Diverse Biological Activity of Benzofuroxan/Sterically Hindered Phenols Hybrids

Combining two pharmacophores in a molecule can lead to useful synergistic effects. Herein, we show hybrid systems that combine sterically hindered phenols with dinitrobenzofuroxan fragments exhibit a broad range of biological activities. The modular assembly of such phenol/benzofuroxan hybrids allows variations in the phenol/benzofuroxan ratio. Interestingly, the antimicrobial activity only appears when at least two benzofuroxan moieties are introduced per phenol. The most potent of the synthesized compounds exhibit high cytotoxicity against human duodenal adenocarcinoma (HuTu 80), human breast adenocarcinoma (MCF-7), and human cervical carcinoma cell lines. This toxicity is associated with the induction of apoptosis via the internal mitochondrial pathway and an increase in ROS production. Encouragingly, the index of selectivity relative to healthy tissues exceeds that for the reference drugs Doxorubicin and Sorafenib. The biostability of the leading compounds in whole mice blood is sufficiently high for their future quantification in biological matrices.


Introduction
Cancer continues to be one of the most serious challenges facing modern science and medicine. Oncological diseases steadily occupy the second place in the list of causes of human death. According to the World Health Organization, by 2030, 15 million people will be dying from this pathology in the world every year [1].
In our search for new highly effective antitumor drugs, we chose a molecular design that can achieve two main goals: (1) to reduce toxicity in a healthy microenvironment In addition, NO is a necessary component of a non-specific defense mechanism against many pathogens, including bacteria, viruses and fungi. A number of publications describe the high fungicidal activity of the so-called "hybrid" benzofuroxan derivatives, which include, in addition to the benzofuroxan moiety, known pharmacophore In summary, our aim is to develop agents with a broad spectrum of biological acti Our basic molecular platform is a substituted sterically hindered phenol, which can reversibly oxidized to the corresponding methylene quinone in cancer cells but exhib antioxidant activity in healthy cells. The auxiliary components include diaminopyridine or diaminophenyl linker for targeted modification, phosphorus-containing fragment for increasing bioavailability, and benzofuroxan as additional pharmacophore with the potential to be both the NO-donor a apoptosis-inducing agent ( Figure 1).

Chemistry
Phosphorylated sterically hindered phenols (SHPs) 2 containing diaminopyridine diaminophenyl fragments were used as a starting point due to their already identif high and selective cytotoxicity [35]. The synthesis of the starting SHPs was carried out accordance with the original method developed by us earlier (Scheme 1, [35]). The m stages of this method include preparation of the corresponding phosphorylated phen its oxidation to methylene quinone 1, and, as the last step, reaction of the methyle quinone with C-nucleophile, i.e., 2,6-diaminopyridine or 1,3-diaminobenzene. T sequence produces the key compounds 2, that possess two amino groups suitable further modifications with heterocyclic fragments. Finally, the amino groups of the two hindered phosphorylated phenols 2 were us in reactions with nitrochlorobenzofuroxans. This highly reactive heterocycle superelectrophilic and readily reacts with the appended aniline moieties. The phenol O group is unreactive under these conditions due to the steric protection provided by two tert-butyl groups. The final products of this synthetic sequence result are previou unknown "hybrid" phosphorus-containing SHPs containing a nitrobenzofurox fragment linked to the SHP fragment through a (hetero)aromatic "linker" (Scheme 2). Scheme 1. The synthesis of the starting SHPs 2 containing diaminopyridine or diaminophenyl fragments.
Finally, the amino groups of the two hindered phosphorylated phenols 2 were used in reactions with nitrochlorobenzofuroxans. This highly reactive heterocycle is superelectrophilic and readily reacts with the appended aniline moieties. The phenol OH group is unreactive under these conditions due to the steric protection provided by the two tertbutyl groups. The final products of this synthetic sequence result are previously unknown "hybrid" phosphorus-containing SHPs containing a nitrobenzofuroxan fragment linked to the SHP fragment through a (hetero)aromatic "linker" (Scheme 2).
Depending on the initial ratio of reagents, it is possible to vary the composition of the final products, leading to the formation of compounds with a composition of 2:1 when two molecules of benzofuroxan interact with one molecule of sterically hindered phenol and compounds of the composition of 1:1, when one molecule of benzofuroxan reacts with one molecule of sterically hindered phenol. The excess phosphorus-containing SHP in both cases was used to neutralize the hydrogen chloride formed during the reaction. Depending on the initial ratio of reagents, it is possible to vary the composition of the final products, leading to the formation of compounds with a composition of 2:1 when two molecules of benzofuroxan interact with one molecule of sterically hindered phenol and compounds of the composition of 1:1, when one molecule of benzofuroxan reacts with one molecule of sterically hindered phenol. The excess phosphorus-containing SHP in both cases was used to neutralize the hydrogen chloride formed during the reaction.
As is known in the literature, benzofuroxan rapidly interconverts in solution between two non-symmetric bicyclic structures through the open dinitroso form (so-called N-1-oxide/N-3-oxide tautomerism, Scheme 3) [36,37]. As a result, the NMR 1 H and 13 C spectra of benzofuroxan derivatives taken at room temperatures are often broadened as they correspond to a fast mutual transition of equivalent non-symmetric forms, while the spectra taken at low temperature are non-symmetric forms [38]. In our study, we also observed our products in the spectra, not as individual compounds but as a mixture of tautomers. In some cases, they were separated into the pure form (as in the case of compounds 4e-g). In other cases, where the content of the second tautomer was insignificant, we limited ourselves to describing only the main structure. The complete assignment of signals in 1 H and 13 C spectra (Figures S1-S45) was accomplished by using the 2D NMR techniques (COSY, 1 H-13 C HSQC and 1 H-13 C HMBC). Examples of 2D spectra for compounds 4d and 4e are presented (Figures S12-S14, S18-S20). For compounds within the series, the assignment of peaks was conducted by analogy, taking into account the effects of neighboring substituents. Some difficulty is that benzofuroxans exist as two tautomers due to oxygen migration. As we showed earlier [39], the presence of only one proton in the tri-substituted benzofuroxan moiety makes it difficult to use two-dimensional NMR experiments. At the same time, the chemical shifts of carbons in these tautomeric forms do not differ significantly. As is known in the literature, benzofuroxan rapidly interconverts in solution between two non-symmetric bicyclic structures through the open dinitroso form (so-called N-1oxide/N-3-oxide tautomerism, Scheme 3) [36,37]. As a result, the NMR 1 H and 13 C spectra of benzofuroxan derivatives taken at room temperatures are often broadened as they correspond to a fast mutual transition of equivalent non-symmetric forms, while the spectra taken at low temperature are non-symmetric forms [38]. In our study, we also observed our products in the spectra, not as individual compounds but as a mixture of tautomers. In some cases, they were separated into the pure form (as in the case of compounds 4e-g). In other cases, where the content of the second tautomer was insignificant, we limited ourselves to describing only the main structure.

Scheme 2. Synthesis of SHP/furoxan hybrids of varying composition.
Depending on the initial ratio of reagents, it is possible to vary the composition of the final products, leading to the formation of compounds with a composition of 2:1 when two molecules of benzofuroxan interact with one molecule of sterically hindered phenol and compounds of the composition of 1:1, when one molecule of benzofuroxan reacts with one molecule of sterically hindered phenol. The excess phosphorus-containing SHP in both cases was used to neutralize the hydrogen chloride formed during the reaction.
As is known in the literature, benzofuroxan rapidly interconverts in solution between two non-symmetric bicyclic structures through the open dinitroso form (so-called N-1-oxide/N-3-oxide tautomerism, Scheme 3) [36,37]. As a result, the NMR 1 H and 13 C spectra of benzofuroxan derivatives taken at room temperatures are often broadened as they correspond to a fast mutual transition of equivalent non-symmetric forms, while the spectra taken at low temperature are non-symmetric forms [38]. In our study, we also observed our products in the spectra, not as individual compounds but as a mixture of tautomers. In some cases, they were separated into the pure form (as in the case of compounds 4e-g). In other cases, where the content of the second tautomer was insignificant, we limited ourselves to describing only the main structure. The complete assignment of signals in 1 H and 13 C spectra (Figures S1-S45) was accomplished by using the 2D NMR techniques (COSY, 1 H-13 C HSQC and 1 H-13 C HMBC). Examples of 2D spectra for compounds 4d and 4e are presented (Figures S12-S14, S18-S20). For compounds within the series, the assignment of peaks was conducted by analogy, taking into account the effects of neighboring substituents. Some difficulty is that benzofuroxans exist as two tautomers due to oxygen migration. As we showed earlier [39], the presence of only one proton in the tri-substituted benzofuroxan moiety makes it difficult to use two-dimensional NMR experiments. At the same time, the chemical shifts of carbons in these tautomeric forms do not differ significantly. The complete assignment of signals in 1 H and 13 C spectra (Figures S1-S45) was accomplished by using the 2D NMR techniques (COSY, 1 H-13 C HSQC and 1 H-13 C HMBC). Examples of 2D spectra for compounds 4d and 4e are presented (Figures S12-S14 and S18-S20). For compounds within the series, the assignment of peaks was conducted by analogy, taking into account the effects of neighboring substituents. Some difficulty is that benzofuroxans exist as two tautomers due to oxygen migration. As we showed earlier [39], the presence of only one proton in the tri-substituted benzofuroxan moiety makes it difficult to use two-dimensional NMR experiments. At the same time, the chemical shifts of carbons in these tautomeric forms do not differ significantly. Nevertheless, the difference between C3a and C7a makes it possible to quite unambiguously attribute the observed shape of the benzofuroxan ring.
The exception is the interaction of benzofuroxan 3 with diaminophenyl derivative 2f containing diethylphosphonate fragment. We found that regardless of the ratio of reagents employed, there is only a formation of a mono-substitution product 4f. The 2:1 product cannot be obtained, probably because of a sterically hindered environment for the NH 2 group located in the ortho-position relative to the methyldiethylphosphonate fragment.

Biological Evaluation
For the synthesized compounds, diverse biological properties were studied: antitumor potential with the determination of the mechanisms of action, hemolytic activity, antibacterial properties and biostability.

Anticancer Activity
To start, a new series of synthesized compounds proposed as potential anticancer agents were tested for cytotoxicity against cancer and normal cell lines (Table 1). Data on cytotoxic activity are represented by IC 50 values (the concentration of compound that causes 50% cell death in the test population). It is interesting to compare the activity of hybrid molecules 4-5 to their building blocks, SHPs 2 and chlorobenzofuroxan 3. As can be seen from the data given in Table 1, the simple benzofuroxan 3 does not show cytotoxicity at these concentrations. At the same time, the initial SHPs 2 have cytotoxic activity, which, as we noted earlier, motivated us to choose them as a starting point of our design. Gratifying, the hybrid compounds 4-5 showed relatively high activity against all cancer lines used in the experiments. In addition, the lead compounds have moderate cytotoxicity against the normal Chang liver cells.
When compared with the starting compounds, the SHP/benzofuroxan hybrid 4c is 40 times more active than SHP 2c with respect to M-HeLa and 20 times more active with respect to MCF-7. The activity of compound 5d exceeds the activity of compound 2d by 3.7 times with respect to M-HeLa and 7.6 times with respect to MCF-7. However, it should be noted that the cytotoxicity towards normal cells of these hybrid leader compounds also increases, which makes these compounds more toxic. These data suggest that finding the proper balance between efficacy and selectivity remains a challenge. From these data, we have identified two lead compounds, 4c and 5d, that show high cytotoxicity against human duodenal adenocarcinoma (HuTu 80), human breast adenocarcinoma (MCF-7) and human cervical carcinoma cell lines. The IC 50 values of compounds 4c and 5d for these lines were either comparable to or exceeded the activity of the reference drugs Doxorubicin and Sorafenib.
The key indicator for evaluating perspective antitumor drugs is the selectivity index (SI), which was calculated as the ratio between the IC 50 value for normal cells and the IC 50 value for cancer cells. The selectivity index values for 5d are shown in Table 1. According to the literature guidelines [40], compounds are considered selective at SI ≥ 3. Therefore, the lead compound 5d can be considered selective for MCF-7 and M-HeLa cell lines at SI = 4. Note that the reference drugs Doxorubicin and Sorafenib are significantly inferior in selectivity compared to the lead compound.
Apoptosis is one of the preferred mechanisms of cytotoxic action for the development of new anticancer agents. Apoptosis-inducing properties of the leading compounds, 4c and 5d, were evaluated by flow cytometry at IC 50 /2 and IC 50 concentrations on the M-HeLa cell line (Figure 2a,b). This assay is convenient for detecting apoptosis and for differentiating its stages. In particular, the viable cells are negative for both PI and Annexin V-Alexa Fluor 647 binding; non-viable, necrotic cells are negative for Annexin V-Alexa Fluor 647 binding and positive for PI uptake; cells in early apoptosis are Annexin V-Alexa Fluor 647 positive and PI negative; cells in late apoptosis are positive for both Annexin V-Alexa Fluor 647 binding and PI uptake. The possibility of apoptosis through the mitochondrial pathway was assessed by flow cytometry using the JC-10 fluorescent dye (in the Mitochondria Membrane Potential Kit). In normal cells, JC-10 accumulates in the mitochondrial matrix, where it forms aggregates identified via their red fluorescence. However, in apoptotic cells, JC-10 diffuses out of the mitochondria, converts to its monomeric form, and emits green fluorescence, which is recorded by a flow cytometer. After treatment with compounds 4c and 5d at concentrations of IC50/2 and IC50, we observed the dissipation of the mitochondrial membrane potential of M-HeLa cells, which became more pronounced in the case of compound 4c (Figure 3a). Figure 3b shows that the intensity of green fluorescence significantly increased relative to the control. These results suggest that the mechanism of action of the studied compounds is associated with the induction of identified via their red fluorescence. However, in apoptotic cells, JC-10 diffuses out of the mitochondria, converts to its monomeric form, and emits green fluorescence, which is recorded by a flow cytometer. After treatment with compounds 4c and 5d at concentrations of IC 50 /2 and IC 50 , we observed the dissipation of the mitochondrial membrane potential of M-HeLa cells, which became more pronounced in the case of compound 4c (Figure 3a). Figure 3b shows that the intensity of green fluorescence significantly increased relative to the control. These results suggest that the mechanism of action of the studied compounds is associated with the induction of apoptosis, which occurs along the internal mitochondrial pathway. Apoptosis can be induced in various ways, including an increase in the production of reactive oxygen species (ROS) in the cell, with subsequent oxidative stress and destruction of membrane lipids, proteins and nucleic acids. Thus, chemical compounds that disturb the redox balance and lead to the production and accumulation of ROS are potential agents for targeting the transformed cells. To understand the possible synergy between the different components of these hybrid agents, we compared the ROS production for compounds 4c, 5d, starting phenols 2с, 2d and furoxan 3 at the concentration of IC50 cytotoxicity on M-HeLa cells (Figure 4). A significant increase in CellROX ® Deep Red fluorescence intensity indicates an increase in ROS production. This increase is especially pronounced in the presence of compound 4c where ROS production significantly exceeds that for the original phenol 2c and furoxan 3. Hence, it is reasonable to suggest that activity of compound 4c benefits from the synergy between the methylene quinones (as producers of superoxide) and furoxans (as the NO donors). Apoptosis can be induced in various ways, including an increase in the production of reactive oxygen species (ROS) in the cell, with subsequent oxidative stress and destruction of membrane lipids, proteins and nucleic acids. Thus, chemical compounds that disturb the redox balance and lead to the production and accumulation of ROS are potential agents for targeting the transformed cells. To understand the possible synergy between the different components of these hybrid agents, we compared the ROS production for compounds 4c, 5d, starting phenols 2c, 2d and furoxan 3 at the concentration of IC50 cytotoxicity on M-HeLa cells (Figure 4). A significant increase in CellROX ® Deep Red fluorescence intensity indicates an increase in ROS production. This increase is especially pronounced in the presence of compound 4c where ROS production significantly exceeds that for the original phenol 2c and furoxan 3. Hence, it is reasonable to suggest that activity of compound 4c benefits from the synergy between the methylene quinones (as producers of superoxide) and furoxans (as the NO donors).
Thus, the phenol-benzofuroxan hybrids exhibit high cytotoxicity. This biological activity is primarily associated with the induction of apoptosis, occurring via the internal mitochondrial pathway and an increase in ROS production.
Thus, the phenol-benzofuroxan hybrids exhibit high cytotoxicity. This biological activity is primarily associated with the induction of apoptosis, occurring via the internal mitochondrial pathway and an increase in ROS production.
Additionally, we evaluated the hemolytic activity (i.e., the ability to destroy human erythrocytes) and biostability in whole mice blood for the leader compounds. The tested compounds do not show hemolytic activity (HC50 >100 µM) and are metabolized relatively slowly (data are presented in supporting information, Figure S46).

Antimicrobial Activity
The synthesized compounds were also tested for antibacterial activity against a number of gram-positive Staphylococcus aureus ATCC 6538P FDA 209P (Sa), Bacillus cereus ATCC 10702 (Bc), Enterococcus faecalis ATCC 29212 (Ef), gram-negative bacteria Escherichia coli ATCC 25922 (Ec) and Pseudomonas aeruginosa ATCC 9027 (Рa), including against methicillin-resistant strains of Staphylococcus aureus MRSA-1 and MRSA-2. Methicillin-resistant strains of S. aureus were provided to us by the Republican Clinical Hospital (Kazan, Russia) from patients with chronic tonsillitis and sinusitis and were highly resistant: MRSA-1-to β-lactams and fluoroquinolones and MRSA-2-only to β-lactams. Antifungal activity was studied on Candida albicans 10231. Neither sterically hindered phenols 2 and benzofuroxan 3 nor their 1:1 hybrids 4 show activity against fungi and bacteria. On the other hand, the introduction of the second benzofuroxan fragment leads to the rise of antimicrobial activity ( Table 2). Compounds 5a, 5с and 5d display selective antimicrobial activity against gram-positive bacteria S. aureus, B. cereus, E. faecalis (at the level of the reference drug Chloramphenicol) and strain MRSA 1. These compounds are less active against MRSA-2. All studied compounds were inactive toward gram-negative bacteria and the yeast Candida albicans 10231. . Induction of ROS production by compounds 2c, 2d, 3, 4c and 5d. The data were obtained with flow cytometry and the CellROX ® Deep Red kit. Concentration for compounds was equivalent to IC 50 cytotoxicity on M-HeLa cells. Data are presented as mean ± SD (n = 3). ** p < 0.01, **** p < 0.0001 compared to control; #### p ≤ 0.0001, compared to 2c; $$$$ p ≤ 0.0001 and compared to 3 (oneway ANOVA).

Antimicrobial Activity
The synthesized compounds were also tested for antibacterial activity against a number of gram-positive Staphylococcus aureus ATCC 6538P FDA 209P (Sa), Bacillus cereus ATCC 10702 (Bc), Enterococcus faecalis ATCC 29212 (Ef ), gram-negative bacteria Escherichia coli ATCC 25922 (Ec) and Pseudomonas aeruginosa ATCC 9027 (Pa), including against methicillinresistant strains of Staphylococcus aureus MRSA-1 and MRSA-2. Methicillin-resistant strains of S. aureus were provided to us by the Republican Clinical Hospital (Kazan, Russia) from patients with chronic tonsillitis and sinusitis and were highly resistant: MRSA-1-to βlactams and fluoroquinolones and MRSA-2-only to β-lactams. Antifungal activity was studied on Candida albicans 10231. Neither sterically hindered phenols 2 and benzofuroxan 3 nor their 1:1 hybrids 4 show activity against fungi and bacteria. On the other hand, the introduction of the second benzofuroxan fragment leads to the rise of antimicrobial activity ( Table 2). Compounds 5a, 5c and 5d display selective antimicrobial activity against gram-positive bacteria S. aureus, B. cereus, E. faecalis (at the level of the reference drug Chloramphenicol) and strain MRSA 1. These compounds are less active against MRSA-2. All studied compounds were inactive toward gram-negative bacteria and the yeast Candida albicans 10231. Table 2. Antimicrobial activity of studied compounds.
7-Chloro-4,6-dinitrobenzofuroxan 3 was synthesized according to the literature [45]. Reaction between sterically hindered phenols 2a-g and 7-chloro-4,6-dinitrobenzofuroxan 3. To a solution of 7-chloro-4,6-dinitrobenzofuroxan 3 (0.8 mmol) in 5 mL of CHCl 3 at room temperature was added a solution of sterically hindered phenols 2 (1.6 mmol (for compounds 4) or 1.2 mmol (for compounds 5)) in 5 mL of CHCl 3 . The reaction was carried out at room temperature and under magnetic stirring, and the conversion was monitored through TLC analysis (eluent: toluene/ethyl acetate, 2:1). The mixture was stirred at room temperature overnight; the crude mixture was precipitated in hexane (10 mL), the obtained solid was filtered off, washed with cold water (100 mL) and dried under vacuum (0.06 mm Hg) at 40 • C temperature to constant weight. In any case, a mixture of compounds 4 and individually) to give the target compound (the second product in this case was isolated in an insignificant amount).
2:1). The mixture was stirred at room temperature overnight; the crude mixture was precipitated in hexane (10 mL), the obtained solid was filtered off, washed with cold water (100 mL) and dried under vacuum (0.06 mm Hg) at 40 °C temperature to constant weight. In any case, a mixture of compounds 4 and 5 was obtained, depending on the conditions, with a high content of one of them. The crude product was purified by column chromatography (eluent in each case was selected individually) to give the target compound (the second product in this case was isolated in an insignificant amount). 2:1). The mixture was stirred at room temperature overnight; the crude mixture was precipitated in hexane (10 mL), the obtained solid was filtered off, washed with cold water (100 mL) and dried under vacuum (0.06 mm Hg) at 40 °C temperature to constant weight. In any case, a mixture of compounds 4 and 5 was obtained, depending on the conditions, with a high content of one of them. The crude product was purified by column chromatography (eluent in each case was selected individually) to give the target compound (the second product in this case was isolated in an insignificant amount).  other tautomer are present in the spectra. 1 H NMR (500 MHz, Acetone-d6): δ 9.08 (s, 1H, H6-Bz1), 8.14 (dd, J = 8.

Cells and Materials
For the experiments, we used tumor cell cultures: M-HeLa clone 11 (epithelioid carcinoma of the cervix, subline HeLa., clone M-HeLa); T 98G-human glioblastoma; PANC-1, human pancreatic carcinoma; HuTu 80, human duodenal adenocarcinoma; MCF7-human breast adenocarcinoma (pleural fluid); A549, human lung carcinoma; WI38, VA 13 subline 2RA, human embryonic lung from the collection of the Institute of Cytology, Russian Academy of Sciences (St. Petersburg); PC3-prostate adenocarcinoma cell line from ATCC (American Type Cell Collection, USA; CRL 1435; human liver cells (Chang liver) from the collection and the Research Institute of Virology of the Russian Academy of Medical Sciences (Moscow). The cells were cultured in a standard Eagle's nutrient medium manufactured at the Chumakov Institute of Poliomyelitis and Virus Encephalitis (PanEco company, Moscow, Russia), and supplemented with 10% fetal calf serum (Biosera, France) and 1% nonessential amino acids (PanEco company, Russia).

Cytotoxicity Assay
The cytotoxic effect on cells was determined using the colorimetric method of cell proliferation-the MTT test. NADP-H-dependent cellular oxidoreductase enzymes can, under certain conditions, reflect the number of viable cells. These enzymes are able to reduce the tetrazolium dye, (MTT)-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, to insoluble blue-violet formazan, which crystallizes inside the cell. The amount of formazan formed is proportional to the number of cells with active metabolism [42]. Cells were seeded on a 96-well Nunc plate at a concentration of 5 × 103 cells per well in a volume of 100 µL of medium and cultured in a CO2 incubator at 37 °C until a monolayer was formed. The process of cell monolayer formation took 24 h. Then, the nutrient medium was removed, and 100 µL of solutions of the test drug in the given dilutions were added to the wells,

Cells and Materials
For the experiments, we used tumor cell cultures: M-HeLa clone 11 (epithelioid carcinoma of the cervix, subline HeLa., clone M-HeLa); T 98G-human glioblastoma; PANC-1, human pancreatic carcinoma; HuTu 80, human duodenal adenocarcinoma; MCF7-human breast adenocarcinoma (pleural fluid); A549, human lung carcinoma; WI38, VA 13 subline 2RA, human embryonic lung from the collection of the Institute of Cytology, Russian Academy of Sciences (St. Petersburg); PC3-prostate adenocarcinoma cell line from ATCC (American Type Cell Collection, USA; CRL 1435; human liver cells (Chang liver) from the collection and the Research Institute of Virology of the Russian Academy of Medical Sciences (Moscow). The cells were cultured in a standard Eagle's nutrient medium manufactured at the Chumakov Institute of Poliomyelitis and Virus Encephalitis (PanEco company, Moscow, Russia), and supplemented with 10% fetal calf serum (Biosera, France) and 1% nonessential amino acids (PanEco company, Russia).

Cytotoxicity Assay
The cytotoxic effect on cells was determined using the colorimetric method of cell proliferation-the MTT test. NADP-H-dependent cellular oxidoreductase enzymes can, under certain conditions, reflect the number of viable cells. These enzymes are able to reduce the tetrazolium dye, (MTT)-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, to insoluble blue-violet formazan, which crystallizes inside the cell. The amount of formazan formed is proportional to the number of cells with active metabolism [46]. Cells were seeded on a 96-well Nunc plate at a concentration of 5 × 103 cells per well in a volume of 100 µL of medium and cultured in a CO 2 incubator at 37 • C until a monolayer was formed. The process of cell monolayer formation took 24 h. Then, the nutrient medium was removed, and 100 µL of solutions of the test drug in the given dilutions were added to the wells, which were prepared directly in the nutrient medium with the addition of 5% DMSO to improve solubility. After 48 h of incubation of the cells with the tested compounds, the nutrient medium was removed from the plates, and 100 µL of the nutrient medium without serum with MTT at a concentration of 0.5 mg/mL was added and incubated for 4 h at 37 • C. Formazan crystals were added 100 µL of DMSO to each well. Optical density was recorded at 540 nm on an Invitrologic microplate reader (Russia). The experiments for all compounds were repeated three times.

Flow Cytometry Assay
Cell Culture. M-HeLa cells at 1 × 10 6 cells/well in a final volume of 2 mL were seeded into six-well plates. After 48 h of incubation, various concentrations of compounds 4c and 5d were added to wells.
Cell Apoptosis Analysis. The cells were harvested at 2000 rpm for 5 min and, then washed twice with ice-cold PBS, followed by resuspension in binding buffer. Next, the samples were incubated with 5 µL of annexin V-Alexa Fluor 647 (Sigma-Aldrich, St. Louis, MO, USA) and 5 µL of propidium iodide for 15 min at room temperature in the dark. Finally, the cells were analyzed by flow cytometry (Guava easy Cyte, Merck, Rahway, NJ, USA) within 1 h. The experiments were repeated three times.
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, Rahway, NJ, USA).
Detection of Intracellular ROS. M-HeLa cells were incubated with compounds at concentrations of IC 50 for 48 h. ROS generation was investigated using flow cytometry assay and CellROX ® Deep Red flow cytometry kit. For this M-HeLa cells were harvested at 2000 rpm for 5 min and then washed twice with ice-cold PBS, followed by resuspension in 0.1 mL of medium without FBS, to which was added 0.2 µL of CellROX ® Deep Red and incubated at 37 • C for 30 min. After three times washing the cells and suspending them in PBS, the production of ROS in the cells was immediately monitored using flow cytometer (Guava easy Cyte, Merck, Rahway, NJ, USA).

Antimicrobial Activity
Antimicrobial activity of test compounds was determined by serial micro dilutions in 96-well plates using Mueller-Hinton broth for bacterial culture and Sabouraud broth for yeast culture [47]. Cultures of gram-positive bacteria were used in the experiment:

Preparation of Spikes and Samples
A substance of 1 mg was dissolved in methanol to achieve the solution of 1 mg/mL. By a series of further dilutions, a sample containing 5000 ng/mL in the same solvent was prepared. A total of 50 µL of the prepared solution was added to 450 µL of whole blood, resulting in a spike containing 500 ng/mL of the compound in the matrix.
Aliquot of 10 µL was taken from the spike and added to a 100 µL of the precipitation solution consisting of a mixture of 0.2 M ZnSO 4 in water and methanol (2:8, v/v). The sample was then vortex mixed for 20-30 s, incubated for 10-15 min, vortex mixed again and centrifuged for 10 min at 13,400 rpm (Eppendorf MiniSpin). Supernatant (100 µL) was transferred into a vial and analyzed.

Conclusions
The synthesis of novel sterically hindered phenols containing benzofuroxan fragments obtained via aromatic nucleophilic substitution reaction of 7-chloro-4,6-dinitrobenzofuroxan is presented. Depending on the initial ratio of reagents, it is possible to vary the composition of the final products, leading to the formation of compounds with a composition of 2:1 or compounds 1:1. Antimicrobial activity and antitumor potential were studied for the phenols/benzofuroxan hybrids. Most substances exhibit high cytotoxicity against human duodenal adenocarcinoma (HuTu 80), human breast adenocarcinoma (MCF-7) and human cervical carcinoma cell lines. The IC 50 values of compounds 4c and 5d for these lines ranged from 0.9 to 5.9 µM and were either comparable to or exceeded the activity of Doxorubicin and Sorafenib. Moreover, the selectivity indices for healthy cells for the compound 5d also exceed those for the reference drugs. A study of the mechanisms of cytotoxicity suggests that the latter can be associated with the induction of apoptosis along the internal mitochondrial pathway and an increase in ROS production. Encouragingly, all tested compounds do not show hemolytic activity (HC 50 >100 µM). The biostability of the leading compounds was evaluated in the whole blood of mice, where the substances remained unchanged for two h. This is a positive sign for their future quantitative determination in biological matrices. When studying antimicrobial activity, we note an interesting trend that the effect only appears when at least two benzofuroxan moieties are introduced per phenol.
Overall, the combination of sterically hindered phenol and benzofuroxan in one molecule leads to a number of positive effects, including increased ROS production and greater cytotoxicity. Compounds 4c and 5d can be considered a promising basis for the development of antitumor drugs.