Design of DNA Intercalators Based on 4-Carboranyl-1,8-Naphthalimides: Investigation of Their DNA-Binding Ability and Anticancer Activity

In the present study, we continue our work related to the synthesis of 1,8-naphthalimide and carborane conjugates and the investigation of their anticancer activity and DNA-binding ability. For this purpose, a series of 4-carboranyl-1,8-naphthalimide derivatives, mitonafide, and pinafide analogs were synthesized using click chemistry, reductive amination, amidation, and Mitsunobu reactions. The calf thymus DNA (ct-DNA)-binding properties of the synthesized compounds were investigated by circular dichroism (CD), UV–vis spectroscopy, and thermal denaturation experiments. Conjugates 54–61 interacted very strongly with ct-DNA (∆Tm = 7.67–12.33 °C), suggesting their intercalation with DNA. They were also investigated for their in vitro effects on cytotoxicity, cell migration, cell death, cell cycle, and production of reactive oxygen species (ROS) in a HepG2 cancer cell line as well as inhibition of topoisomerase IIα activity (Topo II). The cytotoxicity of these eight conjugates was in the range of 3.12–30.87 µM, with the lowest IC50 value determined for compound 57. The analyses showed that most of the conjugates could induce cell cycle arrest in the G0/G1 phase, inhibit cell migration, and promote apoptosis. Two conjugates, namely 60 and 61, induced ROS production, which was proven by the increased level of 2′-deoxy-8-oxoguanosine in DNA. They were specifically located in lysosomes, and because of their excellent fluorescent properties, they could be easily detected within the cells. They were also found to be weak Topo II inhibitors.


Introduction
1,8-Naphthalimide is a class of heterocycles, constituting a π-deficient planar aromatic structure and a versatile pharmacophore with diverse biological applications in pharmaceuticals such as anticancer, antibacterial, antiviral, and analgesic agents [1,2]. Naphthalimides exert antiproliferative activity primarily because of their ability to intercalate into the base pairs of DNA, through DNA-groove binding and topoisomerase inhibition [3,4]. They also exert anticancer activity through inhibition of receptor tyrosine kinases [5], induction of reactive oxygen species production [6], and other mechanisms [7]. The most popular and well-described naphthalimides are mitonafide, pinafide (Figure 1), amonafide, and elinafide. The development of functional 1,8-naphthalimide derivatives as anticancer agents and DNA-targeting agents is a fast-growing area and has resulted in several products.  [8,9].
In our work, we have described thus far the methods used to synthesize naphthalimides modified with carborane or metallacarborane groups through the modification of imide (Figure 1) [8] and bearing the carborane group at position 3 of the ring [9]. We observed that the attachment site of the boron cluster to the naphthalimide moiety, type of the boron cluster, and structure of the linker between the boron cluster and the heteroaromatic ring system influenced various cytotoxic activities against HepG2 cancer cells. The tested conjugates induced cell cycle arrest at the G0/G1 or G2M phase and mainly activated apoptosis. Selected conjugates activated autophagy and ferroptosis. The presence of the carboranyl cluster at position 3 did not promote them as effective topoisomerase II (Topo II) inhibitors. However, the studied compounds were rather weak classical DNA intercalators as compared to mitonafide [8]. Additionally, J. Laskova et al. classically obtained 3-nitro-1,8-naphthalimides bearing the nido-carborane and closo-dodecaborate modifications by reaction of naphthalic anhydride with ammonium derivatives containing a boron cluster. A mitonafide derivative modified with nido-carborane was obtained using the nucleophilic ring-opening reaction of the corresponding cyclic oxonium derivatives [10]. However, physicochemical and biological studies on this group of compounds have not been performed.
In recent years, several studies have described the potential biomedical application of boron clusters, mostly as boron carriers for boron neutron capture therapy, pharmacophores, or modulators of different types of chemical compounds [11][12][13]. The properties of boron clusters that are critical to their use in medicinal chemistry include abiotic origin (these are therefore chemically and biologically orthogonal to native cellular components), the unique interaction properties of boron clusters and their derivatives with biomolecules, lipophilicity, amphiphilicity, hydrophobicity (depending on the chemical structure), chemical stability, susceptibility to functionalization, spherical or ellipsoidal geometry, and rigid three-dimensional arrangement [14,15]. As reported earlier, the introduction of boron clusters into drug molecules could improve the activity toward resistant forms [16][17][18][19] or to obtain hitherto unknown nematicidal activity [20].  [8,9].
In our work, we have described thus far the methods used to synthesize naphthalimides modified with carborane or metallacarborane groups through the modification of imide (Figure 1) [8] and bearing the carborane group at position 3 of the ring [9]. We observed that the attachment site of the boron cluster to the naphthalimide moiety, type of the boron cluster, and structure of the linker between the boron cluster and the heteroaromatic ring system influenced various cytotoxic activities against HepG2 cancer cells. The tested conjugates induced cell cycle arrest at the G0/G1 or G2M phase and mainly activated apoptosis. Selected conjugates activated autophagy and ferroptosis. The presence of the carboranyl cluster at position 3 did not promote them as effective topoisomerase II (Topo II) inhibitors. However, the studied compounds were rather weak classical DNA intercalators as compared to mitonafide [8]. Additionally, J. Laskova et al. classically obtained 3-nitro-1,8-naphthalimides bearing the nido-carborane and closo-dodecaborate modifications by reaction of naphthalic anhydride with ammonium derivatives containing a boron cluster. A mitonafide derivative modified with nido-carborane was obtained using the nucleophilic ring-opening reaction of the corresponding cyclic oxonium derivatives [10]. However, physicochemical and biological studies on this group of compounds have not been performed.
In recent years, several studies have described the potential biomedical application of boron clusters, mostly as boron carriers for boron neutron capture therapy, pharmacophores, or modulators of different types of chemical compounds [11][12][13]. The properties of boron clusters that are critical to their use in medicinal chemistry include abiotic origin (these are therefore chemically and biologically orthogonal to native cellular components), the unique interaction properties of boron clusters and their derivatives with biomolecules, lipophilicity, amphiphilicity, hydrophobicity (depending on the chemical structure), chemical stability, susceptibility to functionalization, spherical or ellipsoidal geometry, and rigid three-dimensional arrangement [14,15]. As reported earlier, the introduction of boron clusters into drug molecules could improve the activity toward resistant forms [16][17][18][19] or to obtain hitherto unknown nematicidal activity [20].
The synthesis of amides remains one of the most significant transformations, and it is one of the more frequently performed reactions. The amide function is unarguably a critical feature, being the constituent of natural and synthetic polymers and found in a wide variety of small bioactive molecules produced in both nature and laboratory [29].
Reductive amination plays a vital role in pharmaceutical and medicinal chemistry owing to its synthetic merits and the ubiquitous presence of amines among biologically active compounds [31]. 8-naphthalimide (33) [28] with an appropriate aldehyde containing ortho-carborane 44 or meta-carborane 45 [32] in anhydrous THF (for the synthesis of compounds 46, 48, 50, and 52), anhydrous AcOEt (for the synthesis of compound 47), or anhydrous MeOH (for the synthesis of compounds 49, 51, and 53) at reflux under an inert (Ar) atmosphere yielded the corresponding Schiff bases and 46-53, but these could not be isolated due to their instability (Scheme 5).
In contrast to the synthesis of 3-aminonaphthalimide derivatives bearing the carborane group [9], the direct attachment of the aldehyde to the amino group at position 4 of the heterocyclic ring system proceeded with low yield (10%). Therefore, it was necessary to prepare suitable naphthalimide substrates containing ethylenediamine or hexylenediamine linkers. Figure 2 shows the crystal structure of an asymmetric unit of one molecule of modified 1,8-naphthalic anhydride 23 ( Figure 2). The packing of molecules in the crystal lattice involves extensive parallel stacking of the heteroaromatic ring systems, while the carborane clusters form separate zones. Notably, the CH group of the carborane clusters form weak hydrogen bonds with one of the carbonyl oxygen atoms of neighboring molecules, thus forming a crystal lattice network. The donor-acceptor distance is 3.16 Å, indicating a relatively strong bond for a carbon atom acting as a donor. This is another confirmation that the carborane groups can participate in weak H-bonding interactions [9,19], and the acidic nature of the C-H group was previously observed for free carboranes [33].

Melting Temperature Measurements
The melting temperature (Tm) of DNA is defined as the temperature at which 50% of the DNA is in form of single strands forming randomly coiled structures [34]. DNA melting is measured by the absorbance of UV light (260 nm) by the DNA solution, where the amount of UV light absorbed is proportional to the fraction of non-bonded base pairs. The majority of DNA-binding small molecules known thus far stabilize duplex DNA against heat denaturation. A high, drug-induced increase in the Tm of DNA is generally viewed as an excellent criterion to select DNA ligands and is a common feature of several anticancer drugs such as intercalators (a significant increase in Tm = 3-8 °C is observed for strong intercalation) and alkylators. The reverse situation (destabilization of DNA to facilitate its denaturation) may be an attractive option to identify therapeutic agents acting on the DNA structure.
Our study conducted Tm measurement to estimate the impact of 1,8-naphthalimides bearing the carborane cluster on DNA stabilization (   The melting temperature (T m ) of DNA is defined as the temperature at which 50% of the DNA is in form of single strands forming randomly coiled structures [34]. DNA melting is measured by the absorbance of UV light (260 nm) by the DNA solution, where the amount of UV light absorbed is proportional to the fraction of non-bonded base pairs. The majority of DNA-binding small molecules known thus far stabilize duplex DNA against heat denaturation. A high, drug-induced increase in the T m of DNA is generally viewed as an excellent criterion to select DNA ligands and is a common feature of several anticancer drugs such as intercalators (a significant increase in T m = 3-8 • C is observed for strong intercalation) and alkylators. The reverse situation (destabilization of DNA to facilitate its denaturation) may be an attractive option to identify therapeutic agents acting on the DNA structure.
Our study conducted T m measurement to estimate the impact of 1,8-naphthalimides bearing the carborane cluster on DNA stabilization ( were the same as those reported in a previous paper [9], with a difference in the position of the substituent (4-substituted vs. 3-substituted). A naphthalimide derivative can stabilize a DNA duplex to a varying degree depending on the substituent attached to the naphthalimide, the position of the substituent, and the sequence of DNA [35]. We observed that 4-substituted derivatives stabilize DNA slightly more than the corresponding 3-substituted naphthalimides. However, naphthalimide derivatives 54 and 61 modified with the carborane cluster through reductive amination revealed a considerable increase in melting temperature. The thermal denaturation experiment conducted for ct-DNA in the absence of the modified naphthalimide revealed a T m value of 74 • C. In contrast, the observed T m of ct-DNA in the presence of 54-61 significantly increased within the range of T m values 81.67-86.33 • C (∆T m = 7.67-12.33 • C, Table 1) with the highest value for pinafide derivative 61 bearing meta-carborane and a (CH 2 ) 6 linker between modification and a 1,8-naphthalimide residue. In comparison to unmodified mitonafide and pinafide (∆T m = 5.17 and 6.50 • C, respectively) [9], this may confirm classical intercalation of the modified compounds as a dominant binding mode. The rich photophysical properties of the 1,8-naphthalimides (which are highly dependent on the nature and substitution pattern of the aryl ring) make them prime candidates as probes for changes in spectroscopic properties such as dichroism, and absorption can be used to monitor their binding to biomolecules.
To understand the interactions of the modified 1,8-naphthalimides with DNA, circular dichroism (CD) measurement was conducted. CD is a technique to investigate the conformational changes in DNA morphology during its interaction with ligands. The CD spectra of the B-form DNA duplex generally display a positive Cotton effect at 270 nm and a negative effect at approximately 250 nm with nearly equal magnitudes of positive longwave bands and negative shortwave bands [36,37].
The CD spectrum of free ct-DNA showed a negative band at 248 nm due to polynucleotide helicity and a positive band at 276 nm due to base stacking, thus confirming the existence of ct-DNA in the right-handed B-form [38]. Treatment with mitonafide or pinafide decreased the negative peak and increased the positive peak [9]. As illustrated in Figures S254, S256-S262, S265, and S266 (ESI), conjugates bearing carborane clusters 6, 7, 11, 14-19, 22-27, 41, and 42 did not cause any appreciable change in the CD spectra of ct-DNA with an increase in concentration. For compounds 8-10, 36-40, and 43, the positive and negative bands were perturbed by the presence of these compounds (Figures S255, S256, and S263-S266, ESI). Thus, simple groove binding and electrostatic interaction of ligands show less or no perturbation on base-stacking and helicity bands, while intercalation enhances the intensities of both the bands, stabilizing the right-handed B conformation of DNA, as observed for the classical intercalator methylene blue [39]. As depicted in Figures S267-S270 (ESI), the CD spectrum of ct-DNA was remarkably perturbed in the presence of conjugates 54-61, resulting in the rapid increase in positive bands without any shift. Negative bands increased slightly with a slight red shift of their maximum. The obtained data for those compounds agree well with their melting curve results. The CD changes are reminiscent of destacking of the DNA base pairs and some degree of local helix destabilization consequent to intercalation of the 1,8-naphthalimide moiety. However, no conformational change from the B-form structure was observed.

UV-Vis Spectra Measurement
The interaction of 1,8-naphthalimides bearing carborane clusters with ct-DNA was also studied by UV-vis absorption titration to better understand the mode of interaction and binding strength. For compounds interacting with DNA by intercalation, bathochromic and hypochromic effects are observed in the absorption spectra [40]. The spectral changes observed in the electronic absorption of 6-11, 16-19, 36-43, and 54-61 in the absence and presence of ct-DNA are illustrated in Figures S271-S296 (ESI). Progressive addition of ct-DNA at the concentration of 1.25-15 µM to a fixed amount of modified naphthalic anhydride or naphthalimide (10 µM concentration) decreased absorbance for almost all the tested modified conjugates, except conjugates 14, 15, and 22-27 for which changes in absorbance were not observed. For all other conjugates, the absorption spectra demonstrated no bathochromic effect (6-10, 16, 18, 36-43, 54, 56, 58, 60, and 61) or slight bathochromic shifts (11,17,19,55, and 59) (1-2 nm). It should be noted that mitonafide also caused a small bathochromic shift, which was confirmed in our study [9] and the literature [41]. Hypochromicity (10-56%) was also observed for all conjugates, with the highest value for compound 40. The isosbestic point within 404-412 nm was observed for 6, 7, 11, 17, and 19, indicating that ligand molecules are in two spectrophotometrically distinguished conditions-bound and free.
To compare the DNA-binding strength of the tested conjugates, we calculated the binding constant K b , as described in the Materials and Methods section. The selected modified compounds (6, 7, 9, 11, 16-19, 36-39, 42, 42, and 54-61) showed a similar K b value as compared to mitonafide (2.54 × 10 5 ) [9], and some of the conjugates (8, 10, 40, and 41) revealed a comparable K b value (Table 1) to that of pinafide (6.60 × 10 4 ) [9]. For conjugates 14, 15, and 22-27, the K b value could not be determined due to the lack of noticeable changes in the UV spectra.

In Vitro Cytotoxic Activity
The obtained 1,8-naphthalimide-carborane conjugates were investigated for their in vitro antitumor activity by examining their cytotoxic effects using the MTT tetrazolium dye assay [42,43] against the human cancer cell line HepG2 established from hepatocellular carcinoma. IC 50 refers to the drug concentration (µM) required to inhibit cell growth by 50%. The IC 50 values determined for the synthesized compounds are summarized in Table 2. Generally, 1,8-naphthalimides modified with carboranes (8-11, 16-19, 24-27, 36-43, and 54-61) exhibited more cytotoxicity than naphthalic anhydrides containing carborane clusters (6, 7, 14, 15, 22, and 23). In compounds 8-11, 16-19, 24-27, and 36-43, the modified mitonafide derivatives were less cytotoxic than the modified pinafide derivatives, and conjugates modified with ortho-carboranes were less active than the corresponding conjugates modified with meta-carborane. Molecules synthesized via "click reaction" 8-11 (triazole ring attached directly to the heteroaromatic system) and 16-19 (triazole ring attached through an oxygen atom to the heteroaromatic system) showed similar cytotoxicity. A comparative analysis of the naphthalimides in the series in terms of their activity showed that these two groups of compounds were the least cytotoxic as compared to the modified 1,8-naphthalimides obtained using Mitsunobu reaction, amidation reaction, and reductive amination. Among the tested compounds, the most cytotoxic were conjugates 54-61 obtained by a reductive amination reaction. In this group of compounds, the conjugates containing a shorter linker between the heteroaromatic system and carborane modification (54)(55)(56)(57) were slightly more cytotoxic than those containing a longer linker between the heteroaromatic system and carborane (58-61). The pinafide analog 57 containing meta-carborane was identified to be the most cytotoxic to the tested tumor cell line at a concentration as low as 3.12 µM. The pinafide analog 56 containing ortho-carborane was less cytotoxic with an IC 50 value of 12.07 µM. The mitonafide analogs bearing meta-carborane 55 (IC 50 = 3.43 µM) or ortho-carborane 54 (IC 50 = 5.49 µM) were slightly less cytotoxic as compared to their pinafide analogs. It is worth mentioning that conjugates 54-61 were rather more active than the mitonafide and pinafide analogs also obtained through reductive amination but containing carborane clusters at position 3 of the heteroaromatic system (IC 50 = 4.77-53.09 µM) [9]. Naphthalic anhydrides 6, 14, 15, and 22 showed low cytotoxicity against the HepG2 cell line (IC 50 = 28.65-71.19 µM), while compounds 7 and 23 were not toxic (IC 50 > 100 µM).
Considering the physicochemical and cytotoxic properties of conjugates 54-61, these compounds were used for further biological assays.

Cell Migration Inhibition Assay
Cell migration is a vital process for various physiological and pathological conditions such as tissue homeostasis, immune response, cancer, and chronic inflammatory diseases [44].
By using the xCELLigence platform, we evaluated migration potential of HepG2 cells after the administration of compounds. We incubated the cells with compounds 54-61 at the concentration corresponding to one-fourth of the IC 50 value for 24 h before the analysis, and the cells were then seeded on a CIM plate. Impedance-based cell index (CI) of HepG2 cells was measured every 30 min for 72 h. Our results indicate that all the analyzed compounds significantly affected the migration potential of HepG2 cells as compared to control. The most significant changes were observed within the first 24 h ( Figure 3A). Thus, to obtain more insight into the altered migration profile caused by the analyzed compounds, we additionally calculated the slope representing the rate of change of the cell index within the first 24 h. Among the tested compounds, pinafide analogs bearing orthoand meta-carborane clusters attached to heterocyclic rings through a (CH 2 ) 2 (56, 57) or (CH 2 ) 6 linker (60 and 61) influenced the migratory activity most. However, the highest impairment was observed after treatment with conjugate 61 as compared to control cells ( Figure 3B).  The migration potential of the cells was monitored using the xCELLigence system. HepG2 cells were incubated with the analyzed compounds for 24 h before the analysis at the concentration corresponding to one-fourth of the IC50 value. The impedance (cell index values) was measured every 30 min for the next 72 h. To eliminate the influence of the solvent, DMSO was added to the cells in a volume that corresponded to the highest compound concentration (A). The slope parameter represents the rate of changes in CI values of HepG2 cells within the first 24 h (B). Statistical significance is indicated by asterisks: (***) p < 0.001, and (****) p < 0.0001.

Cell Cycle Analysis by Flow Cytometry
Recent preclinical and clinical studies of highly selective agents that target various regulators of the mammalian cell cycle demonstrate cell cycle arrest, inhibition of transcription, and apoptotic cell death in models of human cancer [45]. To reveal the mechanism underlying the inhibitory effect of the obtained compounds on cellular viability, we examined cell cycle regulation. For this purpose, HepG2 cells were exposed to compounds The migration potential of the cells was monitored using the xCELLigence system. HepG2 cells were incubated with the analyzed compounds for 24 h before the analysis at the concentration corresponding to one-fourth of the IC 50 value. The impedance (cell index values) was measured every 30 min for the next 72 h. To eliminate the influence of the solvent, DMSO was added to the cells in a volume that corresponded to the highest compound concentration (A). The slope parameter represents the rate of changes in CI values of HepG2 cells within the first 24 h (B). Statistical significance is indicated by asterisks: (***) p < 0.001, and (****) p < 0.0001.

Cell Cycle Analysis by Flow Cytometry
Recent preclinical and clinical studies of highly selective agents that target various regulators of the mammalian cell cycle demonstrate cell cycle arrest, inhibition of transcription, and apoptotic cell death in models of human cancer [45]. To reveal the mechanism underlying the inhibitory effect of the obtained compounds on cellular viability, we examined cell cycle regulation. For this purpose, HepG2 cells were exposed to compounds 54-61 (54 (5.5 µM), 55 (3.4 µM), 56 (12 µM), 57 (3.1 µM), 58 (3.9 µM), 59 (5.4 µM), 60 (31 µM), and 61 (4.8 µM)). The chosen concentration of each of these compounds corresponded to their total IC 50 value.
After exposure, HepG2 cells were examined by flow cytometry, and their DNA content was measured by PI staining. Based on DNA content, it was found that all compounds affected the cell cycle, although the observed effect differed depending on the compound. Compounds 54-59 caused arrest in the G1/G0 phase since we observed a higher number of cells in that phase as compared to control ( Figure 4 and Figure S297, ESI). The most significant effect on cell cycle progression was shown by compound 58, where the G0/G1 cell fraction increased to 70.4% from 60.1% in control. Compounds 60 and 61 arrested cell cycle in the G2M phase, where the accumulation of cells was observed (>18% vs. 11.5% in control cells) ( Figure 4 and Figure S297, ESI). In comparison, mitonafide and pinafide induced cell cycle arrest at the S and G2M phases [8], respectively. Our previous studies showed that 1,8-naphthalimide derivatives with carborane or metallacarborane modification at the N-imide position also caused cell cycle arrest at the G0/G1 phase [8]. In contrast, 1,8-naphthalimide products with carborane modification at position 3 caused cell cycle arrest at the G2M phase [9], similar to pinafide and compounds 60 and 61 from this study.  The graph presents the percentage of cells in the G0/G1, S, and G2M phases, respectively. Data are presented as the mean ± SD of three independent experiments. Statistical significance is indicated by asterisks: (ns) p > 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001.

Oxidative Stress Measurement in HepG2 Cells by Flow Cytometry
To reveal the mechanism responsible for the inhibitory effect of compounds 54-61 on cell viability, we examined their ability to induce ROS production. A previous study showed that 1,8-naphthalimide derivatives could elevate intracellular and mitochondrial ROS production and a remarkable activation of the p38 MAPK pathway [6].
To determine the effect of the compounds on HepG2 cells, we analyzed oxidative status after 24 h of treatment as the pathological level of ROS affects the proper functioning of proteins, lipids, and nucleic acids. The compounds were added to the growth medium at a concentration that corresponds to the total IC50 value. After incubation, the cells were stained with CellROX Deep Red Reagent, which is an oxidative stress indicator. This is a non-fluorescent dye that emits red fluorescent signal upon oxidation by ROS. Flow cytometry analysis indicated that the most potent inductors of ROS were compounds 60 and 61 as the fluorescence intensity in HepG2 treated cells increased by 64% and 65%, respectively, as compared to control ( Figures 5 and S298, ESI). Conjugates 54-59 showed The graph presents the percentage of cells in the G0/G1, S, and G2M phases, respectively. Data are presented as the mean ± SD of three independent experiments. Statistical significance is indicated by asterisks: (ns) p > 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001.

Oxidative Stress Measurement in HepG2 Cells by Flow Cytometry
To reveal the mechanism responsible for the inhibitory effect of compounds 54-61 on cell viability, we examined their ability to induce ROS production. A previous study showed that 1,8-naphthalimide derivatives could elevate intracellular and mitochondrial ROS production and a remarkable activation of the p38 MAPK pathway [6].
To determine the effect of the compounds on HepG2 cells, we analyzed oxidative status after 24 h of treatment as the pathological level of ROS affects the proper functioning of proteins, lipids, and nucleic acids. The compounds were added to the growth medium at a concentration that corresponds to the total IC 50 value. After incubation, the cells were stained with CellROX Deep Red Reagent, which is an oxidative stress indicator. This is a non-fluorescent dye that emits red fluorescent signal upon oxidation by ROS. Flow cytometry analysis indicated that the most potent inductors of ROS were compounds 60 and 61 as the fluorescence intensity in HepG2 treated cells increased by 64% and 65%, respectively, as compared to control ( Figure 5 and Figure S298, ESI). Conjugates 54-59 showed a moderate effect on ROS generation in HepG2 cells. We observed an increase in fluorescence intensity of approximately 22-32% as compared to that of control cells ( Figure 5 and Figure S298, ESI). cell viability, we examined their ability to induce ROS production. A previous study showed that 1,8-naphthalimide derivatives could elevate intracellular and mitochondrial ROS production and a remarkable activation of the p38 MAPK pathway [6].
To determine the effect of the compounds on HepG2 cells, we analyzed oxidative status after 24 h of treatment as the pathological level of ROS affects the proper functioning of proteins, lipids, and nucleic acids. The compounds were added to the growth medium at a concentration that corresponds to the total IC50 value. After incubation, the cells were stained with CellROX Deep Red Reagent, which is an oxidative stress indicator. This is a non-fluorescent dye that emits red fluorescent signal upon oxidation by ROS. Flow cytometry analysis indicated that the most potent inductors of ROS were compounds 60 and 61 as the fluorescence intensity in HepG2 treated cells increased by 64% and 65%, respectively, as compared to control ( Figures 5 and S298, ESI). Conjugates 54-59 showed a moderate effect on ROS generation in HepG2 cells. We observed an increase in fluorescence intensity of approximately 22-32% as compared to that of control cells ( Figures 5  and S298, ESI). The concentration chosen for each compound corresponded to the total IC 50 value. Oxidative stress was evaluated using CellROX Deep Red Reagent by flow cytometry. Data are presented as the mean ± SD of three independent experiments. Statistical significance is indicated with asterisks: (ns) p > 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001.

Analysis of 8-oxo-dG in HepG2 Cells
We also estimated 8-oxo-dG levels in DNA since 2-deoxyguanosine is most susceptible to oxidation among nucleosides and might serve as an oxidative DNA damage marker within the cells. HPLC-UV-ED analysis of DNA revealed that all compounds significantly increased 8-oxo-dG levels in HepG2 cells after 24 h of incubation. The most effective were compounds 60 and 61 as we observed over 57-and 43-fold increase, respectively, in the number of 8-oxo-dG residues per 10 6 dG as compared to control (Table 3). Both compounds generate much higher oxidative disturbance in DNA than mitonafide, resulting in approximately 4-and 6-fold more 8-oxo-dG residues, respectively. Taken together, these results confirm that all the analyzed compounds affect oxidative balance in HepG2 cells and contribute significantly to oxidative stress generation.

Apoptosis/Necrosis Assay by Flow Cytometry
Apoptosis and necrosis are two mechanisms involved in cell death in multicellular organisms. Apoptosis is considered as a naturally occurring physiological process, whereas necrosis is a pathological process caused by external agents such as toxins. In our studies on 1,8-naphthalimides modified with a carboranyl group in position 3, it was found that this type of conjugates strongly promoted apoptosis, which might inhibit the growth of HepG2 cells [9].
To investigate whether compounds 54-61 induced apoptosis in HepG2 cells, we incubated the cells with these compounds for 24 h and performed a flow cytometry analysis. The concentration chosen for each compound corresponded to their total IC 50 values. After incubation, the cells were stained with Annexin V Alexa Fluor 647 conjugate, which enabled detection of the externalization of phosphatidylserine, one of the earliest indicators of apoptosis. To eliminate the influence of the solvent (DMSO), we also analyzed its ability to induce apoptosis in a volume that corresponded to the highest concentration. All the studied compounds induced apoptosis in HepG2 cells after 24 h of treatment. The apoptosis rate is presented in Figure S299 (ESI). The most potent apoptosis inductor was compound 56, where we observed 77.4% of apoptotic cells. In the cells treated with compounds 54, 55, and 60, almost half of the test population underwent apoptosis, with the apoptosis rates of 44.3%, 48.2%, and 46.9%, respectively. The other compounds, 57, 58, 59, and 61, could not induce as high apoptosis as those mentioned above. Approximately one-third of cells targeted apoptotic pathways (33.4%, 39.1%, 23.1%, and 37.1%, respectively) when treated with these compounds (Figure 6). third of cells targeted apoptotic pathways (33.4%, 39.1%, 23.1%, and 37.1%, respectively) when treated with these compounds (Figure 6).

Fluorescence Imaging of Lysosomes
In recent years, considerable efforts have been made to develop 1,8-naphthalimide derivatives as fluorescent probes and fluorescent dyes. Many excellent examples of 1,8naphthalimide-based probes have been reported, and some of them have been successfully applied in live-cell imaging research. A series of multifunctional compounds based on 1,8-naphthalimide moiety were designed and synthesized. They could be used as imaging reagents to detect lysosomes in live human cervical cancer cells (HeLa) by using fluorescence microscopy [46].
Our previous studies showed that the selected 1,8-naphthalimides modified with the carboranyl group at position 3 specifically targeted the lysosomes of living cells with good cell membrane permeability, which enabled the localization of boron/carborane in the cells [9]. The observed autofluorescence of the analyzed compounds 54-61 also enabled They could be used as imaging reagents to detect lysosomes in live human cervical cancer cells (HeLa) by using fluorescence microscopy [46].
Our previous studies showed that the selected 1,8-naphthalimides modified with the carboranyl group at position 3 specifically targeted the lysosomes of living cells with good cell membrane permeability, which enabled the localization of boron/carborane in the cells [9]. The observed autofluorescence of the analyzed compounds 54-61 also enabled us to determine intracellular localization of the analyzed compounds. After 24 h of incubation with the compounds at the concentration corresponding to the total IC 50 value, the cells were stained with the fluorescent dyes that specifically stain cellular organelles. Hoechst 33342 was used to stain nuclei blue, whereas LysoTracker Red DND-99 stained lysosomes. The analyzed compounds emitted strong green fluorescent signals (left panel, Figure 7). Colocalization analysis by confocal microscopy revealed that the autofluorescence of the compounds overlapped with that of DND-99 (red). Because of the observed green and red fluorescence overlap, an orange signal appeared on merged images. These results suggest that the compounds can quickly diffuse through the membranes and can specifically target the lysosomes.

Human Topoisomerase IIα Relaxation Assay
DNA topoisomerases are the enzymes that regulate DNA replication, transcription, and repair. Inhibitors of topoisomerases I and II (Topo I and II, respectively) are effectively used as anticancer agents [47]. Drugs targeting Topo II are divided into two broad classes. The first class, which includes most clinically active agents, leads to an increase in the levels of Topo II-DNA covalent complexes. These drugs have been termed as Topo II poisons. The second class of compounds inhibits Topo II catalytic activity but does not cause an increase in the levels of Topo II covalent complexes. Agents in this second class are thought to kill cells by eliminating the essential enzymatic activity of Topo II and are therefore termed as Topo II catalytic inhibitors. Topo II poisons can be subdivided into intercalating and non-intercalating poisons [48]. Inhibition of topoisomerases, especially Topo II, has been proposed as the primary mechanism by which naphthalimides induce cell cycle arrest and apoptosis in cancer cells [49].
In our earlier study, the presence of the carboranyl cluster at position 3 of 1,8-naphthalimide moieties did not promote them as effective Topo II inhibitors [9].
Based on the results obtained, it was observed that compound 8 revealed inhibitory properties against the tested enzyme with the highest potency at the concentration as low as 0.58 µM. Conjugate 38 was almost 5-fold less active with an IC 50 value of 2.81 µM. It is worth mentioning that compounds 8 and 38 were more active than mitonafide (IC 50 = 5.13 µM). Promising inhibitory properties were also shown by compounds 6, 7, 36, and 37 relative to the standard used (IC 50 = 7.73, 8.78, 7.72, and 7.49 µM, respectively). Conjugate 15 was 12-fold less active than mitonafide (Table 4). Compounds 54-61, which were characterized by their ability to stabilize DNA (Table 1), did not inhibit the activity of this enzyme. As described above, intercalation into DNA is not required to inhibit Topo II.

Chemistry
Most of the chemicals were obtained from the Acros Organics (Geel, Belgium) and were used without further purification unless otherwise stated. 4-Bromo-1,8-naphthalic anhydride (1) was obtained from the TCI (Tokyo, Japan) and used without further purification. Boron clusters were purchased from KATCHEM spol. S.r.o. (Řež/Prague, Czech Republic). All experiments that involved water-sensitive compounds were conducted under rigorously anhydrous conditions and under an argon atmosphere. Flash column chromatography was performed on silica gel 60 (230-400 mesh, Sigma-Aldrich, Steinheim, Germany). R f refers to analytical TLC performed using pre-coated silica gel 60 F254 plates purchased from Sigma-Aldrich (Steinheim, Germany) and developed in the solvent system indicated. Compounds were visualized using UV light (254 nm) or a 0.5% acidic solution of PdCl 2 in HCl/methanol by heating with a heat gun for boron-containing derivatives. The yields are not optimized. 1 H NMR, 13 C NMR, and 11 B NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer equipped with a direct ATM probe. The spectra for 1 H, 13 C, and 11 B nuclei were recorded at 600. 26, 150.94, and 192.59 MHz, respectively. Deuterated solvents were used as standards. The following abbreviations are used to denote the multiplicities: s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublets of doublets, t = triplet, dt = doublet of triplets, q = quartet, quin = quintet, br s = broad singlet, and m = multiplet. J values are given in Hz.
Mass spectra were recorded on a CombiFlash PurIon Model Eurus35 (Teledyne ISCO, Lincoln, NE, USA). The ionization was achieved by Atmospheric-Pressure Chemical Ionization (APCI) ionization in the positive ion mode (APCI+) and negative ion mode (APCI-). The entire flow was directed to the APCI ion source operating in the positive ion mode. Total ion chromatograms were recorded in the m/z range of 100-700. The vaporization and capillary temperature were set at 250-400 and 200-300 • C, respectively. The capillary voltage of 150 V, corona discharge of 10 µA. High-resolution mass spectra (HRMS) were obtained on a Agilent 6546 LC/Q-TOF with ESI ion source spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA). The data are presented for the most abundant mass in the boron distribution plot of the base peak (100%) and for the peak corresponding to the highest m/z value with its relative abundance (%).
The theoretical molecular mass peaks of the compounds were calculated using the "Show Analysis Window" option in the ChemDraw Ultra 12.0 program. The calculated m/z corresponds to the average mass of the compounds consisting of natural isotopes.
Infrared absorption spectra (IR) were recorded using a Nicolet 6700 Fourier-transform infrared spectrometer from Thermo Scientific equipped with a ETC EverGlo* source for the IR range, a Geon-KBr beam splitter, and a DLaTGS/KBr detector with a smart orbit sampling compartment and diamond window. The samples were placed directly on the diamond crystal, and pressure was added to make the surface of the sample conform to the surface of the diamond crystal. UV measurements were performed using a GBC Cintra10 UV-vis spectrometer (Dandenong, Australia). The samples used for the UV experiment were dissolved in 99.8% C 2 H 5 OH. The measurement was performed at ambient temperature.
RP-HPLC analysis was performed on a Hewlett-Packard 1050 system equipped with a UV detector and Hypersil Gold C18 column (4.6 × 250 mm, 5 µm particle size, Thermo Scientific, Runcorn, UK). UV detection was conducted at λ = 340 nm for compounds 6-11, 14-19, and 22-27 and at 380 nm for compounds 36-43 and 54-61. The flow rate was 1 mL min -1 . All analyses were run at ambient temperature. The gradient elution was as follows: gradient A-10 min from 30% to 55% A, 10 min from 55% to 90% A, and 10 min from 90% to 30% A. Buffer A contained 0.1% HCOOH in CH 3 CN, and buffer B contained 0.1% HCOOH in H 2 O; gradient B-10 min from 0% to 25% A, 10 min from 25% to 60% A and 10 min from 60% to 0% A. Buffer A-CH 3 CN contained 0.1% HCOOH, and buffer B-H 2 O contained 0.1% HCOOH. Crystals of 23 were obtained by slow evaporation from MeOH. X-ray diffraction measurements were carried out under cryogenic conditions on an XtaLab Synergy four-circle diffractometer (Oxford Diffraction) equipped with a Cu rotating anode PhotonJet X-ray source and HyPis-6000HE CCD detector. The data were processed with CRYSALISPRO software (Rigaku Oxford Diffraction), the structure was solved with SHELXT and refined with SHELXL programs, as above, via the Olex2 interface [50]. The refinement of atomic positions was unrestrained except for hydrogen atoms which were maintained at riding positions. Table S1 (ESI) summarizes the crystallographic data.

Cytotoxicity Assay
The cytotoxic properties of the synthesized compounds were evaluated using the human hepatocellular carcinoma cell line HepG2 established from hepatocellular carcinoma. The HepG2 cell line was purchased from ECACC (Salisbury, UK). Cells were grown in EMEM medium (Corning ® ) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Corning ® ) and antibiotics (Corning ® ), at 37 • C in a 5% CO 2 atmosphere. Upon reaching 80-90% confluency, cells were detached with the trypsin (Corning ® ) and transferred into 96-well microplates at a density of 12 × 10 3 . After overnight incubation at 37 • C in a humidified atmosphere containing 5% CO 2 , the cells were treated for the next 24 h with compounds. The stock solution of each compound was prepared in DMSO at 10 mM. The cytotoxicity was evaluated by the MTT assay. The final content of DMSO in solutions did not exceed 0.2%, and an additional control group with 0.2% DMSO was included to rule out the effect of solvent. Subsequently, the medium was aspirated and replaced with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye solution (50 µL, 0.5 mg/mL). After 3 h incubation, the resulting MTT formazan crystals were dissolved in DMSO (100 µL). To ensure the complete dissolution of formazan, the plates were shaken on an orbital microplate shaker at 1000 rpm for 15 min (Thermoshaker NeoLab 7-0055, Bionovo, Legnica, Poland). The optical density of each well was then measured on a VICTOR Nivo multimode plate reader at a wavelength of 570 nm. Each experiment consisted of 6 replications of each concentration, and each experiment was repeated three times independently. The results were calculated as a percentage of control group viability. The IC 50 values were determined using a non-linear regression from the plot of % viability against log dose of compounds by using GraphPad Prism 6.0 software (GraphPad, San Diego, CA, USA).

Cell Migration Inhibition Assay
Transwell cell migration experiments were performed using xCELLigence RTCA Analyzer (Roche) in Cell Invasion and Migration (CIM) plates (ACEA Biosciences, San Diego, CA, USA). Each well consisted of an upper and a lower chamber separated by a microporous polyethylene terephthalate (PET) membrane containing randomly distributed 8 µm-pores. Prior to the migration assay, HepG2 cells (15 × 10 3 ) were seeded onto 48-well plates containing the growth medium (EMEM) and incubated until 70-80% confluency and incubated at 37 • C in a humidified atmosphere containing 5% CO 2 . Subsequently, the cells were treated for 24 h with the tested compounds at the final concentration corresponding to the one-fourth of IC 50 value. Next, 160 µL of complete growth medium (supplemented with 10% FBS) was added to the lower chamber and 50 µL of serum-free growth medium to the upper chambers of CIM plate. The plates were incubated at 37 • C and 5% CO 2 saturation for 1 h prior to insertion into the xCELLigence platform. To initiate a transwell migration experiment, cells were detached, resuspended in the serum-free growth medium,