Novel Cytotoxic Pt(IV) Compounds with Improved Safety Profiles

TL;DR

Researchers developed new platinum-based cancer drugs with vitamin E-like structures that are more toxic to cancer cells and less harmful to normal cells.

Contribution

Incorporating antioxidant ligands into platinum(IV) complexes to improve safety and efficacy.

Findings

Novel platinum(IV) complexes showed higher cytotoxicity than cisplatin in cancer cells.

The compounds exhibited less toxicity toward normal fibroblast cells.

Phenolic ligands conferred antioxidant activity, modulating redox processes.

Abstract

Platinum(II)-based drugs, such as cisplatin, are commonly used to treat various types of cancer. However, their clinical use is limited due to a number of side effects and the development of resistance. To overcome these limitations, researchers have explored the development of platinum(IV) complexes as potential prodrugs that can be selectively activated under physiological conditions. In this study, we have incorporated synthetic analogs of vitamin E into the structure of platinum(IV) complexes to further improve their safety profile. The antioxidant properties of the compounds were evaluated using DPPH and CUPRAC assays, as well as lipid peroxidation inhibition models, revealing that incorporation of phenolic ligands confers pronounced antioxidant activity. Cytotoxicity was assessed towards cancer cell lines using the MTT assay, where the novel complexes showed significantly increased cytotoxic activity compared to cisplatin, while also demonstrating less toxicity toward normal fibroblast cells under the same in vitro conditions. These results suggest that the conjugation of antioxidant ligands to platinum(IV) scaffolds can modulate both redox processes and the biological activity of the resulting complexes. This proposed design strategy has the potential to create more effective platinum-based cancer treatments with enhanced biological characteristics.

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Full text

1. Introduction

Since the approval of cisplatin, platinum(II) drugs (Figure 1) have been widely used for the treatment of various types of cancer, including testicular, ovarian, bladder, lung, and colorectal cancer [1,2]. These compounds work by forming covalent bonds with DNA, creating intra- and interstrand crosslinks that disrupt the DNA structure and prevent replication and transcription and ultimately leading to cell death [3,4].

Despite their effectiveness, platinum(II)-based chemotherapeutic agents have some significant side effects. One major concern is their lack of selectivity. These compounds can nonspecifically interact with plasma proteins and other coordinating biomolecules, leading to unspecific toxicity. They can also accumulate in healthy organs, contributing to dose-limiting toxicities such as nephrotoxicity, neurotoxicity, and ototoxicity [5]. Furthermore, the toxicity of these platinum compounds is not solely due to their interaction with biotargets but also to their impact on the redox balance of cells [6]. Studies have shown that nephrotoxicity and ototoxicity are related to increased levels of reactive oxygen species (ROS) and subsequent apoptosis [7].

A promising approach to addressing these challenges is the development of platinum(IV) prodrugs. Several platinum(IV) prodrugs have advanced to clinical trials, showing the potential of this class of compounds as next-generation anticancer agents (Figure 2) [8,9,10,11,12]. The octahedral structure of these complexes provides higher stability, making them less reactive towards biotargets and ensuring prolonged circulation in the bloodstream. Under hypoxic conditions of the tumor environment, platinum(IV) compounds are reduced to active platinum(II), which directly attacks DNA. This process results in lower systemic toxicity than traditional platinum(II) drugs [2,13].

Another advantage of platinum(IV) scaffolds is their capacity to be modified by incorporating functional ligands into their axial positions. This structural flexibility enables the creation of multifunctional compounds that combine the cytotoxic potential of platinum with the other biological properties of the ligands. Following cellular reduction, platinum(IV) complexes transform into active platinum(II) species [13], while the accompanying ligands are released and can exert additional biological effects through interaction with specific targets, thus broadening the therapeutic potential of the compounds [13,14,15,16,17,18]. Numerous studies have explored the use of platinum(IV) complexes functionalized with biologically active ligands in combination with clinically used drugs in order to combine the cytotoxicity of platinum with additional pharmacological activities (Figure 3) [19,20,21,22].

Since platinum-induced side effects can be associated with oxidative stress, introducing a protective antioxidant group as a ligand could be a promising strategy to reduce acute toxicity. Hindered phenols, which are vitamin E mimetics, have been shown to scavenge reactive oxygen species (ROS) and protect cells from oxidative stress damage. When introduced as axial ligands into platinum(IV) scaffolds, such antioxidant moieties may influence the intracellular redox environment after the reductive activation of the prodrug and thus could have a protective effect against oxidative damage.

We have synthesized and evaluated a novel series of platinum(IV) complexes functionalized with antioxidants, including phenosanic acid (the antiepileptic drug Dibuphelone) and its analogs. These complexes combine the stability and in vitro cancer cells selectivity of platinum prodrugs with the potential protective effects of antioxidants.

2. Results and Discussion

2.1. Syntheses and Characterization

To obtain new Pt(IV) complexes, cisplatin and oxaliplatin were first converted to their corresponding Pt(IV) derivatives 1–3 using a well-established method [10]. The Pt(IV) compounds were then reacted with a series of acid chlorides, generated in situ through the reaction of the corresponding carboxylic acids a–d with oxalyl chloride. The reaction between Pt(IV) derivatives 1–3 and the acid chlorides was carried out in acetone, with pyridine acting as an HCl acceptor (Scheme 1). Complexes 1a, 2d and 3d were isolated from the reaction mixture by precipitation by dichloromethane or diethyl ether, while all other complexes were purified via column chromatography on silica gel.

The composition and structure of these compounds were confirmed using nuclear magnetic resonance (NMR) spectroscopy (^1^H, ^13^C), electrospray ionization mass spectrometry (ESI MS), and the purity was also confirmed by elemental analysis (Figures S1–S27). The ^1^H NMR spectra of all Pt(IV) complexes show signals confirming the formation of the complexes. The ^1^H NMR spectrum of complex 2a (Figure 4) shows characteristic signals consistent with its proposed structure. In the high-field region of the spectra, a singlet at 1.36 ppm corresponds to the protons of the tert-butyl group while the signals related to the protons of the cyclohexane ring appear as a multiplet at 2.42 ppm, doublets at 2.09 and 1.47 ppm, and a triplet at 1.07 ppm respectively. Additionally, multiplet signals at 2.44 and 2.67 ppm are observed in the same region and correspond to the CH_2_ groups of the alkyl linker. A singlet at 6.90 ppm in the high-field region is assigned to the aromatic protons of the phenyl group in the ligand. Furthermore, two broad singlets at 8.36 and 8.31 ppm are observed and correspond to the NH protons that are coordinated to the platinum center.

The ESI mass spectra of the synthesized compounds reveal the formation of characteristic ions with a platinum isotopic distribution, including the [M + H]^+^, [M − H]^−^ and [M + Na]^+^ species. The experimental mass spectrum is in good agreement with the simulated one for the target compound (Figure 5). This confirms the molecular composition of the Pt(IV) complexes.

2.2. Antioxidant Activity

The incorporation of the 2,6-di-tert-butylphenol group into the structure of the platinum complexes led to antioxidant activity, as confirmed by the DPPH and CUPRAC assays (Table 1).

The DPPH assay was used to investigate the activity of the complexes in radical transfer reactions. This technique is a valuable tool for estimating the ability of compounds under study to participate in reactions involving hydrogen atom transfer mechanisms. This method relies on the transfer of a hydrogen atom from an antioxidant molecule to a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. As expected, complexes 2d and 3d, which do not contain the phenolic group, showed no detectable antioxidant activity. Likewise, complexes 2c and 3c, containing 3,5-di-tert-butyl-4-hydroxybenzoic acid as the ligand (acid c), also exhibited negligible activity towards the DPPH radical. This lack of activity may be attributed to the shorter linker between the phenolic ring and the platinum center, which could limit the redox accessibility of the phenolic group and thus hinder its radical-scavenging capacity. In contrast, complexes 2a, 2b, 3a, and 3b, which have a methylene spacer between the platinum atom and the phenolic fragment, demonstrated antioxidant activity comparable to or greater than that of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known reference antioxidant. The presence of two antioxidant ligands in each complex led to approximately a twofold increase in antioxidant activity, with complex 2a demonstrating the highest levels of antioxidant activity in this experiment.

The capacity of the compounds to undergo single electron transfer was determined using the CUPRAC assay. This assay is based on the ability of the compounds to reduce Cu^2+^ to Cu^+^ in complex with neocuproine (2,9-dimethyl-1,10-phenanthroline). The results of this assay were in line with those obtained from the DPPH test. Complexes 2d and 3d, which lack a phenolic functional group, did not exhibit any measurable reducing activity. Only minimal activity was observed for complexes 2c and 3c, further confirming the importance of the phenolic group and linker in maintaining redox activity. Among the tested compounds, complex 2b showed the highest reducing capacity, showing twofold increase in reducing capacity relative to the reference antioxidant Trolox. These results suggest that this compound may have the potential to act as a powerful antioxidant.

2.3. Mitochondrial Lipid Peroxidation (LP)

The capacity of novel platinum complexes to act as antioxidants in biological systems was investigated, particularly in terms of their ability to inhibit lipid peroxidation (LP) in rat brain homogenate.

Almost all complexes showed activity against both H_2_O_2_-induced LP and Fe-induced lipid peroxidation (Table 2). As expected, complexes 2d and 3d, which lack a hydroxyl group, showed no antioxidant potential under either condition. Compounds with two phenolic ligands (2a–2b) revealed significantly higher antioxidant potential compared to their monoligand analogs (3a–3b). Among them, complex 2a containing two antioxidant moieties demonstrated the strongest protective effect against rat brain membrane lipid peroxidation. A general trend was also observed, with complexes with shorter linker fragments between the platinum center and the phenolic ring exhibiting somewhat lower antioxidant activity. Overall, these results indicate that these novel platinum complexes have a high potential for protecting cellular lipids of membranes from oxidative damage.

2.4. Antiglycating Activity

Protein glycation is a complex oxidative process that results in the formation of advanced glycation end-products (AGEs). These AGEs contribute to cellular damage and the development of various pathologies. The early stages of glycation involve the formation of reactive oxygen species and carbonyl intermediates, which can be disrupted or delayed by antioxidant molecules. Due to the potential link between antiglycation and antioxidant activity, the antiglycation properties of several Pt(IV) complexes have been evaluated using two complementary models: albumin-glucose and albumin-methylglyoxal (Table 3).

The results showed that complexes 1a, 2b and 3a had measurable antiglycating activity in the glucose-based system. However, none of these complexes showed inhibitory effects in the methylglyoxal-induced glycation model. Overall, the antiglycating effects observed can be described as weak to moderate, although they are still significant in terms of structure.

The selective inhibition observed only in the glucose system provides insight into the possible mechanism. Glucose-driven glycation occurs through several oxidative transformations, generating reactive carbonyl intermediates. Therefore, the inhibition in this system—but not in the methylglyoxal-based model, where reactive dicarbonyl species are supplied directly and in excess—suggests that the complexes act primarily through antioxidant mechanisms, such as interception of early oxidative intermediates or suppression of glucose autoxidation. When the generation of reactive dicarbonyls is not the rate-limiting step (as in the methylglyoxal system), the complexes do not significantly affect the reaction progress.

2.5. Cytotoxic Activity

The cytotoxicity of the compounds was assessed using the MTT assay on several different cell types including lung carcinoma A549, colorectal carcinoma HCT116, breast adenocarcinoma MCF7, ovarian adenocarcinoma A2780, cisplatin-resistant ovarian adenocarcinoma A2780cis and rat dermal fibroblasts (RDF). All the novel platinum(IV) complexes showed significant cytotoxic activity, which was higher than that of both cisplatin and oxaliplatin (Table 4). Complex 2a in particular showed exceptional potency, exceeding the cytotoxicity of the reference platinum drugs across the tested cell lines significantly.

To validate the concept of designing hybrid structures that combine the platinum center with biologically active organic fragments, the individual components were also examined. Precursor acids a–d displayed no measurable cytotoxicity in the MTT assay. The physical mixture of oxaliplatin with acid a did not result in any synergistic enhancement. However, while when these fragments are combined in one molecule, the activity is significantly increased compared to cisplatin and oxaliplatin alone.

To further understand the structural factors that influence cytotoxic potency, the lipophilicity of the synthesized platinum(IV) complexes was evaluated using high-performance liquid chromatography (HPLC). Retention times were measured using mobile phases with varying concentrations of methanol. We then calculated log P values using aniline, nitrobenzene, methyl benzoate, butanone-2, benzonitrile, naphthalene, and phenanthrene as standards. The calculations were carried out according to the previously described method [24,25] (see Table 5).

The data showed a clear and consistent correlation between the structure of the complexes and their lipophilicity as the length of the aliphatic linker connecting the platinum center to the antioxidant fragment increased. The introduction of two antioxidant ligands resulted in an almost twofold increase compared to mono-substituted analogs, which strongly correlates with the cytotoxicity data. Higher lipophilicity is associated with increased cytotoxic activity in all tested cancer cell lines. This suggests that improved membrane permeability and cellular uptake play a significant role in the superior biological activity of the most lipophilic derivatives.

Importantly, several of the new platinum(IV) complexes have shown the potential to overcome cisplatin resistance. Compounds 2a and 2d demonstrated high activity against the cisplatin-resistant ovarian carcinoma cell line A2780cis, with a resistance factor of approximately two times lower than that of cisplatin, which has a resistance factor of at least 5.

In addition to their cytotoxic effects toward tumor cell lines, the synthesized complexes were also evaluated in a non-malignant cell model using rat dermal fibroblasts (RDF). Under these in vitro conditions, most compounds exhibited lower cytotoxicity toward RDF cells compared to cancer cells. A moderate cytotoxic effect was observed only for complex 3d, which lacks a phenolic moiety (Figure 6). This finding supports the hypothesis that incorporation of the antioxidant ligand may influence the cellular response of non-cancerous cells potentially providing some protective properties.

The pro-apoptotic properties of the platinum(IV) complexes were further examined using flow-cytometric analysis (Figure 7). All synthesized platinum(IV) complexes were able to induce apoptosis, with activity profiles similar to that of cisplatin. Interestingly, each complex induced apoptotic cell death at significantly lower concentrations than those required for cisplatin, consistent with their enhanced cytotoxicity observed in the MTT assay. This increased ability to initiate programmed cell death, coupled with improved safety profile, emphasizes the therapeutic potential of these novel structures.

3. Materials and Methods

All solvents were purified and degassed before use [26]. NMR spectra were recorded on a Bruker Avance II 400 spectrometer (Bruker, Rheinstetten, Germany) at room temperature at 400.13 (^1^H) and 100.61 (^13^C [^1^H]) MHz. 2D NMR measurements were carried out using standard pulse programs. Chemical shifts were referenced relative to the solvent signal for ^1^H and ^13^C spectra. The elemental analysis was conducted using the MicroCube Elementar analyzer. The mass spectra were obtained through electrospray ionization (ESI) using the TSQ Endura instrument from Thermo Fisher Scientific, Waltham, MA, USA. Each sample was dissolved in methanol (HPLC grade) and injected directly into the ionization source using a syringe pump. The spectra were recorded for 30 s in the m/z range of 150 to 1400, with both positive and negative ionization modes, and spray voltages of 3.4 and 2.5 kV, respectively.

(OC-6-44)-(Diamine)dichlorido(3,5-di-tert-butyl-4-hydroxyphenylpropionate)(acetate)platinum(IV) (1a)

To a solution of 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid (92 mg, 0.332 mmol) in dichloromethane (15 mL), oxalyl chloride (285 μL, 3.299 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred and boiled for 2 h. The solvent and the excess of oxalyl chloride were removed on a rotary evaporator at reduced pressure. The resulting chloroanhydride was used without further purification. To a suspension of (OC-6-44)-acetato(diamine)dichloridohydroxidoplatinum(IV) (50 mg, 0.133 mmol) in acetone (50 mL), pyridine (54 μL, 0.671 mmol) and a pre-mixed solution of the chloranhydride in acetone (1 mL) were added. The mixture was stirred for 24 h at room temperature without access to light. The resulting clear solution was evaporated to dryness and the resulting solid was treated with dichloromethane (10 mL). Resulting precipitate was separated on glass filter and additionally washed with dichloromethane (2 × 10 mL). The resulting yellow crystalline product was dried under vacuum. The yield was 40 mg (48%). T_dec_ = 228–233 °C. Elemental analysis: for C_19_H_34_Cl_2_N_2_O_5_Pt × 0.3 C_3_H_7_NO calculated (%): C 36.34, H 5.54, N 4.90, found (%): C 36.80, H 5.27, N 4.49. ^1^H NMR (400.13 MHz, [d6]-DMSO, δ ppm): 6.88 (s, 2H; H6, H8), 6.69 (s, 1H; OH), 6.54 (br. s, 6H; NH_3_), 2.65 (m, 2H; H16), 2.44 (m, 2H; H15), 1.90 (s, 3H; H19), 1.34 (s, 18H; H1, H2, H3, H11, H12, H13). ^13^C [^1^H] NMR (100.61 MHz, [d6]-DMSO, δ ppm): 188.69 (C17), 185.48 (C18), 151.94 (C14), 139.22 (C5, C9), 132.11 (C7), 124.16 (C6, C8), 37.98 (C16), 34.50 (C4, C10), 31.53 (C15), 30.46 (C1, C2, C3, C11, C12, C13), 22.89 (C19). ESI-MS: for C_19_H_34_Cl_2_N_2_O_5_Pt: m/z calculated for [M + Na]^+^ 659, found: 659.

(OC-6-33)-(trans-1R,2R-Diaminocyclohexane)-bis(3,5-di-tert-butyl-4-hydroxyphenylpropionate)oxalatoplatinum(IV) (2a)

To a solution of 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid (640 mg, 2.3 mmol) in dichloromethane (20 mL), oxalyl chloride (1991 μL, 22.3 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred and boiled for 2 h. The solvent and the excess of oxalyl chloride were removed on a rotary evaporator at reduced pressure. The resulting chloroanhydride was used without further purification. To a suspension of (OC-6-33)-(trans-1R,2R-diaminocyclohexane)dihydroxido(oxalato)platinum(IV) (200 mg, 0.464 mmol) in acetone (200 mL), pyridine (374 μL, 4.65 mmol) and a pre-mixed solution of the chloranhydride in acetone (3 mL) were added. The mixture was stirred for 24 h at room temperature without access to light. The product was purified by the column chromatography (silica gel, eluent—acetone:hexane (1:1), R_f_ = 0.66). The obtained white crystalline product was dried in vacuo. The yield was 163 mg (74%). T_dec_ = 224–227 °C. Elemental analysis: for C_42_H_64_N_2_O_10_Pt calculated (%): C 52.99, H 6.78, N 2.94, found (%): C 53.37, H 5.77, N 3.07. ^1^H NMR (400.13 MHz, [d6]-DMSO, δ ppm): 8.36 (br. s, 2H; NH_2_), 8.31 (br. s, 2H; NH_2_), 6.90 (s, 4H; H6/H6′, H8/H8′), 6.7 (s, 2H; OH), 2.67 (dd, 4H, J = 9.9, 6.4 Hz; H16/H16′), 2.58–2.41 (m, 6H; H15/H15′, H18, H23), 2.09 (d, 2H, J = 11.3 Hz; H19, H22), 1.47 (d, 2H, J = 7.5 Hz; H19, H20, H21, H22), 1.36 (s, 38H; H1/H1′, H2/H2′, H3/H3′, H11/H11′, H12/H12′, H13/H13′, H20a, H21a), 1.07 (t, 2H, J = 10.2 Hz; H20b, H21b). ^13^C [^1^H] NMR (100.61 MHz, [d6]-DMSO, δ ppm): 180.39 (C17/C17′), 163.35 (C24, C25), 151.92 (C14/C14′), 139.1 (C5/C5′, C9/C9′), 131.76 (C7/C7′), 124.09 (C6/C6′, C8/C8′), 61.03 (C18, C23), 37.84 (C16/C16′), 34.41 (C4/C4′, C10/C10′), 31.23 (C15/C15′), 30.90 (C19, C22), 30.39 (C1/C1′, C2/C2′, C3/C3′, C11/C11′, C12/C12′, C13/C13′), 23.43 (C20, C21). ESI-MS: for C_42_H_64_N_2_O_10_Pt: m/z calculated for [M − H]^−^ 950, found: 950.

(OC-6-33)-(trans-1R,2R-Diaminocyclohexane)-bis(3,5-di-tert-butyl-4-hydroxyphenylacetate)oxalatoplatinum(IV) (2b)

To a solution of 3,5-di-tert-butyl-4-hydroxyphenylacetic acid (306 mg, 1.16 mmol) in dichloromethane (30 mL), oxalyl chloride (996 μL, 11.6 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred and boiled for 2 h. The solvent and the excess of oxalyl chloride were removed on a rotary evaporator at reduced pressure. The resulting chloroanhydride was used without further purification. To a suspension of (OC-6-33)-(trans-1R,2R-diaminocyclohexane)dihydroxido(oxalato)platinum(IV) (100 mg, 0.232 mmol) in acetone (100 mL), pyridine (187 μL, 2.32 mmol) and a pre-mixed solution of the chloranhydride in acetone (2 mL) were added. The mixture was stirred for 24 h at room temperature without access to light. The product was purified by the column chromatography (silica gel, eluent—acetone:hexane (1:1), R_f_ = 0.61). The obtained yellow crystalline product was dried in vacuo. The yield was 70 mg (47%). T_dec_ = 220–223 °C. Elemental analysis: for C_40_H_60_N_2_O_10_Pt calculated (%): C 51.99, H 6.55, N 3.03, found (%): C 52.11, H 6.43, N 3.01. ^1^H NMR (400.13 MHz, [d6]-DMSO, δ ppm): 8.38 (br. s, 2H; NH_2_), 8.19 (br. s, 2H; NH_2_), 6.91 (s, 4H; H6, H6′, H8, H8′), 6.77 (s, 2H; OH), 3.45 (m, 4H; H15/H15′), 2.32 (br. s, 2H; H17, H22), 2.01 (d, 2H, J = 11.6 Hz; H18, H22), 1.41 (d, 4H, J = 8.1 Hz; H18, H21), 1.33 (s, 36H; H1, H1′, H2, H2′, H3, H3′, H11, H11′, H12, H12′, H13, H13′), 0.92 (t, 2H, J = 8.9 Hz; H19, H20). ^13^C [^1^H] NMR (100.61 MHz, [d6]-DMSO, δ ppm): 179.73 (C16), 163.18 (C23, C24), 152.47 (C14/C14′), 138.92 (C5/C5′, C9/C9′), 126.66 (C7/C7′), 125.39 (C6/C6′, C8/C8′), 60.92 (C17, C22), 34.45 (C4/C4′, C10/C10′), 32.28 (C15), 31.41 (C18, C21), 30.35 (C1/C1′, C2/C2′, C3/C3′, C11/C11′, C12/C12′, C13/C13′), 23.45(C19, C20). ESI-MS: for C_40_H_60_N_2_O_10_Pt: m/z calculated for [M − H]^−^ 922, found 922.

(OC-6-33)-(trans-1R,2R-Diaminocyclohexane)-bis(3,5-di-tert-butyl-4-hydroxybenzoate)oxalatoplatinum(IV) (2c)

To a solution of 3,5-di-tert-butyl-4-hydroxyphenylbenzoic acid (145 mg, 0.58 mmol) in dichloromethane (20 mL), oxalyl chloride (498 μL, 5.8 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred and boiled for 2 h. The solvent and the excess of oxalyl chloride were removed on a rotary evaporator at reduced pressure. The resulting chloroanhydride was used without further purification. To a suspension of (OC-6-33)-(trans-1R,2R-diaminocyclohexane)dihydroxido(oxalato)platinum(IV) (50 mg, 0.116 mmol) in acetone (50 mL), pyridine (93 μL, 1.16 mmol) and a pre-mixed solution of the chloranhydride in acetone (2 mL) were added. The mixture was stirred for 24 h at room temperature without access to light. The product was purified by the column chromatography (silica gel, eluent—acetone:hexane (1:1), R_f_ = 0.59). The obtained pale-yellow crystalline product was dried in vacuo. The yield was 31 mg (29%). T_dec_ = 214–217 °C. Elemental analysis: for C_38_H_56_N_2_O_10_Pt × 1 CH_2_Cl_2_ calculated (%): C 47.76, H 5.96, N 2.86, found (%): C 47.36, H 6.23, N 3.21. ^1^H NMR (400.13 MHz, [d6]-DMSO, δ ppm): 8.49 (br. s, 2H; NH_2_), 8.40 (br. s, 2H; NH_2_), 7.69 (s, 4H; H6, H6′, H8, H8′), 7.52 (s, 2H; OH), 2.80 (br. s, 2H; H16, H21), 2.19 (d, 2H, J = 10.6 Hz; H17, H20), 1.55 (m, 4H; H17, H18, H19, H20), 1.37 (s, 36H; H1, H1′, H2, H2′, H3, H3′, H11, H11′, H12, H12′, H13, H13′), 1.23 (t, 2H, J = 6.9 Hz; H18, H19). ^13^C [^1^H] NMR (100.61 MHz, [d6]-DMSO, δ ppm): 173.55 (C15), 164.05 (C22, C23), 157.61 (C14/C14′), 137.77 (C5/C5′, C9/C9′), 126.53 (C7/C7′), 123.59 (C6/C6′, C8/C8′), 61.15 (C16, C21), 34.48 (C4/C4′, C10/C10′), 30.97 (C17, C20), 30.10 (C1/C1′, C2/C2′, C3/C3′, C11/C11′, C12/C12′, C13/C13′), 23.80 (C18, C19). ESI-MS: for C_38_H_56_N_2_O_10_Pt: m/z calculated for [M − H]^−^ 894, found 894.

(OC-6-33)-(trans-1R,2R-Diaminocyclohexane)-bis(3,5-di-tert-butylbenzoate)oxalatoplatinum(IV) (2d)

To a solution of 3,5-di-tert-butylbenzoic acid (272 mg, 1.16 mmol) in dichloromethane (25 mL), oxalyl chloride (996 μL, 11.6 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred and boiled for 2 h. The solvent and the excess of oxalyl chloride were removed on a rotary evaporator at reduced pressure. The resulting chloroanhydride was used without further purification. To a suspension of (OC-6-33)-(trans-1R,2R-diaminocyclohexane)dihydroxido(oxalato)platinum(IV) (100 mg, 0.232 mmol) in acetone (100 mL), pyridine (187 μL, 2.32 mmol) and a pre-mixed solution of the chloranhydride in acetone (2 mL) were added. The mixture was stirred for 24 h at room temperature without access to light. The reaction mixture was evaporated to a minimum volume. The product was precipitated by diethyl ether and washed with diethyl ether (3 × 10 mL). The obtained white crystalline product was dried in vacuo. The yield was 147 mg (73%). T_dec_ = 220–224 °C. Elemental analysis: for C_38_H_56_N_2_O_8_Pt calculated (%): C 52.83, H 6.53, N 3.24, found (%): C 51.91, H 7.13, N 3.39. ^1^H NMR (400.13 MHz, [d6]-DMSO, δ ppm): 8.53 (br. s, 2H; NH_2_), 8.28 (br. s, 2H; NH_2_), 7.71 (s, 4H; H6, H6′, H8, H8′), 7.57 (s, 2H; H14), 2.84 (br. s, 2H; H16, H21), 2.19 (d, 2H, J = 12.2 Hz; H17, H20), 1.57 (m, 4H; H17, H18, H19, H20), 1.28 (s, 36H; H1, H1′, H2, H2′, H3, H3′, H11, H11′, H12, H12′, H13, H13′), 1.13 (m, 2H; H18, H19). ^13^C [^1^H] NMR (100.61 MHz, [d6]-DMSO, δ ppm): 173.23 (C15), 164.08 (C22, C23), 150.18 (C14/C14′), 132.26 (C5/C5′, C9/C9′), 125.71 (C7/C7′), 123.53 (C6/C6′, C8/C8′), 61.19 (C16, C21), 34.61 (C4/C4′, C10/C10′), 31.17 (C1/C1′, C2/C2′, C3/C3′, C11/C11′, C12/C12′, C13/C13′), 30.99 (C17, C20), 23.87 (C18, C19). ESI-MS: for C_38_H_56_N_2_O_8_Pt: m/z calculated for [M + Na]^+^ 886, found 886.

(OC-6-44)-acetato(trans-1R,2R-Diaminocyclohexane)(3,5-di-tert-butyl-4-hydroxyphenylpropionate)oxalatoplatinum(IV) (3a)

To a solution of 3,5-di-tert-butyl-4-hydroxyphenylpropionic acid (147 mg, 0.529 mmol) in dichloromethane (20 mL), oxalyl chloride (454 μL, 5.25 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred and boiled for 2 h. The solvent and the excess of oxalyl chloride were removed on a rotary evaporator at reduced pressure. The resulting chloroanhydride was used without further purification. To a suspension of (OC-6-44)-acetato(trans-1R,2R-diaminocyclohexane)hydroxido(oxalato)platinum(IV) (100 mg, 0.211 mmol) in acetone (100 mL), pyridine (85 μL, 1.06 mmol) and a pre-mixed solution of the chloranhydride in acetone (2 mL) were added. The mixture was stirred for 24 h at room temperature without access to light. The product was purified by the column chromatography (silica gel, eluent—acetone:hexane (3:2), R_f_ = 0.63). The obtained pale-yellow crystalline product was dried in vacuo. The yield was 65 mg (42%). T_dec_ = 187–191 °C. Elemental analysis: for C_27_H_42_N_2_O_9_Pt calculated (%): C 44.20, H 5.77, N 3.82, found (%): C 44.04, H 5.71, N 3.85. ^1^H NMR (400.13 MHz, [d6]-DMSO, δ ppm): 8.36 (br. s, 2H; NH_2_), 8.27 (br. s, 2H; NH_2_), 6.88 (s, 2H; H6, H8), 7.57 (s, 1H; OH), 2.64 (br. s, 2H; H16), 2.55–2.47 (m, 4H; H15, H18, H23), 2.07 (m, 2H; H19, H22), 1.94 (s, 3H; H27), 1.45 (d, 2H, J = 12.4 Hz; H19, H22), 1.34 (s, 20H; H1, H1′, H2, H2′, H3, H3′, H11, H11′, H12, H12′, H13, H13′, H20, H21), 1.08 (t, 2H, J = 7.0 Hz; H20, H21). ^13^C[^1^H] NMR (100.61 MHz, [d6]-DMSO, δ ppm): 180.25 (C17), 178.53 (C26), 163.46 (C24, C25), 151.97 (C14), 139.18 (C5, C9), 131.82 (C7), 124.17 (C6, C8), 61.00 (C18, C23), 37.89 (C16), 34.48 (C4, C10), 31.28 (C15), 30.91 (C19, C22), 30.43 (C1, C2, C3, C11, C12, C13), 23.49 (C20,C21), 23.03 (C27). ESI-MS: for C_27_H_42_N_2_O_9_Pt: m/z calculated for [M + Na]^+^ 756, found 756.

(OC-6-44)-acetato(trans-1R,2R-Diaminocyclohexane)(3,5-di-tert-butyl-4-hydroxyphenylacetate)oxalatoplatinum(IV) (3b)

To a solution of 3,5-di-tert-butyl-4-hydroxyphenylacetic acid (140 mg, 0.53 mmol) in dichloromethane (20 mL), oxalyl chloride (454 μL, 5.3 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred and boiled for 2 h. The solvent and the excess of oxalyl chloride were removed on a rotary evaporator at reduced pressure. The resulting chloroanhydride was used without further purification. To a suspension of (OC-6-44)-acetato(trans-1R,2R-diaminocyclohexane)hydroxido(oxalato)platinum(IV) (100 mg, 0.21 mmol) in acetone (100 mL), pyridine (85 μL, 1.06 mmol) and a pre-mixed solution of the chloranhydride in acetone (2 mL) were added. The mixture was stirred for 24 h at room temperature without access to light. The product was purified by the column chromatography (silica gel, eluent—acetone:hexane (3:2), R_f_ = 0.59). The obtained pale-yellow crystalline product was dried in vacuo. The yield was 52 mg (34%). T_dec_ = 231–234 °C. Elemental analysis: for C_26_H_40_N_2_O_9_Pt × 0.5 CH_2_Cl_2_ calculated (%): C 41.76, H 5.42, N 3.68, found (%): C 41.82, H 5.24, N 3.72. ^1^H NMR (400.13 MHz, [d6]-DMSO, δ ppm): 8.37 (br. s, 2H; NH_2_), 8.22 (br. s, 2H; NH_2_), 6.92 (s, 2H; H6, H8), 6.77 (s, 1H; OH), 3.48–3.38 (m, 2H; H15), 2.54 (m, 2H; H17, H22), 2.04 (m, 2H; H18, H22), 1.94 (s, 3H; H26), 1.43 (m, 2H; H18, H21) 1.34 (s, 20H; H1, H2, H3, H11, H12, H13, H18, H21), 1.09 (m, 2H; H19, H20). ^13^C [^1^H] NMR (100.61 MHz, [d6]-DMSO, δ ppm): 179.61 (C16), 178.59 (C25), 163.31 (C23, C24), 152.48 (C14), 138.91 (C5, C9), 126.65 (C7), 125.39 (C6, C8), 60.82 (C17, C22), 42.80 (C15), 39.51 (C18, C21), 34.44 (C4, C10), 31.02, 30.34 (C1, C2, C3, C11, C12, C13), 23.48 (C19, C20), 23.02 (C26). ESI-MS: for C_26_H_40_N_2_O_9_Pt: m/z calculated for [M − H]^−^ 718, found 718.

(OC-6-44)-acetato(trans-1R,2R-Diaminocyclohexane)(3,5-di-tert-butyl-4-hydroxybenzoate)oxalatoplatinum(IV) (3c)

To a solution of 3,5-di-tert-butyl-4-hydroxyphenylbenzoic acid (132 mg, 0.53 mmol) in dichloromethane (20 mL), oxalyl chloride (454 μL, 5.3 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred and boiled for 2 h. The solvent and the excess of oxalyl chloride were removed on a rotary evaporator at reduced pressure. The resulting chloroanhydride was used without further purification. To a suspension of (OC-6-44)-acetato(trans-1R,2R-diaminocyclohexane)hydroxido(oxalato)platinum(IV) (100 mg, 0.21 mmol) in acetone (100 mL), pyridine (85 μL, 1.1 mmol) and a pre-mixed solution of the chloranhydride in acetone (2 mL) were added. The mixture was stirred for 24 h at room temperature without access to light. The product was purified by the column chromatography (silica gel, eluent—acetone:hexane (3:2), R_f_ = 0.59). The obtained pale-yellow crystalline product was dried in vacuo. The yield was 33 mg (22%). T_dec_ = 194–197 °C. Elemental analysis: for C_25_H_38_N_2_O_9_Pt calculated (%): C 42.55, H 5.43, N 3.97, found (%): C 41.53, H 5.35, N 3.80. ^1^H NMR (400.13 MHz, [d6]-DMSO, δ ppm): 8.44 (br. s, 2H; NH_2_), 8.32 (br. s, 2H; NH_2_), 7.65 (s, 2H; H6, H8), 7.52 (s, 1H; OH), 2.71 (d, 2H, J = 32.2; H16, H21), 2.14 (d, 2H, J = 12.6 Hz; H18, H20), 1.97 (s, 3H; H25), 1.50 (m, 4H; H17, H18, H19, H20), 1.35 (s, 18H; H1, H2, H3, H11, H12, H13), 1.15 (m, 2H; H18, H19). ^13^C [^1^H] NMR (100.61 MHz, [d6]-DMSO, δ ppm): 178.66 (C24), 173.35 (C15), 163.79 (C22, C23), 157.60 (C14), 137.75 (C5, C9), 126.49 (C7), 123.56 (C6, C8), 61.05 (C16, C21), 34.48 (C4, C10), 31.08 (C17, C20), 30.09 (C1, C2, C3, C11, C12, C13), 23.58 (C18, C19), 23.04 (C25). ESI-MS: for C_25_H_38_N_2_O_9_Pt: m/z calculated for [M − H]^−^ 704, found 704.

(OC-6-44)-acetato(trans-1R,2R-Diaminocyclohexane)(3,5-di-tert-butylbenzoate)oxalatoplatinum(IV) (3d)

To a solution of 3,5-di-tert-butylbenzoic acid (124 mg, 0.53 mmol) in dichloromethane (25 mL), oxalyl chloride (454 μL, 5.29 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred and boiled for 2 h. The solvent and the excess of oxalyl chloride were removed on a rotary evaporator at reduced pressure. The resulting chloroanhydride was used without further purification. To a suspension of (OC-6-44)-acetato(trans-1R,2R-diaminocyclohexane)hydroxido(oxalato)platinum(IV) (100 mg, 0.21 mmol) in acetone (100 mL), pyridine (85 μL, 1.06 mmol) and a pre-mixed solution of the chloranhydride in acetone (2 mL) were added. The mixture was stirred for 24 h at room temperature without access to light. The reaction mixture was evaporated to a minimum volume. The product was precipitated by diethyl ether and washed with diethyl ether (3 × 10 mL). The obtained white crystalline product was dried in vacuo. The yield was 55 mg (37%). T_dec_ = 235–238 °C. Elemental analysis: for C_25_H_38_N_2_O_8_Pt × 1.7 C_3_H_6_O calculated (%): C 45.86, H 6.16, N 3.55, found (%): C 46.42, H 5.55, N 3.83. ^1^H NMR (400.13 MHz, [d6]-DMSO, δ ppm): 8.45 (br. s, 2H; NH_2_), 8.22 (br. s, 2H; NH_2_), 7.67 (s, 2H; H6, H8), 7.55 (s, 1H; H14), 2.70 (d, 2H, J = 30,3 Hz; H16, H21), 2.13 (br. s, 2H; H17, H20), 1.98 (s, 3H; H25), 1.51 (m, 4H; H17, H18, H19, H20), 1.27 (s, 18H; H1, H2, H3, H11, H12, H13), 0.84 (m, 2H; H18, H19). ^13^C [^1^H] NMR (100.61 MHz, [d6]-DMSO, δ ppm): 178.63 (C24), 173.05 (C15), 163.85 (C22, C23), 150.15 (C14), 132.31 (C5, C9), 125.65 (C7), 123.47 (C6, C8), 61.07 (C16, C21), 34.59 (C4, C10), 31.16 (C1, C2, C3, C11, C12, C13), 29.59 (C17, C20), 23.59 (C18, C19), 23.02 (C25). ESI-MS: for C_25_H_38_N_2_O_8_Pt: m/z calculated for [M + H]^+^ 690, found 690.

3.1. Antioxidant Activity Assay

The antioxidant activity was studied by DPPH and CUPRAC assays as published previously [27]. The absorbance was measured on a “Feyond-A400” microplate reader (Allsheng, Hangzhou, China) against a reagent blank. The measurements were carried out at 37 °C for 3–4 h and 100 min for DPPH and CUPRAC assays, respectively.

3.2. Antiglycating Activity Assay

The model for studying the effects of antiglycation was based on the process of glycation of bovine serum albumin (BSA) with glucose and methylglyoxal. The reaction mixture consisted of glucose (0.36 M) or methylglyoxal (0.006 M) and BSA (1 mg/mL) dissolved in phosphate buffer solution (PBS, pH 7.4, 0.05 M). The substances under investigation were dissolved in 99% dimethyl sulfoxide (DMSO). The final concentration of all substances in the reaction mixture was 100 μM. An equivalent volume of solvent was added to the control samples. The samples were incubated at 60 °C for 24 h. After incubation, the protein was precipitated with trichloroacetic acid and centrifuged (15,000 rpm, 4 min, 4 °C). The supernatant was discarded, and the protein precipitate was washed with phosphate buffer solution. The precipitate was then dissolved in phosphate buffer solution (pH 10.5). The fluorescence of AGEs was quantified in samples at excitation wavelengths of λex 370 nm and emission wavelengths of λ 440 nm using a spectrofluorimeter M 200 PRO (TECAN). Logarithmic normalization, as delineated in Formula (1), was employed to mitigate false positives potentially arising from compounds that suppress the fluorescence of AGEs, independent of the inhibition of AGEs formation:

$$[eqn]$$

where Flu(lg) denotes the normalized fluorescence intensity of AGEs, while lg(Exp) and lg(Blank) represent the decimal logarithms of the actual fluorescence levels of glycated and corresponding unglycated samples, respectively.

The activity of compounds that neither inhibited the intrinsic fluorescence of AGEs nor exhibited fluorescence at the specified wavelengths was quantified using Formula (2).

$$[eqn]$$

where Flu(lin) represents the fluorescence intensity of AGEs, while Exp and Blank denote the actual fluorescence levels of glycated and corresponding unglycated samples, respectively.

The activity, articulated as the percentage suppression of AGEs fluorescence, was calculated using Formula (3).

$$[eqn]$$

where Flu(Exp) and Flu(Contr) are the fluorescence intensity of AGEs in experimental and control samples, respectively (lg-normalized or non-lg-normalized).

3.3. Cells and Cell Death Assays

The MCF7, A549, HCT116, A2780 and A2780Cis cell lines were obtained from the European collection of authenticated cell cultures (ECACC; Salisbury, UK). Cells were cultured in Dulbecco modified Eagle’s medium (DMEM; Gibco, Paisley, UK) or Roswell Park Memorial Institute medium (RPMI-1640, Servicebio, Wuhan, China) with 10% fetal bovine serum (Gibco, Brazil) and antibiotics (PanEco, Moscow, Russia) in 5% CO_2_, 37 °C. The cytotoxic activity was studied by MTT assays as published previously [28]. Primary rat dermal fibroblasts (RDF) were isolated from rat skin explants. We used white outbred rats aged 24 days, weighing 25 g. Under anesthesia (Nembutal), a 5 mm^2^ flap of skin was isolated, disinfected, and subjected to enzymatic treatment and mechanical separation of the epidermis. The donor animal’s wound was sutured, and complete rehabilitation was carried out. The use of animals was approved by the ethical committee No. IRB 00005839 IORG 0004900 (OHRP) protocol 2021/056 15 June 2021. Explants were placed on a Petri dish to initiate cell migration and growth in complete growth DMEM (90% DMEM with glucose 4.5 g/L, 10% FBS, L-glutamine 0.002 M, PenStrep 50,000 ED, 1% sodium pyruvate, 1% essential amino acids). After a week of incubation, fibroblasts that migrated from the explant were removed with a trypsin solution and used for MTT assay.

For the flow cytometry studies, cells were plated into 6-well plates (Eppendorf, Hamburg, Germany; HCT-116 cells, 4 × 10^5^ cells in 2 mL of DMEM) and incubated for 24 h. Solutions of complexes in DMSO were prepared immediately prior to the day of the experiments. A cisplatin solution was prepared in DMEM without the addition of DMSO. Cells were treated with compounds concentrations corresponding to twofold IC_50_ values based on MTT assays. Cells were incubated for 72 h, collected, washed with PBS, and resuspended in DMEM. Aliquots of cells were processed as recommended in the Muse Annexin V&Dead Cell Kit. Measurements were carried out on a Muse Cell Analyser, Luminex Corp., Austin, TX, USA according to the manufacturer protocol.

3.4. Study of the Effect of Compounds on LP in Rat Brain Homogenate

The antioxidant properties of the synthesized conjugates were evaluated by their ability to inhibit lipid peroxidation in the crude membrane fraction (1500 g) of the rat brain homogenate [29]. Experiments were performed using outbred male rats, 200–220 g. The animals were housed under standard vivarium conditions in a normal day/night cycle and had libitum access to water and food. All animal manipulations were approved by the local bioethics committee of the Institute of Physiologically Active Compounds at Federal Research Center of Problems of Chemical Physics and Medicinal Chemistry (Protocol No. 72 dated 24 April 2023). These experiments were carried out using equipment of the Center for Collective Use of the Institute of Physiologically Active Compounds, Russian Academy of Sciences within the framework of state assignment (subject No. FFSN-2021-0005).

The brain homogenate in the presence of the test compound or an equal volume of solvent (DMSO) was incubated for 1 h in the presence of 10 mM H_2_O_2_ or 0.5 mM FeNH_4_ (SO_4_)2 × 12H_2_O. The degree of LP was assessed by the formation of trimetine complexes of secondary LP products with 2-thiobarbituric acid (TBARs). The data were normalized to control and IC_50_ were calculated with nonlinear regression fit (GraphPad Prism v8.0). Results are presented as the mean ± SD from three independent experiments.

3.5. Log P Determination [24,25]

Log P values of the new compounds were determined by the HPLC method using a Spursil 5 µm C18 column 150 × 4.6 mm using two mobile phases: phase A was 20 mM MOPS, 0.15% decylamine, pH = 7.4; phase B was 0.25% 1-octanol in methanol. Briefly, samples dissolved in methanol with uracil as an internal standard were injected into the column and eluted with mobile phase B between 70%, 80%, and 90% (or 80, 85, and 90%). The log P values were calculated using aniline, nitrobenzene, methyl benzoate, butanone-2, benzonitrile, naphthalene, and phenanthrene as standards. These experiments were repeated three times for each of the compounds.

4. Conclusions

Developing platinum(IV) complexes provides a significant advantage over classical platinum(II)-based drugs, as enhanced stability in the bloodstream reduces the risk of systemic toxicity. The incorporation of phenolic antioxidant fragments into the axial positions of platinum(IV) scaffolds was investigated as a structural strategy to modulate the redox properties and cellular responses of the resulting complexes after reductive activation. The results obtained indicate that the presence of antioxidant moieties can influence the biological activity of platinum(IV) compounds in vitro and may contribute to reduced cytotoxic effects toward a non-malignant cell line (fibroblast) under the tested conditions. In addition, the modification of the platinum(IV) core with organic ligands leads to the development of complexes that exhibit enhanced anticancer activity compared to cisplatin in selected tumor cell lines. Among the synthesized compounds, complex 2a showed pronounced cytotoxic activity toward cancer cells, while displaying lower cytotoxicity toward non-malignant cells in MTT assays. Despite these observations being limited to in vitro models and not allowing conclusions regarding systemic toxicity, these findings support the potential of axial ligand functionalization as a design strategy for tuning the biological properties of platinum(IV) complexes and provide a basis for further in vivo studies.