Self-Assembling Metallocomplexes of the Amphiphilic 1,4-Diazabicyclo[2.2.2]octane Derivative as a Platform for the Development of Nonplatinum Anticancer Drugs.

New 1-cetyl-4-aza-1-azoniabicyclo[2.2.2]octane bromide complexes with copper(II) bromide and lanthanum(III) nitrate were characterized using dynamic light scattering and transmission electron microscopy, with self-assembly and the morphological behavior elucidated. For the lanthanum(III) nitrate complex, the 3D crystal structure was characterized using X-ray diffractometry. These metallosurfactants were tested as antitumor agents, and a high cytotoxic effect comparable with doxorubicin was revealed against the M-HeLa and A-549 cell lines. Both complexes were 2 times more active toward the MCF-7 cell line than the breast cancer drug tamoxifen. The cytotoxic mechanism of complexes is assumed to be related to the induction of apoptosis through the mitochondrial pathway.

Introduction

Amphiphilic compounds provide a versatile platform for the development of polyfunctional nanosystems that find wide application in modern nanotechnologies.[1−6] This is due to their ability to form nanosized aggregates in solutions, which can bind a variety of active molecules, thereby modifying their stability and functionality. This is of particular interest for the fabrication of drug and gene nanocarriers. The introduction of functional substituents (including metals) into amphiphilic compounds leads to the appearance of new surfactants with properties potentially useful for biomedicine. According to the literature data, the development of supramolecular systems including liposomes based on surfactants chelated to paramagnetic or radioactive ions Gd(III), 64Cu(II), and 111In(III) as tumor-specific magnetic resonance and single-photon emission tomography contrast agents is of urgent interest.[7−11] At the same time, supramolecular architectures are used to enhance the relaxation of agents.[9,12,13] The ruthenium(II) metallocomplexes based on surfactants with chelating aromatic head groups are fluorescent due to their rich redox/photophysical properties.[14] This opens up a good prospect of using metallosurfactants as fluorescent labels.

The biomedical aspect of the use of metallosurfactants is also associated with an increase in the antimicrobial activity of amphiphiles upon their complexation with metals.[15−22] In this case, surfactants of both anionic and cationic nature serve as ligands.[15−22] Typically, long-chain linear or heterocyclic amines (en, triene, bipyridine, and phenanthroline), amides, and quaternary ammonium salts containing an ester fragment are used as cationic ligands,[16−22] with Co(III), Co(II), Cu(II), Sn(II), Ni(II), La(III), Ru(II), and so forth serving as the metal centers.

One of the actual problems of modern pharmacology is the difficulty in treatment and prevention of oncological diseases unresponsive to various drugs. As a result, it is necessary to search for new safe chemical compounds that will become a drug candidate. It is known that organometallic compounds are widely used in oncological practice.[23,24] Moreover, varying the structure of these compounds (metal type and ligand structure) allows us to search for the most effective drugs and reduce negative side effects.[23−25] The analogues of cisplatin of the second and third generation can serve as a vivid example of varying the structure of ligands.[23] The nonplatinum metals in the complexes of iron, cobalt, copper, molybdenum, ruthenium, tin, lanthanum, osmium, gold, silver, and so forth are used in oncological pharmacy.[23,25] There are currently a number of nanocarriers including macromolecular structures such as cyclodextrins, dendrimers, liposomes, mesoporous materials, carbon nanotubes, polymer structures, micellar aggregates, and so forth that can be used for the functionalization of metallosurfactants with the goal of targeted drug delivery and protecting them from in vivo degradation.[23] At the same time, metallodrugs can be transformed into nanostructures by means of hydrogen bonds, covalent bonds, and hydrophobic and electrostatic interactions. These types of bonds also facilitate the release of the metallocomplex at the desired site.[23] Metal-containing surfactants are attractive in the field of chemotherapy due to their composition and specific properties.[26−29] The increased interest in these compounds is associated with their subsequent use in therapeutic, instrumental, and diagnostic purposes.

The anticancer activity of micelle-forming complexes based on various metals and ligands has been investigated in a number of publications.[26−28,30−33] For these metallosurfactants, literature data are available on the significant role of structural factors (ligand composition, hydrophobicity, and nature of the metal) responsible for the anticancer activity of the complexes. Thus, for the human cervical cancer cell line (M-HeLa), the inhibitory concentration (IC50) for the double-chain surfactant–ruthenium(II) complexes is lower than that for the single-chain complexes.[30]

The objects of our study were new metallosurfactants based on a monoalkylated (cetyl) derivative of 1,4-diazabicyclo[2.2.2]octane (1-cetyl-4-aza-1-azoniabicyclo[2.2.2]octane bromide, D16Br) (Figure ), as well as Cu(II) bromide (1) and La(III) nitrate (2) composition surfactant: metal = 1:1 ([Cu(D16)Br3]) and 2:1 ([La(D16)2Br2(NO3)3]), respectively. Previous tensiometric, conductometric, and fluorimetric studies showed that these metal complexes were micelle-forming surfactants. Moreover, their aggregation ability was higher than that of the D16Br ligand and conventional surfactants with the head group of acyclic and monocyclic types (cetyltrimethylammonium bromide and cetylpyridinium bromide).[22,34,35] In addition, the spectrophotometric method showed that supramolecular systems based on metallocomplexes exhibit high solubilization activity with respect to the hydrophobic agent Orange OT dye,[30,31] as well as poorly soluble drugs quercetin and furadonin.[36,37] This allows us to achieve a significant increase in the content of drugs in an aqueous medium (up to 1–2 orders of magnitude) and thereby increase their bioavailability. In addition, a biologically important property of the studied metallosurfactants is their ability to complex with biomolecules (oligonucleotide),[35] as well as the manifestation of antimicrobial and antifungal activity.[21,22] A wide range of functional applications of metallosurfactants require a deeper study of their structural and physicochemical features, as well as functional activity. We established the structure of the lanthanum complex crystal, the size of complex aggregates in solution, and the type of supramolecular structures of both complexes formed in the aqueous medium using X-ray diffractometry (XRD), dynamic light scattering (DLS), and transmission electron microscopy (TEM) methods, respectively. The biological activity of the complexes was studied. These metallosurfactants were tested also as anticancer agents.

Figure 1

Structural formula of D16Br.

Results and Discussion

Determination of the Spatial Structure of the La(III) Complex Crystals

The crystal structure of the La(III) complex was determined by X-ray single-crystal diffraction. The La ion in the crystal is coordinated by five nitro groups and one water molecule. The asymmetric part of the unit cell (Figure ) also contains three D16 cations and one bromine ion, the latter being disordered over three positions, all near the D16 head groups.

Figure 2

Asymmetric part of the unit cell and La coordination in D16Br with the lanthanum(III) nitrate complex crystal.

Additionally, seven D16 molecules oriented by the head groups to the coordination center complete the second coordination sphere and form the complex crystal packing (Figure ). This arrangement results in the breakage of the trans-conformation of the alkyl chains which change their direction around the 6th–7th carbon atoms starting from the quaternized nitrogen atom. The crystal has a structure composed of hydrophilic and lipophilic layers (see a fragment of the crystal packing of the complex, Figure S1).

Figure 3

Fragment of the crystal packing of D16Br with the lanthanum(III) nitrate complex showing the [La(NO3)5H2O]2– anion coordination sphere.

Single-Crystal X-ray Investigations of the Structure of the D16Br Ligand

In addition to the complex described above, we obtained the crystals of pure D16Br and their structure was also studied by the single-crystal X-ray diffraction method. According to the data obtained, this compound forms crystals with solvent water molecules. There are two pairs of D16+ and Br– ions and one water molecule in the asymmetric part of the monoclinic unit cell (Figure ). D16Br molecules have a fully elongated linear alkyl part, which, however, is characteristic of most crystallizing molecules with extended hydrocarbon fragments. The C–N bond lengths in the diazabicyclo[2.2.2]octane fragments of both independent molecules have close values and range from 1.441 to 1.510 Å. As can be seen from the shape and size of the ellipsoids of the thermal vibrations of atoms, D16Br molecules do not experience noticeable vibrations or disordering in the crystal.

Figure 4

ORTEP view of D16Br in the crystal. Nonhydrogen atoms are represented by probability ellipsoids of thermal vibrations (p = 50%), hydrogen atoms—by spheres of arbitrary radii.

The supramolecular structure in the crystals of D16Br monohydrate is determined by two factors: the strict localization, in accordance with the previously described rules,[38,39] the hydrophilic and hydrophobic fragments of the molecules in the crystal, and the implementation of many hydrogen bonds C–H···O, CH···Br, OH···N, and CH···N types. Together, these two factors lead to the formation of two-dimensional bilayer supramolecular structures in the crystal (X-ray crystallographic data in Figure S2) located in the crystallographic plane 0ab. The inner hydrophilic part of these bilayers is stabilized by numerous intermolecular interactions involving solvate water molecules.

Parallel stacking of such 2D-structures along the crystallographic direction 0c is carried out in such a way that only van der Waals interactions are observed between alkyl chains (presented in Figure S2). With such mutual packing of the molecules in the crystal, there are practically no voids potentially accessible for the solvent, but the packing of the molecules is not dense. The calculated packing index of the molecules, equal to 0.696, approaches the medium of the range characteristic of the crystals of organic compounds (0.65–0.75).[40]

It is interesting to note that this compound crystallizes in the Sohncke space group P21, although it does not have a chiral center in its composition. Apparently, the manifestation of chirality for the crystals of this compound is related to the nature of the packing of molecules in the crystal, since, as can be seen in Figure S2b, two independent molecules form their own layers in the crystal, consisting only of this molecule. Obviously, in an enantiomeric crystal, the same layers will have the opposite position.

D16Br with the La(III) complex was also characterized by powder X-ray diffraction. In addition to this sample, X-ray diffractograms were also obtained for the starting D16Br sample, which made it possible to control the purity and stability of the systems. According to the powder diffraction data (Figure S3), the calculated and experimental diffraction patterns for the D16Br sample are generally consistent with each other, which indicates that the D16Br powder sample does not contain other crystalline components and is structurally consistent with the D16Br monohydrate crystals. The powder sample of complex 2 is characterized by the presence of some additional diffraction lines. An analysis of the data supports the presence of unreacted residues of crystalline D16Br in the sample complex. In this case, some line shifts in the diffractograms are explained by the difference in the temperature of the powder and single-crystal experiments.

Determination of the Size of Metallocomplex Aggregates by DLS

The method of DLS was used to determine the hydrodynamic diameter of aggregates (Dh, nm) in the aqueous solutions of complex 2 and the composition for comparison containing the ligand–lanthanum nitrate mixture. It has been established that in various concentration ranges of the amphiphile, the formation of supramolecular structures of various types occurs. For metallosurfactant solutions, the aggregates with a size of 70–90 nm are predominantly formed in the critical micelle concentration (CMC) region. There are also a small number of particles (up to 2.5%) of a larger size (310–410 nm). Presumably, these may be the structures of a vesicular or vermiform micellar type. When the CMC is twice exceeded, the small associates (4–6 nm) are mainly formed, which can be attributed to spherical micelles (additional data in Table S1 and Figures S4 and S5). Aggregates of medium diameter are formed in the mixed solutions of D16Br/La(NO3)3·6H2O (2:1) in the CMC region as in the complex solutions but with a wider size range of 60–130 nm and up to 1.2% particles with a size of 260–400 nm. In the region of higher concentrations, the formation of spherical micelles with a diameter of 5–7 nm occurs (additional data in Table S1 and Figures S4 and S5). Such size distribution of particles in metal-containing systems is close distribution in the solutions of the ligand itself. According to ref (22), the particles of various types are formed there. Aggregates of the order of 80 nm and larger particles in the range of 150–300 nm are formed in the CMC region. An increase in the surfactant content also leads to the appearance of small particles (1–9 nm) and a relative increase in their number.

Study of the Structure of Metallosurfactant Aggregates by TEM

The data obtained by TEM are consistent with the results of the DLS on the variety of formed supramolecular structures in solutions. The TEM photographs of the lanthanum complex showed elongated formations with a width of the order of 66–127 nm and a length of usually 169–330 nm and an aspect ratio of structures of the order of 2.5 (in some cases 3.5–5), as well as a rare population of sphere-like particles from <10 to 100 nm in size (Figure d,e). The TEM images of the copper complex reflected the formation of individual clearly defined sphere-like particles of 20–50 nm and smaller spherical particles of about 3–7 nm (Figure a,b). A combination of small (1–6 nm) and large (120–240 nm) particles of complex 1 (PdI 0.31–0.69 in the CMC region and 0.27–0.47 in the post micellar region) was also observed according to DLS.[22]

Figure 5

TEM images of copper complex 1 (a,b) and lanthanum complex 2 (d,e) nanoparticles and size distribution curves for complex 1 (c) and 2 (f).

The size distribution curves are shown in Figure c,f. For the lanthanum complex, the predominant particle size is about 100 nm, while for the copper complex, the predominant size of the aggregates is in the range of 7 nm.

Cytotoxicity Assay

Taking into account the numerous literature data describing the high biological activity and various fields of application of metallosurfactants, the study of their cytotoxic properties may be of considerable interest. The evaluation of cytotoxic activity in vitro is an important stage in the creation of new biologically active compounds. In this regard, we studied the cytotoxic effect of the test compounds 1 and 2 against the lines of normal and human cancer cells at concentrations of 1–100 μM. Table shows the IC50 values of the tested compounds. It is seen that both compounds showed high toxicity against all human cancer cell lines. However, compound 1 was found to be more active. Its anticancer activity against the cell lines M-HeLa and A-549 was at the level of the reference drug doxorubicin. Compound 2 showed such high anticancer activity only on the M-HeLa cell line. In relation to the MCF-7 cell line, both compounds were 2 times higher in activity than the reference drug tamoxifen. Importantly, the test compounds were less toxic to normal human cell lines (WI-38) compared to doxorubicin, showing some selectivity toward cancer cells.

Table 1

Cytotoxic Effects of Compounds 1 and 2 on the Cancer and Normal Human Cell Linesa

	IC50 (μM)
	cancer line			normal line
compounds	M-Hela	MCF-7	A549	Chang liver	Wi-38
1	4.0 ± 0.3	14 ± 1.2	2.7 ± 0.2	6.0 ± 0.3	16 ± 0.9
2	4.1 ± 0.3b	13 ± 1.1	14 ± 1.3	5.0 ± 0.4b	5.0 ± 0.3
doxorubicin	3.0 ± 0.1	3.0 ± 0.2	3.0 ± 0.1	3.0 ± 0.1	1.3 ± 0.1
tamoxifen	28 ± 2.5	25 ± 2.2		42.1 ± 3.5	46.2 ± 3.5

The experiments were repeated three times.

Data from ref (41).

Induction of Apoptotic Effects by the Test Compounds

One of the most significant mechanisms of the cytotoxic activity of compounds is the induction of apoptosis, and its assessment is a necessary component of fundamental and applied research in experimental biology. Therefore, using the example of the leading compound 1, the ability to induce apoptosis in the human cancer cell line M-HeLa was studied. Figures and 7 show that after 24 h of treatment with compound 1, apoptotic effects were observed in the cells, most pronounced in the stage of early apoptosis. It can be seen that at a concentration of 10 μM, similar to 2 × IC50, compound 1 caused early and late apoptosis in 1.7% and 2.3% of M-HeLa cells, respectively, and with an accretion in the concentration of the test compound to 25 μM, the number of cells in the early apoptosis increased by about 5 times. The results obtained indicate that the mechanism of the cytotoxic action of compound 1 in M-HeLa cells may be due to the apoptotic pathway.

Figure 6

Apoptotic effects of compound 1 on M-HeLa cells.

Figure 7

Histogram for the number of cells (% of total) in the early and late stages of apoptosis for the control and experimental groups. The values are presented as the mean ± SD (n = 3); (*) P < 0.01 compared to control.

Effects on the Mitochondrial Membrane Potential (Δψm)

There are two main pathways through which apoptotic signals are transmitted in the form of cellular regulators such as hormones, antigens, monoclonal antibodies, and other molecules. This is the mitochondrial (or internal) pathway, as well as through special transmembrane proteins—the so-called death receptors (external pathway). In both cases, in order to trigger apoptosis, special initiator apoptotic complexes must be formed. The mitochondrial pathway is initiated as a result of intense exposure of the cell to a number of damaging factors, including chemical agents.[42]

The ability of the studied compounds to induce apoptosis proceeding along the mitochondrial pathway, causing a decrease in the mitochondrial membrane potential (Δψm) in M-HeLa culture cells, was evaluated using the example of compound 1. Studies were performed by flow cytometry methods using the JC-10 reagent. In normal cells with a high mitochondrial membrane potential, the dye JC-10 forms aggregates (J-aggregate) near the mitochondrial membranes. When the membrane potential falls due to the stimulation of apoptosis, JC-10 is evenly distributed in the cell as a monomer (J-monomer). JC-10 aggregates in normal cells have red fluorescence, while JC-10 monomers are green. The ratio between orange-red and green fluorescence can be used to judge the onset of apoptosis.

Figure a shows that in control samples with a high mitochondrial membrane potential, red to orange luminosity is dominant. A similar staining was observed in the tested samples after exposure to a lower concentration (10 μM), which indicates a partial preservation of the mitochondrial membrane potential and the stage of early apoptosis in M-HeLa cells. In experimental samples treated with the test compound at a concentration of 25 μM, green fluorescence prevailed, demonstrating the process of depolarization of the mitochondrial membrane and the stage of late apoptosis. The decrease in Δψm was also confirmed by flow cytometry analysis (Figure b). The intensity of red fluorescence decreased depending on the concentration of the test compound.

Figure 8

Effects on Δψm in M-HeLa cells that were treated with compound 1 at the indicated concentrations for 24 h and then analyzed by confocal laser scanning microscopy and flow cytometry after JC-10 staining. (a) Confocal laser scanning microscopy images of M-HeLa cells treated with compound 1. (b) Flow cytometry analysis of M-HeLa cells treated with 1.

The obtained results testify that the mechanism of the cytotoxic action of the tested compounds may be due to the induction of apoptosis, proceeding along the mitochondrial pathway.

Conclusions

Thus, new amphiphilic complexes of monoquaternized bicyclic diamine 1,4-diazabicyclo[2.2.2]octane with transition metals [copper(II) and lanthanum(III) ions] were obtained and characterized by physicochemical methods (XRD, DLS, and TEM). The 3D crystal structure of metallosurfactants and their ability to self-associate in solution were described. A variety of morphological forms of amphiphilic complexes were established. It was shown that along with high aggregation, adsorption, solubilization properties, and antimicrobial activity, the studied complexes exhibited a cytotoxic effect. The anticancer activity of metallosurfactants to M-HeLa, A-549, and MCF-7 cell lines was at the level or 2 times higher than the test drugs doxorubicin and tamoxifen. Moreover, the studied amphiphiles were less toxic in relation to normal human cell lines compared with doxorubicin. Using flow cytometry, laser confocal microscopy, and fluorimetry on the Cytell Cell Imaging system, it was shown that metallocomplexes actively induced apoptosis in M-HeLa cells and caused a significant decrease in the mitochondrial membrane potential at the tested concentrations. This suggests that the mechanism of the cytotoxic action of the tested compounds may be due to the induction of apoptosis, proceeding along the mitochondrial pathway. According to the literature,[23,24] it can be noted that this study also lays the foundation for future work on the development of new nonplatinum anticancer drugs with increased selectivity for cancer cells.

Experimental Section

Materials

D16Br (ligand) was prepared by the quaternization of 1,4-diazabicyclo[2.2.2]octane (97%, Acros Organics, USA) with cetyl bromide (98%, Alfa Aesar, Germany) by the procedure.[43] Complexes 1 and 2 were synthesized in methanol and acetone by the procedure.[34,36] The compositions of the products were confirmed by IR and 1H NMR spectroscopy, elemental analysis, and for the crystals of metallocomplex 1 by single-crystal XRD (in Figure S6).[31] La(NO3)3·6H2O (98%) (Acros-Organics, USA) was used as received. Water for preparing the solutions was purified using a Millipore Direct-Q 5 UV system.

Dynamic Light Scattering

The sizes of the aggregates were determined by DLS measurements using a Malvern Zetasizer Nano nanoparticle characterization system (UK). A He–Ne gas laser with 4 mW power and 633 nm wavelength was used as a source of laser radiation. All measurements were performed at 173° light scattering angle. The effective hydrodynamic radius was calculated according to the Einstein–Stokes equation. The diffusion coefficient was measured three times for each sample. The average error of measurements did not exceed 4%.

Transmission Electron Microscopy

TEM was performed on a Hitachi HT7700 (tungsten filament, HV = 80 kV). The samples were deposited on a 300 mesh copper grid with a continuous carbon-formvar support film.

X-ray Crystallography

A Bruker Kappa Apex II CCD diffractometer was used to collect single-crystal data for D16Br and its complex with lanthanum(III) nitrate (ω- and φ-scan modes, graphite monochromated Mo Kα [λ = 0.71073 Å) radiation, 150(2) K]. The data were corrected for the absorption effect using the SADABS program.[44] The features of the structure decoding process are given in the Supporting Information.

Crystal data for D16Br: formula 2(C22H45N2)+, 2(Br)−, H2O, M = 853.03 g/mol, monoclinic, space group P21 (no. 4), Z = 2, a = 5.82828(16) Å, b = 6.9310(19) Å, c = 56.791(15) Å, β = 92.934(3)°, V = 2291(1) Å3, ρcalc = 1.237 g cm–3, μ = 1.805 mm–1, 19,689 reflections collected (−7 ≤ h ≤ 7, −9 ≤ k ≤ 8, −74 ≤ l ≤ 74), θ range = 2.154 to 27.999°, 10,132 independent (Rint = 0.0648) and 7467 observed reflections [I⩾2σ(I)], 468 refined parameters, R = 0.0549, wR2 = 0.0891, goodness of fit S = 0.943, and maximum (minimum) residual electron density 0.691 (−0.611) e Å–3.

Crystal data for the complex of D16Br with lanthanum(III) nitrate: formula [La(NO3)5·H2O]2–·3D16+·Br– (C66H137BrLaN11O16), M = 1559.68 g/mol, monoclinic, space group P21/c (no. 14), Z = 4, a = 29.689(5) Å, b = 14.194(2) Å, c = 20.253(3) Å, β = 108.716(2)°, V = 8083(2) Å3, ρcalc = 1.282 g cm–3, μ = 1.088 mm–1, 165,523 reflections collected (−39 ≤ h ≤ 39, −19 ≤ k ≤ 19, −27 ≤ l ≤ 27), θ range = 1.607° to 28.569°, 20,511 independent (Rint = 0.0800) and 13,962 observed reflections [I⩾2σ(I)], 910 refined parameters, R = 0.0471, wR2 = 0.1201, and maximum residual electron density 1.047 (−0.920) e Å–3. Bromine ion and alkyl groups are disordered; the maximum residual density is located near the Br atom.

X-ray Powder Diffraction Experiments

Powder X-ray diffraction measurements were performed on a Bruker D8 ADVANCE diffractometer equipped with Vario attachment and Vantec linear PSD using Cu radiation (40 kV, 40 mA) monochromated by a curved Johansson monochromator (λ Cu Kα1 1.5406 Å). Room-temperature data were collected in the reflection mode. The samples were loaded on a standard zero-diffraction silicon plate, which was kept spinning (15 rpm) throughout the data collection. The samples were preliminarily ground to a homogeneous state and then, without pressing, were applied onto a silicon plate, which reduces background scattering. The patterns were recorded in the 2Θ range between 3 and 70° in 0.008° steps with a step time of 0.1–1.0 s. The processing of the obtained data was performed using EVA software packages.[45]

Biological Study

Cell Lines

M-HeLa clone 11 human, epithelioid cervical carcinoma, strain of HeLa, clone of M-HeLa, human breast adenocarcinoma cells (MCF-7), human lung adenocarcinoma (A 549), WI-38 VA-13 cell culture, and subline 2RA (human normal embryonic lung cells) were purchased from the Type Culture Collection of the Institute of Cytology (Russian Academy of Sciences). Human normal liver cells (Chang liver) were purchased from the N. F. Gamaleya Research Center of Epidemiology and Microbiology.

Cytotoxicity Assay

The cytotoxic activity of metallosurfactants against cancer and normal human cell lines was assessed using the Cell Viability Bio application of the Cytell multifunctional cell imaging system (GE Health Care Life Science, Sweden). This system makes it possible to estimate the cell viability from the fluorescence intensity. In the experiments, an intercalating dye 4′,6-diamidine-2-phenylindole (DAPI) was used for the intravital staining of cell nuclei in blue and a high-molecular intercalating dye propidium iodide, which penetrates only dead cells, staining them yellow. As a result, the living cells were colored blue, while the dead cells were colored yellow. DAPI and propidium iodide were purchased from Sigma-Aldrich (Merck). The cells were cultured in a standard Eagle’s nutrient medium, produced at the Institute of Poliomyelitis and Viral Encephalitis, Chumakov (PanEko company), with the addition of 10% fetal calf serum and 1% nonessential amino acids. The cells were seeded in a 96-well plate (Eppendorf) in 150 μL of the medium at an amount of 1.5 × 104 cells/well and cultured in a CO2 incubator at 37 °C. After a 24 h incubation, the spent medium was removed and 150 μL of the test compound was added in a pre-established dilution, 150 μL in each well. Dilutions of the compounds were prepared directly in a nutrient medium. To improve the solubility, 5% DMSO was added to the medium, which does not induce cell inhibition at this concentration. Intact cells cultured simultaneously with experimental cells were used as controls.

Cell Viability Bio application was used to calculate the cell viability as a percentage of control. The results of the cytotoxicity assay are presented as the concentration of the test compound that inhibits cell growth by 50% (IC50). Doxorubicin and tamoxifen, purchased from Sigma-Aldrich (Merck), were used as the standard drugs.

Flow Cytometry Assay

Cell Culture

M-HeLa cells at 1 × 106 cells/well were added to 6-well plates, 2 mL in each well. For the confocal laser scanning microscopy assay, a cover glass was placed at the bottom of each well. After a 24 h incubation, the spent medium was removed and solutions of lead compound 1 were added at concentrations 10 and 25 μM. Then, the samples were incubated for 24 h in a CO2 incubator at 37 °C.

Cell Apoptosis Analysis

The cells were detached using a mixture of trypsin–Versene (1:3), suspended in phosphate-buffered saline (PBS) and centrifuged at 2000 rpm for 5 min, and then washed twice with ice-cold PBS, followed by resuspension in the binding buffer. Next, the samples were incubated with 5 μL of annexin V-FITC (Sigma-Aldrich, Merck) and 5 μL of propidium iodide for 15 min at room temperature in the dark. Finally, the cells were analyzed by flow cytometry (Guava easyCyte, Merck, USA).

Mitochondrial Membrane Potential

The M-HeLa cells were detached using a mixture of trypsin–Versene (1:3), suspended in PBS and centrifuged at 2000 rpm for 5 min, and then washed twice with ice-cold PBS, followed by resuspension in PBS. Further, the samples were added with 10 μg/mL of the JC-10 Mitochondrial Membrane Potential Assay Kit (Sigma-Aldrich, Merck) and incubated at 37 °C for 10 min. After the cells were rinsed three times and suspended in PBS, JC-10 fluorescence was observed by flow cytometry.

Confocal Laser Scanning Microscopy Assay

The M-HeLa cells grown on cover glasses were treated with lead compound 1 at concentrations of 10 and 25 μM and stained with a fluorescent dye JC 10 in the dark for 15 min. Then, the samples were washed with the buffer provided by the manufacturer as part of the JC-10 Mitochondrial Membrane Potential Assay Kit (Sigma-Aldrich, Merck). Confocal microscopy studies were carried out using a laser confocal microscope LSM 780.

Statistical Analysis

IC50 calculation was performed with an internet tool: MLA—“Quest Graph IC50 Calculator.” AAT Bioquest, Inc., December 17, 2020, https://www.aatbio.com/tools/ic50-calculator.[46] The data in the tables and graphs are given as the mean ± standard error. All assays were performed in triplicate.