₆): δ 3.36–3.39 (m, 4H, H3/H5-Pz), 3.64–3.66 (m, 4H, H2/H6-Pz), 6.826 (dd, J = 7.8, 7.8 Hz, 1H, H4-Ph), 7.031 (d, J = 7.8 Hz, 2H, H2/H6-Ph), 7.20 (dd, J_HF = 9.0 Hz, J_HH = 8.7 Hz, 2H, H3/H5–Ar), 7.18–7.24 (br, 2H, H5/H6–Bi), 7.264 (dd, J = 7.8 Hz, J = 7.8 Hz, 2H, H3/H5-Ph), 7.38 (d, J = 2.8 Hz, 1H, H5-Q), 7.51–7.57 (brm, 2H, H4/H7–Bi), 7.61 (dd, J_HH = 8.7 Hz, J_HF = 5.9 Hz, 2H, H2/H6–Ar), 7.88 (dd, J = 9.7, 2.8 Hz, 1H, H7-Q), 8.02 (d, J = 9.7 Hz, 1H, H8-Q). The signal of NH-Bi has not been observed. ¹³C NMR (126 MHz, DMSO-d₆): δ 47.2 (C2/C6-Pz), 48.1 (C3/C5-Pz), 107.6 (C5-Q), 162.6 (d, J_CF = 245.6 Hz, C4–Ar), 114.6 (d, J_CF = 21.7 Hz, C3/C5–Ar), 115.7 (C2/C6-Ph), 119.3 (C4-Ph), 122.6 (C5/C6–Bi), 123.8 (C7-Q), 129.1 (C3/C5-Ph), 129.2 (C8-Q), 131.6 (d, J_CF = 8.7 Hz, C2/C6–Ar), 134.2 (C3a-Bi), 135.4 (d, J_CF = 2.2 Hz, C1–Ar), 136.1 (C8a-Q), 141.7 (C3-Q), 143.1 (C4a-Q), 149.7 (C2–Bi), 150.8 (C1-Ph), 152.0 (C6-Q), 152.3 (C2-Q). The signals of C4/C7–Bi, C7a-Bi have not been observed. IR (KBr, ν, cm^–1): 3433, 3053, 2822, 1615, 1599, 1492, 1387, 1331, 1221, 1157, 957, 837, 821, 743, 539, 528. LRMS (MALDI-TOF) m/z: [M + H]⁺ 501.2.

3-(Benzimidazol-2-yl)-2-(4-fluorophenyl)-6-(piperidin-1-yl)quinoxaline (13dc) and 2-(Benzimidazol-2-yl)-3-(4-fluorophenyl)-6-(piperidin-1-yl)quinoxaline (14dc)

A mixture of regioisomers 13dc/14dc was obtained from quinoxalin-2(1H)-one 9d (0.48 g, 1.80 mmol) and benzene-1,2-diamine 12c (0.41 g, 2.16 mmol). The crude mixture of regioisomers 13dc/14dc was chromatographed on silica gel using EtOAc–c-hexane (7/1) as eluent. An analytically pure mixture of regioisomers 13dc/14dc and individual regioisomers 13dc, 14dc were obtained. 13dc/14dc: yield 0.41 g (54%), bright yellow solid, mp 243–245 °C. 13dc (48%): ¹H NMR (500 MHz, DMSO-d₆): δ 1.62–1.70 (m, 6H, H3/H5, H4–Pd), 3.46–3.49 (m, 4H, H2/H6–Pd), 7.20 (dd, J_HF = 9.1 Hz, J_HH = 8.7 Hz, 2H, H3/H5–Ar), 7.16–7.22 (br, 2H, H5/H6–Bi), 7.27 (d, J = 2.5 Hz, 1H, H5-Q), 7.50–7.56 (m, 2H, H4/H7–Bi), 7.58 (dd, J = 8.7 Hz, J_HF = 5.8 Hz, 2H, H2/H6–Ar), 7.81 (dd, J = 9.4 Hz, J = 2.4 Hz, 1H, H7-Q), 7.95 (d, J = 9.3 Hz, 1H, H8-Q), 13.02 (br s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 24.2 (C4–Pd), 25.2 (C3/C5–Pd), 49.0 (C2/C6–Pd), 107.1 (C5-Q), 114.9 (d, J_CF = 21.6 Hz, C3/C5–Ar), 123.2 (C7-Q), 123.4 (C5/C6–Bi), 129.5 (C8-Q), 131.8 (d, J_CF = 8.5 Hz, C2/C6–Ar), 134.8 (C8a-Q), 135.7 (d, J_CF = 2.2 Hz, C1–Ar), 142.3 (C4a-Q), 143.2 (C3-Q), 148.2 (C2-Q), 150.1 (C2–Bi), 153.0 (C6-Q). The signals of C4/C7–Bi, C3a/C7a-Bi, C4–Ar have not been observed. 14dc (52%): ¹H NMR (500 MHz, DMSO-d₆): δ 1.62–1.70 (m, 6H, H3/H5, H4–Pd), 3.49–3.52 (m, 4H, H2/H6–Pd), 7.18 (dd, J_HF = 9.1 Hz, J_HH = 8.7 Hz, 2H, H3/H5–Ar), 7.16–7.22 (br, 2H, H5/H6–Bi), 7.27 (d, J = 2.5 Hz, 1H, H5-Q), 7.50–7.56 (m, 2H, H4/H7–Bi), 7.60 (dd, J_HH = 8.7 Hz, J_HF = 5.8 Hz, 2H, H2/H6–Ar), 7.80 (dd, J = 9.4, 2.4 Hz, 1H, H7-Q), 7.97 (d, J = 9.3 Hz, 1H, H8-Q), 12.95 (br s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 24.2 (C4–Pd), 25.2 (C3/C5–Pd), 48.8 (C2/C6–Pd), 106.9 (C5-Q), 114.9 (d, J_CF = 21.6 Hz, C3/C5–Ar), 123.2 (C7-Q), 123.4 (C5/C6–Bi), 129.6 (C8-Q), 131.8 (d, J_CF = 8.5 Hz, C2/C6–Ar), 135.8 (d, J_CF = 2.2 Hz, C1–Ar), 136.1 (C8a-Q), 138.6 (C2-Q), 143.6 (C4a-Q), 150.2 (C2–Bi), 152.6 (C6-Q), 152.7 (C3-Q). The signals of C4/C7–Bi, C3a/C7a-Bi, and C4–Ar have not been observed. IR (KBr, ν, cm^–1): 3438–2851, 1616, 1493, 1225, 1124, 841, 742, 548. HRMS (MALDI-TOF) m/z: [M + Cs]⁺ calcd for C₂₆H₂₂N₅FCs 556.0908, found 556.0886. 13dc: yield: 90.9 mg (12%), R_f 0.60 (CHCl₃/n-C₆H₁₄/MeOH, 6:3:1), bright yellow solid, mp 148–150 °C. ¹H NMR (500 MHz, DMSO-d₆): δ 1.64–1.70 (m, 6H, H3/H5–Pd, H4–Pd), 3.47–3.49 (m, 4H, H2/H6–Pd), 7.16 (ddd, J = 8.0, 8.0, 1.1 Hz, 1H, H5–Bi), 7.18 (dd, J_HF = 8.9 Hz, J_HH = 8.8 Hz, 2H, H3/H5–Ar), 7.26 (dd, J = 8.0, 8.0 Hz, 1H, H6–Bi), 7.27 (d, J = 2.5 Hz, 1H, H5-Q), 7.52 (d, J = 8.0 Hz, 1H, H4–Bi), 7.57 (dd, J = 8.0, 1.1 Hz, 1H, H7–Bi), 7.58 (dd, J_HH = 8.8 Hz, J_HF = 5.4 Hz, 2H, H2/H6–Ar), 7.81 (dd, J = 9.4, 2.8 Hz, 1H, H7-Q), 7.95 (d, J = 9.4 Hz, 1H, H8-Q), 13.03 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 23.9 (C4–Pd), 24.8 (C3/C5–Pd), 48.6 (C2/C6–Pd), 106.7 (C5-Q), 111.8 (C7–Bi), 114.4 (d, J_CF = 21.5 Hz, C3/C5–Ar), 119.6 (C4–Bi), 121.7 (C5–Bi), 123.3 (C6–Bi), 122.8 (C7-Q), 129.2 (C8-Q), 131.4 (d, J_CF = 8.5 Hz, C2/C6–Ar), 134.3 (C7a-Bi), 135.4 (d, J_CF = 3.0 Hz, C1–Ar), 135.7 (C8a-Q), 141.9 (C4a-Q), 142.9 (C3-Q), 143.4 (C3a-Bi), 147.9 (C2–Bi), 149.8 (C2-Q), 152.5 (C6-Q), 162.2 (d, J_CF = 245.3 Hz, C4–Ar). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 79.6 (N1–Pd), 150.3 (N1–Bi), 320.9 (N4-Q), 329.3 (N1-Q). The signal of N3–Bi has not been observed. IR (KBr, ν, cm^–1): 3442–2849, 1615, 1511, 1484, 1451, 1334, 1226, 1124, 823, 743, 552, 538. HRMS (MALDI-TOF) m/z: [M + Na]⁺ calcd for C₂₆H₂₂N₅FNa 446.1751, found 446.1764. 14dc: yield: 60.8 mg (8%), R_f 0.48 (CHCl₃/n-C₆H₁₄/MeOH, 6:3:1), bright yellow solid, mp 284–285 °C. ¹H NMR (500 MHz, DMSO-d₆): δ 1.63–1.69 (m, 6H, H3/H5–Pd, H4–Pd), 3.49–3.52 (m, 4H, H2/H6–Pd), 7.14 (dd, J = 8.0, 8.0 Hz, 1H, H5–Bi), 7.19 (dd, J_HF = 8.9 Hz, J_HH = 8.7 Hz, 2H, H3/H5–Ar), 7.23 (dd, J = 8.0, 8.0 Hz, 1H, H6–Bi), 7.27 (d, J = 2.8 Hz, 1H, H5-Q), 7.49 (d, J = 8.0 Hz, 1H, H4–Bi), 7.56 (d, J = 8.0 Hz, 1H, H7–Bi), 7.60 (dd, J_HH = 8.7 Hz, J_HF = 5.6 Hz, 2H, H2/H6–Ar), 7.79 (dd, J = 9.4, 2.8 Hz, 1H, H7-Q), 7.963 (d, J = 9.4 Hz, 1H, H8-Q), 12.94 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 23.9 (C4–Pd), 24.9 (C3/C5–Pd), 48.4 (C2/C6–Pd), 106.9 (C5-Q), 111.6 (C7–Bi), 114.4 (d, J_CF = 21.6 Hz, C3/C5–Ar), 119.4 (C4–Bi), 121.5 (C5–Bi), 122.9 (C7-Q), 123.0 (C6–Bi), 129.0 (C8-Q), 131.5 (d, J_CF = 8.5 Hz, C2/C6–Ar), 134.3 (C8a-Q), 134.3 (C7a-Bi), 135.5 (d, J_CF = 2.2 Hz, C1–Ar), 138.7 (C2-Q), 143.1 (C4a-Q), 143.3 (C3a-Bi), 149.9 (C2–Bi), 152.1 (C3-Q), 162.4 (d, J_CF = 245.6 Hz, C4–Ar), 152.5 (C6-Q). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 81.3 (N1–Pd), 141.9 (N1–Bi), 323.1 (N4-Q), 326.1 (N1-Q). The signal of N3–Bi has not been observed. IR (KBr, ν, cm^–1): 3444–2850, 1617, 1494, 1443, 1341, 1226, 1124, 841, 744, 547, 531. HRMS (MALDI-TOF) m/z: [M + Cs]⁺ calcd for C₂₆H₂₂N₅FCs 556.0908, found 556.0915.

3-(5-Chlorobenzimidazol-2-yl)-2-(4-fluorophenyl)-6-(piperidin-1-yl)quinoxaline (13fc) and 2-(5-Chlorobenzimidazol-2-yl)-3-(4-fluorophenyl)-6-(piperidin-1-yl)quinoxaline (14fc)

A mixture of regioisomers 13 fc/14 fc was obtained from quinoxalin-2(1H)-one 9f (0.54 g, 1.80 mmol) and benzene-1,2-diamine 12c (0.42 g, 2.16 mmol). Eluent: c-hexane–EtOAc (4/1). Yield: 0.61 g (75%), bright orange solid, mp 146–148 °C. 13fc (52%): ¹H NMR (500 MHz, DMSO-d₆): tautomer 13fc (26%): δ 1.64–1.68 (m, 6H, H3/H5, H4–Pd), 3.46–3.50 (m, 4H, H2/H6–Pd), 7.16 (dd, J = 8.5, 2.0 Hz, 1H, H5–Bi), 7.18 (dd, J_HF = 9.1 Hz, J_HH = 8.7 Hz, 2H, H3/H5–Ar), 7.27 (d, J = 2.7 Hz, 1H, H5-Q), 7.50 (d, J = 8.5 Hz, 1H, H4–Bi), 7.52–7.56 (m, 1H, H7–Bi), 7.58 (dd, J_HH = 8.7 Hz, J_HF = 5.8 Hz, 2H, H2/H6–Ar), 7.80 (dd, J = 9.4 Hz, J = 2.7 Hz, 1H, H7-Q), 7.95 (d, J = 9.3 Hz, 1H, H8-Q), 13.09 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): tautomer 13fc (26%): δ 23.9 (C4–Pd), 24.9 (C3/C5–Pd), 48.6 (C2/C6–Pd), 106.7 (C5-Q), 111.3 (C7–Bi), 114.5 (d, J_CF = 21.6 Hz, C3/C5–Ar), 120.7 (C4–Bi), 122.2 (C5–Bi), 123.2 (C7-Q), 127.4 (C7a-Bi), 129.2 (C8-Q), 131.5 (d, J_CF = 8.5 Hz, C2/C6–Ar), 134.3 (C8a-Q), 135.0 (C6–Bi), 135.4 (d, J_CF = 2.2 Hz, C1–Ar), 141.9 (C4a-Q), 142.2 (C3a-Bi), 142.4 (C3-Q), 147.9 (C2-Q), 151.1 (C2–Bi), 152.2 (C6-Q), 162.2 (d, J_CF = 245.2 Hz, C4–Ar). ¹H NMR (500 MHz, DMSO-d₆): tautomer 13′fc (26%): δ 1.64–1.68 (m, 6H, H3/H5, H4–Pd), 3.46–3.50 (m, 4H, H2/H6–Pd), 7.26 (dd, J = 8.5, 2.0 Hz, 1H, H6–Bi), 7.18 (dd, J_HF = 9.1 Hz, J_HH = 8.7 Hz, 2H, H3/H5–Ar), 7.27 (d, J = 2.7 Hz, 1H, H5-Q), 7.55–7.59 (m, 1H, H7–Bi), 7.58 (dd, J_HH = 8.7 Hz, J_HF = 5.8 Hz, 2H, H2/H6–Ar), 7.58 (d, J = 2.0 Hz, 1H, H4–Bi), 7.80 (dd, J = 9.4, 2.7 Hz, 1H, H7-Q), 7.95 (d, J = 9.3 Hz, 1H, H8-Q), 13.16 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): tautomer 13′fc (26%): δ 23.9 (C4–Pd), 24.9 (C3/C5–Pd), 48.6 (C2/C6–Pd), 106.7 (C5-Q), 113.1 (C7–Bi), 114.5 (d, J_CF = 21.6 Hz, C3/C5–Ar), 118.7 (C4–Bi), 123.2 (C7-Q), 123.4 (C6–Bi), 126.1 (C7a-Bi), 129.2 (C8-Q), 131.5 (d, J_CF = 8.5 Hz, C2/C6–Ar), 133.1 (C5–Bi), 134.3 (C8a-Q), 135.4 (d, J_CF = 2.2 Hz, C1–Ar), 141.9 (C4a-Q), 142.4 (C3-Q), 144.2 (C3a-Bi), 147.9 (C2-Q), 151.4 (C2–Bi), 152.2 (C6-Q), 162.2 (d, J_CF = 245.2 Hz, C4–Ar). 14fc (48%): ¹H NMR (500 MHz, DMSO-d₆): tautomer 14fc (24%): δ 1.64–1.68 (m, 6H, H3/H5, H4–Pd), 3.56–3.50 (m, 4H, H2/H6–Pd), 7.18 (dd, J = 8.5 Hz, J = 2.0 Hz, 1H, H5–Bi), 7.20 (dd, J_HF = 9.1 Hz, J_HH = 8.7 Hz, 2H, H3/H5–Ar), 7.27 (d, J = 2.7 Hz, 1H, H5-Q), 7.29 (d, J = 8.5 Hz, 1H, H4–Bi), 7.52–7.56 (m, 1H, H7–Bi), 7.60 (dd, J_HH = 8.7 Hz, J_HF = 5.8 Hz, 2H, H2/H6–Ar), 7.83 (dd, J = 9.4, 2.7 Hz, 1H, H7-Q), 7.97 (d, J = 9.3 Hz, 1H, H8-Q), 13.18 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): tautomer 14fc (24%): δ 23.9 (C4–Pd), 24.9 (C3/C5–Pd), 48.6 (C2/C6–Pd), 106.8 (C5-Q), 111.5 (C7–Bi), 114.4 (d, J_CF = 21.6 Hz, C3/C5–Ar), 120.9 (C4–Bi), 122.0 (C5–Bi), 124.1 (C7-Q), 127.7 (C7a-Bi), 129.1 (C8-Q), 131.4 (d, J_CF = 8.5 Hz, C2/C6–Ar), 135.0 (C6–Bi), 135.4 (d, J_CF = 2.2 Hz, C1–Ar), 135.8 (C8a-Q), 138.0 (C2-Q), 142.3 (C3a-Bi), 143.2 (C4a-Q), 150.9 (C2–Bi), 152.2 (C3-Q), 152.6 (C6-Q), 162.4 (d, J_CF = 245.2 Hz, C4–Ar). ¹H NMR (500 MHz, DMSO-d₆): tautomer 14′fc (24%): δ 1.64–1.68 (m, 6H, H3/H5, H4–Pd), 3.46–3.50 (m, 4H, H2/H6–Pd), 7.18 (dd, J = 8.5 Hz, J = 2.0 Hz, 1H, H5–Bi), 7.20 (dd, J_HF = 9.1 Hz, J_HH = 8.7 Hz, 2H, H3/H5–Ar), 7.27 (d, J = 2.7 Hz, 1H, H5-Q), 7.28 (dd, J = 8.5, 2.0 Hz, 1H, H6–Bi), 7.58 (d, J = 2.0 Hz, 1H, H4–Bi), 7.57–7.61 (m, 1H, H7–Bi), 7.60 (dd, J_HH = 8.7 Hz, J_HF = 5.8 Hz, 2H, H2/H6–Ar), 7.83 (dd, J = 9.4 Hz, J = 2.7 Hz, 1H, H7-Q), 7.97 (d, J = 9.3 Hz, 1H, H8-Q), 13.25 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): tautomer 14′fc (24%): δ 23.9 (C4–Pd), 24.9 (C3/C5–Pd), 48.4 (C2/C6–Pd), 106.7 (C5-Q), 113.2 (C7–Bi), 114.5 (d, J_CF = 21.6 Hz, C3/C5–Ar), 118.9 (C4–Bi), 123.2 (C6–Bi), 124.1 (C7-Q), 126.2 (C7a-Bi), 129.1 (C8-Q), 131.5 (d, J_CF = 8.5 Hz, C2/C6–Ar), 133.1 (C5–Bi), 135.4 (d, J_CF = 2.2 Hz, C1–Ar), 135.8 (C8a-Q), 138.0 (C2-Q), 143.2 (C4a-Q), 144.3 (C3a-Bi), 151.6 (C2–Bi), 152.2 (C3-Q), 152.6 (C6-Q), 162.4 (d, J_CF = 245.2 Hz, C4–Ar). IR (KBr, ν, cm^–1): 2925, 2849, 1613, 1488, 1444, 1229, 1123, 840, 822, 535. HRMS (MALDI-TOF) m/z: [M + H]⁺ calcd for C₂₆H₂₂N₅FCl 458.1542, found 458.1541.

4-(3-(Benzimidazol-2-yl)-2-(4-fluorophenyl)quinoxalin-6-yl)morpholine (13dd) and 4-(2-(Benzimidazol-2-yl)-3-(4-fluorophenyl)quinoxalin-6-yl)morpholine (14dd)

A mixture of regioisomers 13dd/14dd was obtained from quinoxalin-2(1H)-one 9d (0.48 g, 1.80 mmol) and benzene-1,2-diamine 12d (0.42 g, 2.16 mmol). Eluent: c-hexane–EtOAc (3/1). Yield: 0.56 g (73%), yellow solid, mp 235–236 °C. 13dd (48%): ¹H NMR (500 MHz, DMSO-d₆): δ 3.44–3.46 (m, 4H, H2/H6-M), 3.81–3.83 (m, 4H, H3/H5-M), 7.15 (dd, J = 8.1, 8.1 Hz, 1H, H6–Bi), 7.20 (dd, J_HF = 9.0 Hz, J_HH = 8.9 Hz, 2H, H3/H5–Ar), 7.24 (dd, J = 8.1, 8.1 Hz, 1H, H5–Bi), 7.34 (d, J = 2.7 Hz, 1H, H5-Q), 7.50 (d, J = 8.1 Hz, 1H, H7–Bi), 7.56 (d, J = 8.1 Hz, 1H, H4–Bi), 7.57–7.63 (m, 2H, H2/H6–Ar), 7.829 (dd, J = 9.4, 2.7 Hz, 1H, H7-Q), 8.03 (d, J = 9.4 Hz, 1H, H8-Q), 13.06 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 47.4 (C2/C6-M), 65.9 (C3/C5-M), 107.5 (C5-Q), 111.7 (C4–Bi), 114.5 (d, J_CF = 21.7 Hz, C3/C5–Ar), 119.4 (C7–Bi), 121.6 (C6–Bi), 122.4 (C7-Q), 123.1 (C5–Bi), 129.0 (C8-Q), 131.4 (d, J_CF = 8.5 Hz, C2/C6–Ar), 134.3 (C3a-Bi), 134.8 (C8a-Q), 135.3 (d, J_CF = 3.2 Hz, C1–Ar), 142.8 (C4a-Q), 143.1 (C2-Q), 143.4 (C7a-Bi), 148.5 (C3-Q), 149.7 (C2–Bi), 152.6 (C6-Q), 162.2 (d, J_CF = 245.4 Hz, C4–Ar). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 74.4 (N1-M), 149.3 (N1–Bi), 323.7 (N4-Q), 327.6 (N1-Q). The signal of N3–Bi has not been observed. 14dd (52%): ¹H NMR (500 MHz, DMSO-d₆): δ 3.41–3.43 (m, 4H, H2/H6-M), 3.81–3.83 (m, 4H, H3/H5-M), 7.16 (dd, J = 8.1, 8.1 Hz, 1H, H6–Bi), 7.19 (dd, J_HF = 9.0 Hz, J_HH = 8.9 Hz, 2H, H3/H5–Ar), 7.26 (dd, J = 8.1, 8.1 Hz, 1H, H5–Bi), 7.33 (d, J = 2.7 Hz, 1H, H5-Q), 7.53 (d, J = 8.1 Hz, 1H, H7–Bi), 7.57 (d, J = 8.1 Hz, 1H, H4–Bi), 7.57–7.63 (m, 2H, H2/H6–Ar), 7.834 (dd, J = 9.4, 2.7 Hz, 1H, H7-Q), 8.01 (d, J = 9.4 Hz, 1H, H8-Q), 12.98 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 47.6 (C2/C6-M), 65.9 (C3/C5-M), 107.2 (C5-Q), 111.8 (C4–Bi), 114.5 (d, J_CF = 21.7 Hz, C3/C5–Ar), 119.6 (C7–Bi), 121.8 (C6–Bi), 123.2 (C7-Q), 123.3 (C5–Bi), 129.3 (C8-Q), 131.6 (d, J_CF = 8.5 Hz, C2/C6–Ar), 134.3 (C3a-Bi), 135.4 (d, J_CF = 3.2 Hz, C1–Ar), 136.1 (C8a-Q), 139.4 (C3-Q), 141.5 (C4a-Q), 143.4 (C7a-Bi), 149.2 (C2–Bi), 152.2 (C2-Q), 152.3 (C6-Q), 162.4 (d, J_CF = 247.8 Hz, C4–Ar). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 72.5 (N1-M), 150.6 (N1–Bi), 323.7 (N4-Q), 329.2 (N1-Q). The signal of N3–Bi has not been observed. IR (KBr, ν, cm^–1): 3438, 1717, 1510, 1492, 1221, 1114, 842, 741 548. HRMS (MALDI-TOF) m/z: [M + H]⁺ calcd for C₂₅H₂₁N₅FO 426.1725, found 426.1705.

3-(Benzimidazol-2-yl)-2-(2-fluorophenyl)-6-(4-methylpiperazin-1-yl)quinoxaline (13ea), 2-(Benzimidazol-2-yl)-3-(2-fluorophenyl)-6-(4-methylpiperazin-1-yl)quinoxaline (14ea), 2-(4-Methylpiperazin-1-yl)benzo[4′,5′]imidazo[1′,2′:1,2]quinolino[3,4-b]quinoxaline (15ea), and 3-(4-Methylpiperazin-1-yl)benzo[4′,5′]imidazo[1′,2′,1,2]quinolino[3,4-b]quinoxaline (16ea)

A mixture of quinoxalin-2(1H)-one 9e (0.48 g, 1.80 mmol) and benzene-1,2-diamine 12a (0.45 g, 2.16 mmol) in AcOH (15 mL) was heated at reflux for 4 h. The solvent was evaporated under reduced presser. The residue was chromatographed on silica gel using CHCl₃–MeOH (50/0.75) as eluent to afford a mixture of regioisomers 13ea/14ea and 15ea/16ea. 13ea/14ea: yield: 0.49 g (63%), R_f 0.50 (CHCl₃/n-hexane/MeOH, 6:3:1), orange solid, mp 228–230 °C. 13ea (48%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.277 (s, 3H, CH₃–Pz), 2.50–2.55 (m, 4H, H3/H5-Pz), 3.44–3.48 (m, 4H, H2/H6-Pz), 7.12–7.29 (m, 4H, H5/H6–Bi, H3–Ar, H5–Ar), 7.32 (d, J = 2.8 Hz, 1H, H5-Q), 7.48–7.64 (m, 4H, H4/H7–Bi, H4–Ar, H6–Ar), 7.83 (dd, J = 9.5 Hz, J = 2.8 Hz, 1H, H7-Q), 7.99 (d, J = 9.5 Hz, 1H, H8-Q), 13.06 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.3 (CH₃–Pz), 46.9 (C2/C6-Pz), 54.0 (C3/C5-Pz), 107.6 (C5-Q), 111.9 (C4–Bi), 114.7 (d, J_CF = 21.7 Hz, C3–Ar), 119.6 (C7–Bi), 121.7 (C6–Bi), 123.2 (C7-Q), 123.5 (C5–Bi), 124.3 (d, J_CF = 0.7 Hz, C5–Ar), 128.0 (d, J_CF = 14.8 Hz, C1–Ar), 129.2 (C8-Q), 130.8 (d, J_CF = 8.3 Hz, C4–Ar), 131.3 (d, J_CF = 2.7 Hz, C6–Ar), 134.5 (C3a-Bi), 134.9 (C8a-Q), 139.6 (C3-Q), 143.0 (C4a-Q), 143.7 (C7a-Bi), 148.7 (C2-Q), 149.8 (C2–Bi), 152.3 (C6-Q), 159.8 (d, J_CF = 245.0 Hz, C2–Ar). 14ea (52%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.281 (s, 3H, CH₃–Pz), 2.50–2.55 (m, 4H, H3/H5-Pz), 3.47–3.51 (m, 4H, H2/H6-Pz), 7.12–7.29 (m, 4H, H5/H6–Bi, H3–Ar, H5–Ar), 7.31 (d, J = 2.8 Hz, 1H, H5-Q), 7.48–7.64 (m, 4H, H4/H7–Bi, H4–Ar, H6–Ar), 7.86 (dd, J = 9.5 Hz, J = 2.8 Hz, 1H, H7-Q), 8.01 (d, J = 9.5 Hz, 1H, H8-Q), 12.97 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.4 (CH₃–Pz), 47.1 (C2/C6-Pz), 54.1 (C3/C5-Pz), 107.3 (C5-Q), 111.9 (C4–Bi), 114.8 (d, J_CF = 21.7 Hz, C3–Ar), 119.7 (C7–Bi), 121.8 (C6–Bi), 123.4 (C5–Bi), 123.5 (C7-Q), 124.3 (d, J_CF = 0.7 Hz, C5–Ar), 128.0 (d, J_CF = 14.8 Hz, C1–Ar), 129.4 (C8-Q), 130.5 (d, J_CF = 8.3 Hz, C4–Ar), 131.4 (d, J_CF = 2.7 Hz, C6–Ar), 134.5 (C3a-Bi), 136.1 (C8a-Q), 142.0 (C4a-Q), 143.4 (C3-Q), 143.7 (C7a-Bi), 144.6 (C2-Q), 149.6 (C2–Bi), 152.3 (C6-Q), 160.0 (d, J_CF = 244.5 Hz, C2–Ar). IR (KBr, ν, cm^–1): 3426, 2935, 2793, 1612, 1485, 1449, 1334, 1221, 1139, 1006, 751. LRMS (MALDI-TOF) m/z: [M + H]⁺ 439.2. 15ea/16ea: yield: 0.12 g (16%), R_f 0.46 (CHCl₃/n-hexane/MeOH, 6:3:1), dark orange solid, mp 180–182 °C. 15ea (40%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.278 (s, 3H), 2.52–2.56 (m, 4H), 3.53–3.57 (m, 4H), 7.38 (d, J = 2.7 Hz, 1H), 7.55–7.62 (m, 2H), 7.69 (dd, J = 7.7, 7.5 Hz, 1H), 7.902 (dd, J = 7.7, 7.5 Hz, 1H), 7.95–7.99 (m, 1H), 8.00–8.07 (m, 1H), 8.13 (d, J = 9.4 Hz, 1H), 8.65–8.69 (m, 1H), 8.79 (d, J = 7.7 Hz, 1H), 9.10 (dd, J = 8.0, 1.6 Hz, 1H). 16ea (60%): ¹H NMR (500 MHz, DMSO-d₆): δ 2.280 (s, 3H), 2.52–2.56 (m, 4H), 3.51–3.55 (m, 4H), 7.44 (d, J = 2.7 Hz, 1H), 7.55–7.62 (m, 1H), 7.68 (dd, J = 7.7, 7.5 Hz, 1H), 7.904 (dd, J = 7.7, 7.5 Hz, 1H), 7.95–7.99 (m, 1H), 8.00–8.07 (m, 1H), 8.11 (d, J = 9.4 Hz, 1H), 8.68–8.72 (m, 1H), 8.79 (d, J = 7.7 Hz, 1H), 9.05 (dd, J = 8.0, 1.6 Hz). IR (KBr, ν, cm^–1): 2933, 2840, 2794, 1612, 1447, 1349, 1219, 1137, 754. LRMS (MALDI-TOF) m/z: [M + Na]⁺ 441.2. The purified mixture of regioisomers 13ea/14ea (0.40 g) was chromatographed on silica gel using c-hexane–EtOAc (2.5/2.0) → EtOAc–i-PrOH (25/1) as eluent to afford an analytically pure isomer 13ea (38.8 mg, 9.7%) and a mixture of regioisomers 13ea/14ea (60/40, 0.35 g, 88%). 13ea: yellow solid, mp 140–141 °C, R_f 0.36 (CHCl₃/n-hexane/MeOH, 6:3:1). ¹H NMR (500 MHz, DMSO-d₆): δ 2.26 (s, 3H, CH₃–Pz), 2.52–2.55 (m, 4H, H3/H5-Pz), 3.44–3.47 (m, 4H, H2/H6-Pz), 7.12 (dd, J_HF = 8.0, J_HH = 8.0, 1.0 Hz, 1H, H3–Ar), 7.13 (dd, J = 8.5, 8.0 Hz, 1H, H6–Bi), 7.25 (dd, J = 8.0, 8.0 Hz, 1H, H5–Bi), 7.33 (d, J = 1.7 Hz, 1H, H5-Q), 7.36 (ddd, 1H, J_HH = 7.5, 7.4 Hz, J_HF = 1.0 Hz, 1H, H5–Ar), 7.41 (d, J = 8.0 Hz, 1H, H7–Bi), 7.47–7.50 (m, 1H, H6–Ar), 7.58 (d, J = 8.0 Hz, 1H, H4–Bi), 7.67 (ddd, J_HH = 7.5, 7.4 Hz, J_HF = 1.0 Hz, 1H, H4–Ar), 7.83 (dd, J = 9.4 Hz, J = 2.7 Hz, 1H, H7-Q), 7.98 (d, J = 9.4 Hz, 1H, H8-Q), 13.11 (s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 45.7 (CH₃–Pz), 47.3 (C2/C6-Pz), 54.3 (C3/C5-Pz), 107.7 (C5-Q), 111.9 (C4–Bi), 119.7 (d, J_CF = 21.7 Hz, C6–Ar), 119.7 (C7–Bi), 121.7 (C6–Bi), 123.5 (C7-Q), 123.5 (C5–Bi), 124.2 (d, J_CF = 2.8 Hz, C3–Ar), 127.9 (d, J_CF = 15.1 Hz, C2–Ar), 129.3 (C8-Q), 130.4 (d, J_CF = 8.4 Hz, C4–Ar), 131.5 (d, J_CF = 3.1 Hz, C1–Ar), 134.4 (C3a-Bi), 136.1 (C8a-Q), 142.0 (C4a-Q), 143.4 (C3-Q), 143.7 (C7a-Bi), 144.5 (C2-Q), 149.5 (C2–Bi), 152.4 (C6-Q), 159.9 (d, J_CF = 244.1 Hz, C5–Ar). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 35.9 (N4-Pz), 74.5 (N1-Pz), 148.6 (N1–Bi), 316.5 (N4-Q), 333.6 (N1-Q). The signal of N3–Bi has not been observed. IR (KBr, ν, cm^–1): 3060, 2941, 2835, 2813, 1619, 1485, 1457, 1334, 1219, 1143, 757. HRMS (MALDI-TOF) m/z: [M + Cs]⁺ calcd for C₂₆H₂₃N₆FCs 571.1017, found 571.0999.

4-(3-(Benzimidazol-2-yl)-2-(2-fluorophenyl)quinoxalin-6-yl)morpholine (13ed), 4-(2-(Benzimidazol-2-yl)-3-(2-fluorophenyl)quinoxalin-6-yl)morpholine (14ed), 4-(Benzo[4′,5′]imidazo[1′,2′:1,2]quinolino[3,4-b]quinoxalin-2-yl)morpholine (15ed), and 4-(Benzo[4′,5′]imidazo[1′,2′:1,2]quinolino[3,4-b]quinoxalin-3-yl)morpholine (16ed)

A mixture of quinoxalin-2(1H)-one 9e (0.48 g, 1.80 mmol) and benzene-1,2-diamine 12d (0.42 g, 2.16 mmol) in AcOH (15 mL) was heated at reflux for 4 h. The solvent was evaporated under reduced pressure. The residue was chromatographed on silica gel using c-hexane–EtOAc (2/1) → EtOAc–i-PrOH (25/1) as eluent to afford a mixture of regioisomer 13ed and a mixture of regioisomers 13ed/14ed, 15ed/16ed. 13ed: yield 91.2 mg (12%), bright yellow solid, mp 210–211 °C, R_f 0.40 (CHCl₃/n-hexane/MeOH, 6:3:1). ¹H NMR (500 MHz, DMSO-d₆): δ 3.40–3.44 (m, 4H, H2/H6-M), 3.80–3.84 (m, 4H, H3/H5-M), 7.128 (dd, J_HF = 8.0, J_HH = 8.0, 1.0 Hz, 1H, H3–Ar), 7.133 (dd, J = 8.5, 8.0 Hz, 1H, H5–Bi), 7.25 (dd, J = 8.5, 8.0 Hz, 1H, H6–Bi), 7.341 (ddd, J_HH = 7.6, 7.5 Hz, J_HF = 0.7 Hz, 1H, H5–Ar), 7.342 (d, J = 2.8 Hz, 1H, H5-Q), 7.43 (d, J = 8.0 Hz, 1H, H4–Bi), 7.47–7.52 (m, 1H, H6–Ar), 7.58 (d, J = 8.0 Hz, 1H, H7–Bi), 7.68 (ddd, J_HH = 7.6, 7.5 Hz, J_HF = 1.5 Hz, 1H, H4–Ar), 7.83 (dd, J = 9.4, 2.8 Hz, 1H, H7-Q), 8.01 (d, J = 9.4 Hz, 1H, H8-Q), 13.12 (br s, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 47.5 (C2/C6-M), 65.9 (C3/C5-M), 107.1 (C5-Q), 111.8 (C7–Bi), 114.8 (d, J_CF = 21.6 Hz, C4–Ar), 127.8 (d, J_CF = 8.2 Hz, C1–Ar), 129.3 (C8-Q), 130.4 (d, J_CF = 8.2 Hz, C3–Ar), 131.4 (d, J_CF = 2.5 Hz, C5–Ar), 134.4 (C7a-Bi), 136.2 (C8a-Q), 141.8 (C4a-Q), 159.9 (d, J_CF = 244.7 Hz, C2–Ar), 143.4 (C2-Q), 143.7 (C3a-Bi), 152.5 (C6-Q), 149.5 (C2–Bi), 144.7 (C3-Q), 119.7 (C4–Bi), 121.7 (C5–Bi), 123.1 (C7-Q), 123.5 (C6–Bi), 124.2 (d, J_CF = 2.1 Hz, C6–Ar). ¹⁵N NMR (51 MHz, DMSO-d₆): δ 73.1 (N1-M), 148.9 (N1–Bi), 250.3 (N3–Bi), 316.6 (N4-Q), 333.8 (N1-Q). IR (KBr, ν, cm^–1): 3379–2799, 1611, 1485, 1455, 1398, 1331, 1213, 1115, 948, 757, 735, 643, 609, 429. HRMS (MALDI-TOF) m/z: [M + Na]⁺ calcd for C₂₅H₂₀FN₅ONa 448.1544, found 448.1532. 13ed/14ed: yield 0.36 g (48%), R_f 0.36 (CHCl₃/n-hexane/MeOH, 6:3:1), yellow solid, mp 170–172 °C. 13ed (48%): ¹H NMR (500 MHz, DMSO-d₆): δ 3.42–3.46 (m, 4H, H2/H6-M), 3.82–3.86 (m, 4H, H3/H5-M), 7.11 (dd, J = 8.0, 8.0 Hz, 1H, H5–Bi), 7.16–7.20 (m, 2H, H3–Ar, H6–Bi), 7.32–7.36 (m, 1H, H5–Ar), 7.349 (d, J = 2.8 Hz, 1H, H5-Q), 7.46–7.52 (m, 1H, H6–Ar, H4/H7–Bi), 7.68 (ddd, J_HH = 7.6, 7.5 Hz, J_HF = 1.5 Hz, 1H, H4–Ar), 7.85 (dd, J = 9.4, 2.8 Hz, 1H, H7-Q), 8.02 (d, J = 9.4 Hz, 1H, H8-Q), 13.12 (br, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 47.8 (C2/C6-M), 65.8 (C3/5-M), 107.1 (C5-Q), 111.8 (C7–Bi), 114.8 (d, J_CF = 21.6 Hz, C4–Ar), 119.7 (C4–Bi), 159.8 (d, J_CF = 244.7 Hz, C2–Ar), 121.5 (C5–Bi), 122.8 (C7-Q), 123.2 (C6–Bi), 124.1 (d, J_CF = 2.1 Hz, C6–Ar), 127.7 (d, J_CF = 8.2 Hz, C1–Ar), 129.3 (C8-Q), 130.4 (d, J_CF = 8.2 Hz, C3–Ar), 131.2 (d, J_CF = 2.5 Hz, C5–Ar), 134.4 (C7a-Bi), 135.0 (C8a-Q), 139.7 (C4a-Q), 142.9 (C3-Q), 143.7 (C3a-Bi), 152.5 (C6-Q), 148.6 (C2-Q), 149.5 (C2–Bi). 14ed (52%): ¹H NMR (500 MHz, DMSO-d₆): δ 3.42–3.46 (m, 4H, H2/H6-M), 3.82–3.86 (m, 4H, H3/H5-M), 7.13 (dd, J = 8.0, 8.0 Hz, 1H, H5–Bi), 7.16–7.20 (m, 2H, H3–Ar, H6–Bi), 7.32–7.36 (m, 1H, H5–Ar), 7.348 (d, J = 2.8 Hz, 1H, H5-Q), 7.46–7.52 (m, 3H, H4/H7–Bi, H6–Ar), 7.67 (ddd, J_HH = 7.6, 7.5 Hz, J_HF = 1.5 Hz, 1H, H4–Ar), 7.87 (dd, J = 9.4, 2.8 Hz, 1H, H7-Q), 8.06 (d, J = 9.4 Hz, 1H, H8-Q), 13.02 (br, 1H, NH-Bi). ¹³C NMR (126 MHz, DMSO-d₆): δ 47.4 (C2/C6-M), 65.8 (C3/C5-M), 107.5 (C5-Q), 111.7 (C7–Bi), 114.8 (d, J_CF = 21.6 Hz, C4–Ar), 119.5 (C4–Bi), 121.7 (C5–Bi), 123.1 (C7-Q), 123.5 (C6–Bi), 124.1 (d, J_CF = 2.1 Hz, C6–Ar), 127.9 (d, J_CF = 8.2 Hz, C1–Ar), 129.1 (C8-Q), 130.6 (d, J_CF = 8.2 Hz, C3–Ar), 131.3 (d, J_CF = 2.5 Hz, C5–Ar), 134.4 (C7a-Bi), 136.2 (C8a-Q), 141.8 (C4a-Q), 143.4 (C2-Q), 143.7 (C3a-Bi), 144.7 (C3-Q), 149.6 (C2–Bi), 152.5 (C6-Q), 159.9 (d, J_CF = 244.7 Hz, C2–Ar). IR (KBr, ν, cm^–1): 3419, 3059–2800, 1612, 1485, 1332, 1215, 1116, 828, 756, 736, 632. HRMS (MALDI-TOF) m/z: [M + H]⁺ calcd for C₂₅H₂₁FN₅O 426.1725, found 426.1705. 15ed/16ed: yield: 94.3 mg (13%), R_f 0.32 (CHCl₃/n-hexane/MeOH, 6:3:1), red solid, mp 170–171 °C. 15ed (70%): ¹H NMR (500 MHz, DMSO-d₆): δ 3.51–3.55 (m, 4H), 3.83–3.87 (m, 4H), 7.39 (d, J = 2.7 Hz, 1H), 7.55–7.62 (m, 2H), 7.68 (dd, J = 7.7, 7.5 Hz, 1H), 7.89–7.98 (m, 2H), 8.00–8.07 (m, 1H), 8.15 (d, J = 9.4 Hz, 1H), 8.65–8.72 (m, 1H), 8.79 (d, J = 7.7 Hz, 1H), 9.10 (dd, J = 8.0, 1.6 Hz, 1H). 16ed (30%): ¹H NMR (500 MHz, DMSO-d₆): δ 3.49–3.53 (m, 4H), 3.83–3.87 (m, 4H), 7.45 (d, J = 2.7 Hz, 1H), 7.55–7.62 (m, 2H), 7.68 (dd, J = 7.7, 7.5 Hz, 1H), 7.89–7.98 (m, 2H), 8.00–8.07 (m, 1H), 8.14 (d, J = 9.4 Hz, 1H), 8.65–8.72 (m, 1H), 8.79 (d, J = 7.7 Hz, 1H), 9.05 (dd, J = 8.0, 1.6 Hz, 1H). IR (KBr, ν, cm^–1): 2959–2852, 1685, 1613, 1447, 1351, 1220, 1114, 752. HRMS (MALDI-TOF) m/z: [M + H]⁺ calcd for C₂₅H₂₀N₅O 406.1662, found 406.1679.

Cytotoxicity Assay

Cytotoxic effects of the test compounds on human cancer and normal cells were estimated by means of the multifunctional Cytell Cell Imaging system (GE Health Care Life Science, Sweden) using the Cell Viability Bio App, which precisely counts the number of cells and evaluates their viability from fluorescence intensity data.⁶² Two fluorescent dyes—DAPI and propidium iodide—were used in the experiments. DAPI and propidium iodide were purchased from Sigma. The M-HeLa clone 11 human, epithelioid cervical carcinoma, strain of HeLa, clone of M-HeLa; human breast adenocarcinoma cells (MCF-7); glioblastoma cell line (T98G); human duodenal cancer cell line (HuTu 80); PANC-1 is a human pancreatic cancer cell line; Human Lung Adenocarcinoma (A549); Wi38 VA-13 cell culture, subline 2RA (human embryonic lung) from the Type Culture Collection of the Institute of Cytology (Russian Academy of Sciences), and PC-3 human Caucasian prostate adenocarcinoma from Type Culture Collection (ATCC, Manassas, VA, USA) were used in the experiments. The cells were cultured in a standard Eagle’s nutrient medium manufactured at the Chumakov Institute of Poliomyelitis and Virus Encephalitis (PanEco company) and supplemented with 10% fetal calf serum and 1% nonessential amino acids. The cells were plated into a 96-well plate (Nunc) at a concentration of 1 × 10⁵ cells/mL, 150 μL of medium per well, and cultured in a CO₂ incubator at 37 °C. Twenty-four hours after seeding the cells into the wells, the test compound was added in a preset dilution, with 150 μL in each well. The dilutions of the compounds were prepared immediately in nutrient media; 5% DMSO that does not induce the inhibition of cells at this concentration was added for better solubility. The experiments were repeated three times. Intact cells cultured in parallel with experimental cells were used as a control.

Hemolytic Activity

The hemolytic activity of the test compounds was assessed by comparing the optical density of a solution containing the test compound with the optical density of blood at 100% hemolysis. The object of the study was a 10% suspension of human erythrocytes. The studies were carried out according to the previously described method.⁶³

Real-Time Monitoring of A 549 Cell Proliferation

XCELLigence S16 Real-time Analysis System RTCA (Acea Biosciences, USA) was used to evaluate the proliferation and growth rate of A549 cells. For this assay, cells were maintained in a standard Eagle’s nutrient medium manufactured at the Chumakov Institute of Poliomyelitis and Virus Encephalitis (PanEco company) and supplemented with 10% fetal calf serum and 1% nonessential amino acids. First, 125 μL of medium was added to each well of a 16-well E-Plate (Acea Biosciences, USA), and background measurements were made. Then, 62.5 μL of A549 suspensions were inoculated, 5 × 10⁵ cells/well, into the E-Plates. The 16-well E-Plate was placed in an xCELLigence RTCA and cultured in a CO₂ incubator at 37 °C.

The device was set to take a measurement every 15 min. Approximately 24 h after inoculation, at the first third of the cell proliferation phase, 62.5 μL of different concentrations of mriBIQ 13da/14da were added to the media. Eagle’s medium (125 μL) was added to the control groups. The measurements continued at 15 min intervals for 72 h. Graphs of concentration and time-dependent cell index values were plotted using RTCA Software 2.0. All experiments were conducted in triplicate and repeated three separate times.

Flow Cytometry Assay

Cell Culture

A549 cells at 1 × 10⁶ cells/well in a final volume of 2 mL were seeded into 6-well plates. After 24 h of incubation, various concentrations of mriBIQ 13da/14da were added to wells.

Cell Cycle Analysis

The DNA content and cell-cycle distribution after mriBIQ 13da/14da treatment were estimated by flow cytometry. After washing with PBS, treated cells were suspended in 150 μL of PBS; then 0.5 mL of phosphate-citrate buffer (0.05 M, pH 4.0) was added, and the suspension was incubated at room temperature for 5 min to facilitate the extraction of low molecular weight DNA. Following centrifugation, the cells were resuspended in 150 μL of DNA staining solution (20 μg/mL propidium iodide, 200 μg/mL DNase (RNase-free) and incubated in the CO₂ incubator (37 °C for 30 min). The distribution of the cell cycle was determined by fluorescence analysis of A549 cells stained with propidium iodide using Guava EasyCyte (Guava easy Cyte, MERCK).⁶⁴

Mitochondrial Membrane Potential

Cells were harvested at 2000 rpm for 5 min and then washed twice with ice-cold PBS, followed by resuspension in JC-10 (10 μg/mL) and incubation at 37 °C for 10 min. After the cells were rinsed three times and suspended in PBS, the JC-10 fluorescence was observed by flow cytometry (Guava easy Cyte, MERCK, USA). The experiments were repeated three times.

Induction of the Production of Intracellular Reactive Oxygen Species (ROS)

Fluorescence Microscopy

A549 cells in an amount of 1 × 10⁶ cells/well in a final volume of 2 mL were seeded in 6-well plates with coverslips at the bottom of each well. After 24 h of incubation, various concentrations of mriBIQ 13da/14da were added to wells and cultured for 24 h in a CO₂ incubator. Then, after treatment with test compounds, A549 cells were fixed and stained with DAPI (blue).

Intracellular induction of ROS production was investigated using diacetate 2′,7′-dichlorofluorescein (DCFH-DA). 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) is a chemically reduced form of fluorescein used as an indicator for reactive oxygen species (ROS) in cells. After cleavage of acetate groups by intracellular esterases and oxidation, nonfluorescent H2DCFDA is converted to highly fluorescent 2′,7′-dichlorofluorescein (DCF).

To detect ROS A549, cells were harvested at 2000 rpm for 5 min and then washed twice with ice-cold PBS, followed by resuspension in 0.5 mL of growth medium without fetal bovine serum (FBS) containing 5 μM DCFH-DA and incubated at 37 °C for 1 h. After washing the cells three times in PBS, the ROS production in the cells was immediately monitored using a Nikon Eclipse Ci-S fluorescence microscope (Nikon, Japan) at a magnification of 1000×. The fluorescence intensity (DCFH-DA) was determined using a specialized computer program NISE lements, designed for the analysis and processing of information obtained from a Nikon Eclipse Ci-S microscope.

Multiplex Analysis of Markers DNA Damage/Genotoxicity

The studies were carried out according to the standard protocol. A549 cells were incubated for 24 h with the test substance. Cells were lysed in MILLIPLEX MAP Lysis buffer containing protease inhibitors. Twenty micrograms of total protein of each lysate diluted in MILLIPLEX MAP Assay Buffer 2 was analyzed according to the analysis protocol (the lysate was incubated at 4 °C overnight). The mean fluorescence intensity (MFI) was detected using the Luminex system, MERCK, USA.

Statistical Analysis

IC₅₀ was calculated using an online tool: MLA–“Quest Graph IC₅₀ Calculator.” AAT Bioquest, Inc., accessed 2021-02-16, https://www.aatbio.com/tools/ic50-calculator.⁶⁵ The cytometric results were analyzed by the Cytell Cell Imaging multifunctional system using the Cell Viability BioApp. The data in the tables and graphs are given as the mean ± standard error.

Glossary

Abbreviations

AcOH

acetic acid

DMF

N,N-dimethylformamide

EtOAc

ethyl acetoacetate

MeOH

methanol

Pd/C

palladium on carbon

DMSO

dimethyl sulfoxide

DNA

DNA

MDC1

mediator of DNA Damage Checkpoint 1

H2AX

H2A histone family member X

BRCA1

breast cancer 1

SMC1

structural maintenance of chromosomes protein 1A

MDM2

mouse double minute 2

P53

protein 53

DAPI

4′,6-diamidino-2-phenylindole

PBS

phosphate-buffered saline

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00118.

• Molecular formula strings (CSV)

• ¹H NMR, ¹³C NMR, and qHNMR spectra (PDF)

• Crystallographic data for 13da in CIF format (PDF)

• Crystallographic data for 14da in CIF format (PDF)

• X-ray crystallographic data of compounds 13da, 14da; powder X-ray diffraction data of mixture regioisomers 13da and 14da (PDF)

The authors declare no competing financial interest.