Heavy‐Atom‐Free Dual‐Action Dual PACT/PDT action Pt(IV) Prodrug With BODIPY Dimer in Axial Position

Abstract

Photodynamic therapy (PDT) is a clinically approved therapeutic modality with great potential for the treatment of cancers due to its excellent spatiotemporal selectivity and noninvasivenes. A combination of light‐controlled chemotherapy (PACT) and PDT in one molecule has the potential to overcome crucial drawbacks of both Pt‐based chemotherapy and PDT via a synergetic effect. Herein, we report a Pt(IV)‐BODIPY agent for dual light‐controlled chemophotodynamic therapy, a Pt(IV) prodrug with a BODIPY orthogonal dimer in the axial position. The orthogonal arrangement of the fluorophores results in a long‐lived, “heavy‐atom‐free” triplet state of the dimeric fluorophore. The high quantum yield of singlet oxygen for the dimer fluorophore and the Pt(IV) prodrug based on it was confirmed, and the formation of the triplet state was demonstrated by flash photolysis. The excellent photosensitive properties of the Pt(IV) prodrug on SK‐BR‐3 and MCF‐7 tumor cells demonstrate photocontrolled toxicity and potential for the development of dual therapy conjugates.

We report herein Pt(IV)‐BODIPY prodrug with PACT/PDT activity, which possesses the potential to overcome crucial drawbacks of both Pt‐based chemotherapy and PDT via a synergetic effect.

Abbreviations

BODIPY

boron dipyrromethene

DCC

N,N′‐dicyclohexylcarbodiimide

DCE

dichloroethane

DCM

dichloromethane

DDQ

2,3‐dichloride‐ 5,6‐dicyano‐p‐benzoquinone

DMSO

dimethyl sulfoxide

DMF

N,N‐dimethylformamide

DPBF

1,3‐Diphenylisobenzofuran

ESI

electrospray ionization

HRMS

high‐resolution mass spectrometry

HPLC

high‐performance liquid chromatography

IRF

instrument response function

MTT

3‐[4,5‐dimethylthiazol‐2yl]‐2,5‐diphenyl‐tetrazolium bromide

NMR

nuclear magnetic resonance

PACT

photoactivated chemotherapy

PDT

photodynamic therapy

PS

photosentitizer

ROS

reactive oxygen species

SOC

spin–orbit coupling

TLC

thin‐layer chromatography

TFA

trifluoroacetic acid

TLC

thin layer chromatography

TMS

tetramethylsilane

TCSPC

time‐correlated single photon counting

1. Introduction

Platinum(II) drugs are the cornerstones for chemotherapeutics of various neoplasms [1]. Platinum‐based anticancer drugs are effective in inducing apoptosis of tumor cells by damaging DNA [2]. However, long‐term Pt‐based chemotherapy usually leads to resistance via several mechanisms [3, 4]. Platinum(IV) complexes are considered as alternative to Pt(II) complexes, since they are less prone to side binding and also due to the possibility of regulating their properties, such as lipophilicity, hydrolysis rate, and redox potential, via the modification of hydroxido axial ligands [5, 6] Photoactive Pt(IV) prodrugs designed by conjugation of photoabsorbers to the axial positions of oxidized form of Pt(II) drugs possess significant advantages due to the ability of their activation on demand by applying light directly to tumor tissues, where light‐driven chemotherapeutic agent release occur [7, 9]. To wit, photoabsorber acts as photoremovable protecting group for Pt(II) drug, thereby implementing the photoactivated chemotherapy (PACT) approach. Although various photoactivatable Pt(IV) prodrugs have already been reported, research is still ongoing.

A combination of PACT and photodynamic therapy (PDT) is high relevant [10]. Integration of oxygen‐dependent PDT and oxygen‐independent PACT techniques may yield synergistic cytotoxic effects and significantly increase the effectiveness of therapy compared to using these two powerful approaches separately, since causing two fundamentally different therapeutic effects in a highly localized manner [11]. Recently, Mitchell et al. reported that PDT treatment is primarily associated with mitochondrial dysfunction, while PACT had little impact on cellular respiration [12]. Thus, PDT and PACT may enhance each other, and development of “two‐in‐one” molecules that combine the features of PDT with PACT is of great interest [13].

Boron dipyrromethene (BODIPY) dyes are widely‐used chromophore scaffolds due to their exceptional chemical versatility, which allows for accessibility, diversity, and photonic adaptability [14]. Also, BODIPY may act as excellent photosensitizers if they contain heavy halogen or sulfur atoms [15]. Furthermore, orthogonal BODIPY dimers are of high interest as effective heavy‐atom‐free photosensitizers [16, 18].

BODIPY‐caged metallodrugs that act as PACT and visualization agents are designed to combine therapeutic action with intrinsic fluorescence for real‐time bioimaging, as well as dual PACT/PDT action [19]. Despite the conjugation of BODIPY with Pt(IV) scaffold yielding a photoactive prodrug have repeatedly been reported [20, 22], only few of those prodrugs can behave as dual PACT/PDT agents. In 2022, Bera et al. reported a RED‐light activated cisplatin‐based Pt(IV)‐BODIPY prodrug which demonstrated a singlet oxygen quantum yield (ΦΔ) value of 0.28 [23]. In 2025, we reported a two green‐light activated cisplatin‐based Pt(IV) prodrugs with thienopyrrole‐based BODIPYs in axial position, which demonstrated a significant synglet oxygen quantum yields 0.53 and 0.56, and were stable and nontoxic in the dark [24]. Also in 2025, we reported a GreenPt, a cisplatin‐based Pt(IV)‐BODIPY brominated prodrug with dual PACT/PDT action, with an extremely high synglet oxygen quantum yield of 0.63 [25].

Herein, we proposed the design and investigation of photoactivable Pt(IV) prodrug with orthogonal BODIPY dimer in axial position in order to obtain an effective heavy‐atom‐free Pt(IV) prodrug with dual PACT and PDT action.

2. Results and Discussion

2.1. Design and Synthesis

β‐meso dimer 3 was obtained by sequential formylation of BODIPY 1 at position 3 and condensation of the resulting 2‐formyl‐BODIPY 2 with 2,4‐dimethylpyrrole, followed by complexation with boron trifluoride etherate. To conjugate a PDT‐active BODIPY with the Pt(IV) scaffold, Cu‐catalyzed click‐reaction was used, and Pt(IV) prodrug Pt‐4 was obtained with 33% yield (Figure 1). The structures of all compounds obtained were confirmed by ¹H, ¹³C, ¹⁹F, ¹⁹⁵Pt nuclear magnetic resonance (NMR), and high‐resolution mass spectra (HRMS) (Figures S1–S10). Purity of Pt(IV) prodrug Pt‐4 (>95%) was confirmed via high‐performance liquid chromatography (HPLC) (Figures S11–S14).

FIGURE 1.

Synthesis scheme for Pt(IV) prodrug Pt‐4 with β‐meso BODIPY dimer 3 moieties as an axial ligand.

2.2. Photophysical Properties

Absorption and emission spectra for the β‐meso dimer 3 and Pt(IV) prodrug Pt‐4 were studied in acetonitrile. Both compounds demonstrated absorption/emission maxima at 505 and 546 nm, which correlate well with previously reported data (Table 1, Figure S15A,B) [26]. Notably, the absorption of the dimer is slightly redshifted compared to the monomer BODIPY 1 (Figure S15C). Identical absorption and emission profile of the compounds indicates an almost complete absence of interference of the Pt(IV) scaffold on photophysical properties due to the absence of direct conjugation between Pt(IV) and BODIPY scaffolds. The absence of direct conjugation between the photoabsorber and the Pt(IV) scaffold, in turn, means that the prodrug will be stable in the absence of irradiation and will not exhibit phototoxicity in the dark.

TABLE 1.

Photophysical data of BODIPY dimer 3 Pt(IV) prodrug Pt‐4.

	Absorption/emission	Φ Δ (DPBFa/phosphorescenceb)	S1/T1 lifetime
BODIPY dimer 3	505/546 nm	0.65/0.15	3.8 ns/2.8 ms
Pt(IV) prodrug Pt‐4	505/546 nm	0.40/0.13	3.7•ns/2.7 ms

^a Measured in chloroform.

^b Measured in acetonitrile.

2.3. Study of Photodynamic Activity and Triplet States

To study the PDT activity of the obtained dimer 3 and Pt(IV) prodrug Pt‐4 based on it, we used singlet oxygen trap DPBF. Quantum yields of singlet oxygen (chloroform, 510 nm, H₂TPP‐standard, DPBF‐trap) for dimer 3 were of ˜0.65 and ˜0.40 for Pt(IV) prodrug Pt‐4 (Table 1, Figure S15D–F). The singlet oxygen‐generating ability has also been additionally verified by detecting singlet oxygen luminescence. Acetonitrile solutions of dimer 3 and Pt(IV) complex Pt‐4 were excited at 500 nm, TPP was employed as a standard. The estimated singlet oxygen generation efficiency determined by this method was 0.15 and 0.13 for 3 and Pt(IV) complex Pt‐4, respectively (Figure S15G). The difference in the singlet oxygen quantum yields determined by two independent methods may be due to different solvents used which could alter the intersystem crossing efficiency. At the same time chemical trap DPBF is not selective toward singlet oxygen and reacts with other reactive oxygen species (ROS); therefore, the obtained results on singlet oxygen may be overestimated [27].

Based on reduced singlet oxygen quantum yield of Pt(IV) prodrug Pt‐4 compared to dimer 3, it can be inferred that the conjugation of the Pt(IV) scaffold with the orthogonal dimer led to a decrease in the quantum yield of singlet oxygen due to the possible energy expenditure on transfer to the Pt(IV) center during photoreduction. However, Pt(IV) prodrug Pt‐4 possesses high quantum yield of singlet oxygen, which imparts to it the properties of a dual‐action prodrug.

The photoreduction of Pt(IV) prodrugs with BODIPY in the axial position can occur via phototransfer of an electron from the photoabsorber to the Pt(IV) center [27]. Upon transitioning to an excited state, BODIPY either accepts an electron from the external environment (from a reducing agent or solvent) and forms an anion‐radical BODIPY^.‐, which subsequently transfers an electron to Pt(IV) [28, 29], or BODIPY transfers its own electron to the Pt(IV) center via forming a cation‐radical BODIPY^.+. The decrease in the quantum yield of the singlet oxygen of Pt(IV) prodrug 4 compared to the dimer 3, along with the absence of fluorescence of both the dimer and the complex, may indirectly indicate energy transfer from the triplet state of the dimer to the Pt(IV) center.

2.4. Investigation of Excited States for Dimer 3 and Pt(IV) Prodrug Pt‐4

Since Pt(IV) prodrug Pt‐4 is the first example of a Pt(IV) complex with BODIPY dimer 3 as a light‐sensitive axial ligand, the lifetimes of its singlet and triplet excited states were of interest. Hence, the singlet excited state lifetimes for complex Pt‐4 and dimer 3 were studied in acetonitrile using time‐correlated single photon counting (TCSPC). Both dimer 3 and Pt(IV) prodrug Pt‐4 were demonstrated identical lifetimes of S₁ excited state 3.7 and 3.8, respectively (Table 1, Figure S16A). This indicates that no direct energy transfer occurs between S₁ excited state of axial BODIPY dimer moiety and Pt(IV) center.

Next, triplet state of the compounds was studied in degassed acetonitrile solutions using flash photolysis technique. After excitation at 480–550 nm, for both BODIPY 3 and complex Pt‐4 the characteristic triplet‐state absorption was observed at 400–450 nm and singlet state photobleaching at 460–570 nm. Thus, the kinetics of triplet state decay were tracked at 420 nm. Both dimer 3 and Pt(IV) prodrug Pt‐4 demonstrated long‐lived triplet excited state 2.8 and 2.7 ms, which is significantly higher than the values reported previously for this type of dimer (Figure S16B,C) [30]. Notably, identical triplet state lifetimes indicates that no energy or electron transfer occurs between the axial BODIPY moiety and Pt(IV) atom and that external electron source may need, which is in line with the previously published data [25, 31]. Overall, the data of triplet state decay for Pt(IV) complex Pt‐4 supports the outstanding singlet oxygen‐generating capabilities since long triplet state lifetime indicate that the energy transfer between the triplet excited state of Pt(IV) prodrug Pt‐4 and oxygen is likely to occur even in low oxygen level environment.

2.5. Light‐Induced Cisplatin Release From the Pt(IV) Prodrug

To confirm the ability of Pt(IV)‐BODIPY prodrug Pt‐4 to act as PACT agent, the light‐induced release of cisplatin from complex Pt‐4 was directly detected and quantitatively monitored by HPLC‐MS analysis. Photoreduction of complex Pt‐4 in MeOH:DMSO:H₂O = 6:3:1 solution under green light irradiation (530 nm, 5 mW/cm²) was monitored; an irradiation of solution with green light was accompanied by a time‐dependent increase in the cisplatin‐DMSO adduct concentration, directly indicating the light‐induced release of the active Pt(II) species from the Pt(IV) prodrug (Figure S15). In addition, photoreduction of complex Pt‐4 was studied in the presence of sodium ascorbate NaAsc (5 equiv.); expectedly, an acceleration of prodrug’s photoreduction and cisplatin accumulation was observed. Acceleration of photoreduction in the presence of sodium ascorbate indicates the formation of the BODIPY anion radical as a photoreduction intermediate. These data are in good agreement with those previously published for the photoreduction of Pt(IV) prodrugs [31, 33].

The photoreduction of the Pt‐4 complex was further studied using ¹H and ¹⁹⁵Pt NMR spectroscopy. A comparison of the ¹H NMR of the Pt‐4 complex before and after irradiation with green light reveals the disappearance of proton resonances of the ammine coordinated to the Pt(IV) center in the Pt‐4 complex at ˜6.5 ppm, as well as a change in chemical shifts of methylene protons of the triazole ring from 5.21 and 5.17 ppm to 5.16 and 5.06 ppm, and an increase in the integrated intensity of the signal at 1.90 ppm, which may indicate the decomposition of the Pt‐4 complex with the formation of acetic acid (Figure 2A,B).

FIGURE 2.

¹H NMR spectrum of Pt‐4 complex (A) before and (B) after LED irradiation (530 nm, 191 mW/cm², 8 h). (C) ¹⁹⁵Pt NMR spectrum of the Pt‐4 complex (C) before and (D) after LED irradiation (530 nm, 191 mW/cm², 8 h) in the range from 1300 to 3100 ppm. (E) ¹⁹⁵Pt NMR spectrum of Pt‐4 complex after LED irradiation (530 nm, 191 mW/cm², 8 h) in the range from −2850 to −3180 ppm.

Comparison of the ¹⁹⁵Pt NMR spectrum of the Pt‐4 complex before and after irradiation with green light reveals the disappearance of the Pt chemical shift at 1229 ppm, which is characteristic for diacetylated Pt(IV) complexes [34, 35], and the appearance of three new chemical shifts at –3030, –3050, and –3133 ppm, which are characteristic for Pt(II) complexes with Pt─S bond, which is likely due to ligand exchange with DMSO d₆ (Figure 2C–E).

The spin–spin coupling constant for the triplet at –3030 ppm is 230 Hz, characteristic of the interaction of ¹⁹⁵Pt ¹⁴N [36]. Taken together, these data indicate the reduction of the Pt‐4 complex with the formation of Pt(II) complexes.

2.6. Intracellular Localization

The intracellular localization of the dimer 3 and the Pt(IV) complex Pt‐4 was analyzed using confocal microscopy, and organelle‐specific fluorescent dyes were used as controls. Both dimer 3 and complex Pt‐4 were able to penetrate into cell membranes and to accumulate in cytoplasm (Figure 3). On the contrary, there was no accumulation of drugs in cell nucleuses that were stained with Hoechst 33342. It was found that there was partly colocalization with mitochondria labeled by MitoTracker Red, and Pearson`s correlation coefficients measured with Coloc‐2 plugin (Fiji software, USA) were evaluated as 0.8–0.9. This generally indicates clear colocalization; however, in the current case, there could be partial overlap of dimers and labels in the spectra. On the contrary, the Pearson`s correlation coefficients for colocalization of the studied compounds with lysosomes‐specific LysoTracker Red at the same conditions were lower (0.5–0.6), so the preferential accumulation in mitochondria in compression to lysosomes could be stated. Mitochondria are an attractive target for Pt‐BODIPY conjugates with dual PACT/PDT activity, since PACT action inside mitochondria can lead to the release of cisplatin and its interaction with mitochondrial DNA, which is an important target for Pt(IV) prodrugs [37], while PDT action inside mitochondria can lead to disruption of cellular respiration and cell death [38].

FIGURE 3.

Intracellular localization of ligand (β‐meso dimer 3) and complex (Pt(IV) prodrug 4) in human breast adenocarcinoma MCF‐7 cells, 10 µM, 1 h of incubation, in green. Cells were additionally stained with LysoTracker Red (left panel) and MitoTracker Red (right panel) for lysosomes and mitochondria, respectively. Scale bar is 20 µm.

2.7. Antiproliferative Properties In Vitro

Antiproliferative properties of the Pt(IV) prodrug Pt‐4 were investigated on human breast adenocarcinoma Sk‐Br‐3 and MCF‐7 cells, with cisplatin and dimer 3 used as positive controls. Both the Pt(IV) prodrug Pt‐4 and the dimer 3 are expected to act as phototherapeutic agents, with minimal toxicity in the absence of light and strong antiproliferative effects under visible light irradiation. Indeed, no effect on cell viability up to 20 μM was observed for BODIPY dimer 3 and the Pt(IV) prodrug Pt‐4 in the dark; notably, no cytotoxicity in the employed conditions was also observed for cisplatin (Figures S18 and S19, Table 2). Under the green light irradiation (530 nm, 1 J/cm²), both dimer 3 and Pt(IV) prodrug Pt‐4 demonstrated pronounced cytotoxicity with IC₅₀ values in the low micromolar range (Table 2). Considering high values of singlet oxygen quantum yields for dimer 3 and Pt(IV) prodrug Pt‐4 (0.65 and 0.40, respectively), the observed cytotoxicity of both compounds could be explained with light‐induced generation of cytotoxic ROS.

TABLE 2.

Antiproliferative activity of BODIPY dimer 3, Pt(IV) prodrug 4 and cisplatin in the dark or under 530 nm light (1 J/cm²) on breast adenocarcinoma Sk‐Br‐3 and MCF‐7 cell lines.

	IC50, µMa
	Sk‐Br‐3			MCF‐7
Compound	Dark	Light	PI	Dark	Light	PI
BODIPYdimer 3	>20	1.5	>13.1	>20	1.08	−>18.5
Pt(IV) prodrug 4	>20	3.2	>6.2	>20	2.67	>7.5
Cisplatin	>20	—	—	>20

^a PI, Phototoxicity index, IC₅₀ (dark)/IC₅₀ (530 nm light).

To confirm the high light response of dimer 3 and prodrug 4, and to compare their intracellular light responses, we also conducted a photoinduced toxicity study on MCF‐7 cells, reducing the light dose from 1.6 to 0.2 J/cm². According to confocal microscopy data, prodrug Pt‐4 accumulates less in cells than dimer 3. However, the cytotoxicity’s of ligand 3 and prodrug Pt‐4 were similar at doses of 2 and 10 μM, since prodrug Pt‐4 acts not solely through its PDT activity. But also, via release of cisplatin. This effect is clearly observed at high doses; thus, at a dose of 10 μM, the phototoxicity of dimer 3 does not change with a light doses of 0.6, 1, and 1.6 J/cm², while the toxicity of prodrug Pt‐4 continues to increase with an increase in the light dose, due to the release of cisplatin (Figure 4A).

FIGURE 4.

Photoinduced toxicity of Pt(IV) prodrug 4 and dimer 3 on (A) MCF‐7 cells and (B) cisplatin‐sensitive fibroblasts at different doses of green light irradiation.

It should be noted that the toxicity of dimer 3 is 2–3 times higher than that of Pt(IV) prodrug Pt‐4;, for example,, the IC₅₀ values for MCF‐7 cells were 1.08 ± 0.10 µM and 2.67 ± 0.99 µM, respectively. This could be explained with lower intracellular accumulation of Pt(IV) prodrug Pt‐4 (Figure 4). At the same time, the superior of Pt(IV) prodrug Pt‐4 in phototoxicity index demonstrates its potential in terms of safety/efficacy ratio due to its dual PACT/PDT action. To directly confirm a high dual chemo/PDT activity of Pt(IV) prodrug Pt‐4, a cisplatin‐sensitive fibroblasts [9] were studied under low‐dose irradiation, which is typical for deep biotissues. When using the cisplatin‐sensitive line, the toxicity of prodrug Pt‐4 to irradiation exceeds that of dimer 3, due to the fact that irradiation of prodrug 3 with even a low dose of green light leads not only to the generation of ROS within cells but also to the release of cisplatin (Figure 4B). Thus, both dimer 3 and prodrug Pt‐4 are potent PDT agents, but prodrug Pt‐4 also acts as a PACT agent. Also, the limitations of PDT agents should be considered; despite the ability of PDT agents to eradicate tumor cells, PDT should always be accompanied by systemic chemotherapy, which affects not only the tumor site but also metastases and free tumor cells in the bloodstream. For this reason, both PDT and photoactivated cisplatin release are required for tumor eradication, inhibiting metastasis and preventing tumor recurrence in vivo [31].

3. Conclusion

In conclusion, we have designed and synthesized a novel phototherapeutic agent comprising a Pt(IV) prodrug Pt‐4 conjugated to a BODIPY dimer 3 that acts as dual PACT/PDT agent/ Cisplatin‐based Pt(IV) prodrug Pt‐4 acts as a heavy‐atom‐free PDT photosensitizer with a high singlet oxygen quantum yield, as well as via light‐induced cisplatin release. By utilizing a heavy‐atom‐free BODIPY as an axial ligand, this approach eliminates potential side effects linked to halogenated PDT agents [39]. Investigation of excited singlet and triplet states revealed that the conjugation of BODIPY dimer with the Pt(IV) complex has no effect on the singlet and triplet excited state lifetimes, indicating the absence of direct energy transfer.

In antiproliferative assay, the complex showed no toxicity in the dark but exhibited strong phototoxicity upon low‐dose green light irradiation (530 nm, 1 J/cm²). Given its high fluorescence and lack of dark cytotoxicity, prodrug Pt‐4 also holds promise as a fluorescent imaging agent, enabling theranostic applications that combine therapy and diagnostics in a single molecule.

Overall, this study highlights the potential of designing Pt(IV) prodrugs with light‐activated antitumor activity without heavy atoms in the fluorophore. It paves the way for developing highly effective cancer therapies capable of targeting malignant tumors in vivo while minimizing toxic side effects.

4. Experimental Section

4.1. Materials and Methods

Acetic acid, 2‐azidoacetic acid, boron trifluoride etherate (BF₃·Et₂O), cisplatin, 2,3‐dichloride‐ 5,6‐dicyano‐p‐benzoquinone (DDQ), 2,4‐dimethyl‐1H‐pyrrole, N, N′‐dicyclohexylcarbodiimide (DCC), hydrogen peroxide (30% w/w in water), 4‐hydroxybenzaldehyde, propargyl bromide, potassium carbonate (K₂CO₃), sodium azide (NaN₃), sodium ascorbate, trifluoroacetic acid (TFA), triethylamine (Et₃N), 4‐Nitrobenzaldehyde, pentafluorobenzaldehyde were used without purification. Acetone, acetonitrile (CH₃CN), dichloromethane (DCM, CH₂Cl₂), diethyl ether (Et₂O), methanol (MeOH), dimethylformamide (DMF), acetone, Pluronic F127 (F127), DSPE‐PEG₂₀₀₀‐OMe, dimethyl sulfoxide (DMSO) and petroleum ether were purchased from commercial sources and purified following the described procedures [40].

[Pt(OAc)(2‐azidoacetate)(Cl₂(NH₃)₂)] was synthesized according to previous work [32].

Analytical thin‐layer chromatography (TLC) was performed on Merck silica gel aluminum plates with F‐254 indicator. Compounds were visualized by irradiation with UV light (254, 365 nm).

Preparative column chromatography was performed using Acros brand silica gel (60–200 mesh).

The NMR spectra were carried out on a Bruker–Avance 400 MHz and Agilent MR‐400 spectrometer in DMSO‐d6 and CDCl3 with TMS as an internal standard for ¹H and ¹³C and K2PtCl6 for ¹⁹⁵Pt NMR.

HRMS were recorded with a G3 QTof quadrupole‐time‐of‐flight (Waters, USA) equipped with an electrospray ionization source (ESI) (ionization: ESI, capillary temperature: 300 °C, voltage on the capillary: 5500 V for positive mode, −4500 V for negative mode, declastering potential: 90 V. Mass‐spectra were recorded in m/z range 150–3000, with an accumulation time 250 ms.

Purity of the compounds was assessed using HPLC‐DAD‐MS. HPLC‐DAD‐MS system consisting of a Vanquish liquid chromatograph (Thermo Fisher Scientific, USA) and an Orbitrap Fusion Lumos Tribrid (Thermo Fisher Scientific, USA) mass spectrometer was used. A Shim‐pack GIST C18‐Aq liquid chromatography reversed‐phase column (3 x 150 mm, 3 μm, Shimadzu, Japan) was used.

HPLC‐DAD‐MS system consisting of a Vanquish liquid chromatograph (Thermo Fisher Scientific, USA) and an Orbitrap Fusion Lumos Tribrid (Thermo Fisher Scientific, USA) mass spectrometer was used. A Shim‐pack GIST C18‐Aq liquid chromatography reversed‐phase column (3 × 150 mm, 3 μm, Shimadzu, Japan) was used.

HPLC‐HRMS analysis was performed with Vanquish liquid chromatograph (Thermo Fisher Scientific, USA) and a high‐resolution mass spectrometer based on the G3 QTof quadrupole‐time‐of‐flight (Waters, USA) equipped with an ESI was used. To separate the components of the analyzed solution, a Shim‐pack GIST C18‐Aq chromatographic column (3 x 150 mm, 3 μm, Shimadzu, Japan) filled with a reverse‐phase sorbent with polar‐endcapping was used. The column temperature was maintained at 35 °C throughout the analysis using a thermostat. A 0.1% aqueous solution of formic acid (A) and acetonitrile (B) were used as eluents. An isocratic elution mode was used with a ratio of eluents A and B of 15:85%. The flow rate of the mobile phase was 0.4 ml/min. The injected sample volume was 1.0 µl. The analysis time was 10 min. The ESI‐MS conditions on the Orbitrap Fusion Lumos Tribrid were as follows: mode for recording positively and negatively charged molecules; the capillary voltage of the ionization source was 3500 V for the positive mode and−2500 V for the negative mode; ion source chamber temperature ‐ 350 °C; ion transfer interface temperature ‐ 325 °C; gas pressure for solvent atomization in the ion source (nitrogen) ‐ 50, auxiliary gas pressure ‐ 10, curtain gas pressure ‐ 1. Scanning range m/z: 200–1700 Da. The resolution of the mass spectrometer for analysis is not less than 15 000. The mass spectrometer was calibrated immediately prior to sample analysis using CalMix PierceTM calibration mixtures (Thermo Scientific, USA). Data processing was performed using Xcalibur 4.6 software (Thermo Scientific, USA).

4.2. Absorption Spectroscopy

UV–vis absorption spectra were recorded on a Shimadzu UV‐3101PC spectrophotometer in the range from 300 to 800 nm in quartz cells with an optical pathlength of 1 cm, unless otherwise noted.

4.3. Fluorescence Spectroscopy

Fluorescence measurement experiments were performed with a FluoTime 300 fluorescence lifetime spectrometer (PicoQuant GmbH, Berlin, Germany). As a reference standard for fluorescence quantum yield determination (calculated according to the work [41]) Rhodamine 6G in ethanol (Φ_fl = 0.95) [42] was employed. Optical absorbance of samples and standards were matched (A < 0.1) at the excitation wavelength (λ _ex = 490 nm). Fluorescence lifetime measurements were made using picosecond timecorrelated single‐photon counting (TCSPC) and a pulsed laser light source. The instrument response function was recorded with the use of a Ludox probe. For singlet oxygen luminescence detection experiments, a spectrometer equipped with the NIR PMT Module H10330‐45 (Hamamatsu, Japan) coupled to the single‐photon counter TimeHarp TCSPC (PicoQuant Gmbh, Berlin, Germany) was used.

Singlet oxygen luminescence experiments were performed using a quartz cuvette with a 1 cm optical path. The excitation source used was a Xe lamp (500 nm). Tetraphenylporphyrin in acetonitrile was used as a reference standard (Φ_Δ = 0.60) [43].

4.4. Flash Photolysis

The transient absorption spectra of radical intermediates were measured using a conventional flash photolysis setup (LLC “MELZ”, Moscow, Russia), optical path length 20 cm, excitation (80 J/15 µs) performed through yellow– blue optical absorption filters (transmission 480–550 nm). Signals were recorded using a PMT‐38 photomultiplier (LLC “MELZ”, Moscow, Russia) at 400–650 nm.

4.5. Determination of Singlet Oxygen Quantum Yield Using DPBF

Quantum yields of singlet oxygen generation (Ф_Δ) were determined in accordance with comparative method described previously in the literature [44]. To the solutions of BODIPY’s and 5,10,15,20‐tetraphenylporphirine (H₂TPP, standard) in CHCl₃ (2 mL) with optical density at irradiation wavelength (515 nm) ca. 0.1 the singlet oxygen trap (DPBF) was added. These solutions were irradiated at 515 nm and DPBF degradation at 440 nm was monitored. During the irradiation the constant temperature (25 °C) and continuous stirring (2000 rpm) were provided. Singlet oxygen quantum yields (Ф_Δ) were calculated using the next equation

$${\mathrm{Ф}}_{\mathrm{\Delta }}={\mathrm{Ф}}_{\mathrm{\Delta }}^{\mathrm{st}}\times \frac{R_{\mathrm{s}}}{R_{\mathrm{st}}}\times \frac{1-{10}^{-A_{\mathrm{st}}}}{1-{10}^{-A_{\mathrm{s}}}}$$

where Ф_Δ is the quantum yields of singlet oxygen generation (for the H₂TPP Ф_Δ = 0.55 in CHCl₃); R is rate of photobleaching of trap under irradiation; A is optical density at 515 nm; indices s and st correspond to analyzed sample and standard, respectively.

4.6. Photoreduction Studies

All experiments were carried out at room temperature. Complex Pt‐4 was dissolved in a mixture of MeOH:DMSO:H₂O (6:3:1) to the resulting concentration of 10⁻³. During light activation, the Pt‐4 solution in a transparent vial (d = 1 cm) was placed in front of the light source (λ = 530 nm) so that irradiation power density was equal to 5 mW/cm².

In all experiments a 30 µl aliquot of the Pt(IV) prodrugs solution was taken and diluted 16 times with MeOH at the chosen periods of time. HPLC‐MS‐analysis of the probes was performed and repeated three times. Cisplatin amount in the probes was calculated from the peak area on the chromatogram. Cisplatin concentration was determined by integrating m/z 340.5–345.5 Da peak, which corresponds to Pt(NH₃)₂Cl(C₂H₆SO) ion.

4.7. Intracellular Localization Study

Human breast adenocarcinoma cells were seeded onto 8‐well glass chamber slide (10⁵ cells per well) and incubated overnight. Then, 10 µM of ligand 3 or 10 µM of complex 4 were added to the wells in full DMEM. After 1 h of incubation the wells were washed twice with PBS and stained with 100 nM of MitoTracker Red for 30 min according to the manufacturer’s protocol. Finally, the cells were washed with PBS and fixed in 4% paraformaldehyde solution. The cell nucleuses were additionally stained with Hoechst 33342 (1 µg/ml, 15 min). The intracellular fluorescence was evaluated; the images were analyzed in ImageJ software.

4.8. MTT Assay

Human breast adenocarcinoma Sk‐Br‐3/MCF‐7 cells, or hTERT‐immortalized foreskin fibroblast BJ‐5ta cells, were seeded in 96‐well plates (10⁴ cells per well) in complete DMEM medium. The following day, the medium in some wells was replaced with 100 μl of fresh medium with 3 and Pt‐4 (including control points and negative control). The cells were incubated with nanoparticles for 1 h, after which they were irradiated at a wavelength of 530 nm for 4 min 20 s (for 1 J, or less, for other light doses); nonirradiated cells served as a control. Cell viability was assessed after 72 h using the MTT assay. For this, an MTT solution (0.5 mg/ml) was added to each well. After 2 h, the medium was removed and 100 μl of DMSO (99%) was added. Optical density was recorded at 565 nm on an Infinite M Nano reader (Tecan, Switzerland). The IC₅₀ values were determined using GraphPad Prism 10.3.0 software.

4.9. Synthetic Procedures

5,5‐difluoro‐1,3,7,9‐tetramethyl‐10‐(4‐(prop‐2‐yn‐1‐yloxy) phenyl)‐5H‐4λ ⁴,5λ ⁴‐dipyrrolo[1,2‐c:2′, 1′‐f [1–3] diazaborinine 1 was synthesized according to previous reports [32]. To a solution of 0.704 g (4.4 mmol, 1 equiv.) of 4‐(prop‐2‐yn‐1‐yloxy)benzaldehyde and 1 ml (9.7 mmol, 2.2 equiv.) of 2,4‐dimethylpyrrole in 72 ml of THF was added 25 μl of TFA under an argon atmosphere. The mixture was stirred at r.t. overnight in an argon atmosphere, and the solution of 1.1 g (4.9 mmol, 1.1 equiv.) of DDQ in 48 ml of THF was added. The resulting mixture was stirred for another 5 h. Then, the reaction mixture was cooled with an ice‐water bath, and 9.5 ml of Et₃N and 14.3 ml of BF₃·Et₂O were added dropwise successively. The resulting mixture was kept stirring at r.t. overnight, then it was filtered through a band of silica gel. The precipitate was washed with CH₂Cl₂ and the combined filtrate was evaporated under reduced pressure. The residue was redissolved in CH₂Cl₂ and the solution was washed with 15% aqueous NaHCO₃ solution and with water. The organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The crude product was purified by flash chromatography using CH₂Cl₂ as eluent. BODIPY 1 was obtained as an orange powder. Yield: 1.02 g (61%).

¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.20 (d, J = 8.7 Hz, 2H, H = 3,4), 7.09 (d, J = 8.6 Hz, 2H, H = 1,2), 5.98 (s, 2H, H = 2,6‐BP), 4.76 (d, J = 2.4 Hz, 2H, H = CH₂), 2.55 (m, 7H, H = 3,5‐Me, CH), 1.42 (s, 6H, H = 1,7‐Me).

5,5‐difluoro‐1,3,7,9‐tetramethyl‐10‐(4‐(prop‐2‐yn‐1‐yloxy)phenyl)‐5H‐4λ ⁴,5λ ⁴‐dipyrrolo[1,2‐c:2′, 1′‐f [1–3] diazaborinine‐2‐carbaldehyde 2. BODIPY 2 was synthesized according to previous reports [45] with slight modifications. To a flask was added dry DMF (3 mL) in an ice bath under N₂ protection firstly POCl₃ (1.8 mL) was added to the mixture and stirred at r.t. for 30 min for preparation of Vilsmeier reagent. Then a DCE solution containing 1 (300 mg, 0.79 mmol) was added to Vilsmeier reagent, which were stirred at r.t. overnight. The reaction mixture was added to saturated Na₂CO₃ and stirred for 3 h and then extracted with DCM, washed water and dried over anhydrous Na₂SO₄. The crude product was purified by flash‐chromatography (CH₂Cl₂). BODIPY 2 was obtained as a red powder. Yield: 0.3 g (93 %).

1H NMR (400 MHz, CDCl₃, δ, ppm): 10.02 (s, 1H, H = CHO), 7.23 – 7.18 (m, 2H, H = 3,4), 7.16 – 7.11 (m, 2H, H = 1,2), 6.15 (s, 2H, H = 6‐BP), 4.78 (d, J = 2.4 Hz, 2H, H = CH₂), 2.82 (s, 3H, H = 3‐Me), 2.61 (s, 3H, H = 5‐Me), 2.56 (t, J = 2.4 Hz, 1H, H = CH), 1.71 (s, 3H, H = 1‐Me), 1.48 (s, 3H, H = 7‐Me).

¹³C NMR (101 MHz, CDCl₃, δ, ppm): 185.52, 162.11, 161.15, 158.04, 155.97, 146.90, 142.97, 142.41, 134.00, 129.65, 128.61, 126.59, 125.79, 123.58, 115.55, 55.62, 36.07, 30.99, 14.65, 12.58, 11.33.

HRMS: calc. for C₂₃H₂₂BF₂N₂O₂ ⁺ (2+H)⁺, 407,1742; found C₂₃H₂₂BF₂N₂O₂ ⁺ (2+H)⁺, 407,1739.

5,5,5′, 5′‐tetrafluoro‐1,1′, 3,3′, 7,7′, 9,9′‐octamethyl‐10‐(4‐(prop‐2‐yn‐1‐yloxy)phenyl)‐5H,5′H‐4λ4,4′λ4,5λ4,5′λ4‐2,10′‐bidipyrrolo[1,2‐c:2′, 1′‐f [1–3] diazaborinine 3. Dimer BODIPY 3 was synthesized according to previous reports [46] with slight modifications. To a solution of 0.363 g (0.89 mmol, 1 equiv.) of BODIPY 2 and 0.183 ml (1.79 mmol, 2 equiv.) of 2,4‐dimethylpyrrole in 81 ml of DCM was added 20 μl of TFA under an argon atmosphere. The mixture was stirred at r.t. overnight in an argon atmosphere, and the solution of 0.202 g (0.89 mmol, 1.1 equiv.) of DDQ was added. The resulting mixture was stirred for another 5 h. Then, the reaction mixture was cooled with an ice‐water bath, and 3.25 ml of Et₃N and 3.25 ml of BF₃·Et₂O were added dropwise successively. The resulting mixture was kept stirring at r.t. overnight, then it was washed with saturated Na₂CO₃ and water. The organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (CH₂Cl₂ : petroleum ether = 1:1). BODIPY 3 was recrystallized from Et₂O and obtained as an orange‐red powder. Yield: 0.43 g (58%).

¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.21 (d, J = 8.5 Hz, 2H, H = 3,4), 7.10 (d, J = 8.4 Hz, 2H, H = 1,2), 6.07 (s, 1H, H = 6‐BP), 5.98 (s, 2H, H = 2′, 6′‐BP), 4.75 (d, J = 2.4 Hz, 2H, H = CH₂), 2.59 (s, 3H, H = 5‐Me), 2.53 (s, 7H, H = 3′, 5′‐Me, CH), 2.41 (s, 3H, H = 3‐Me), 1.70 (s, 6H, H = 1′, 7′‐Me), 1.47 (s, 3H, H = 7‐Me), 1.25 (s, 3H, H = 1‐Me).

¹³C NMR (101 MHz, CDCl₃, δ, ppm): 158.72, 158.47, 155.83, 150.59, 145.63, 142.56, 142.40, 138.39, 133.85, 132.91, 131.97, 131.41, 129.22, 127.59, 125.74, 122.57, 121.33, 116.10, 78.00, 77.36, 76.15, 65.98, 56.17, 15.40, 14.93, 14.75, 14.06, 12.84, 12.41.

¹⁹F NMR (376 MHz, CDCl₃, δ, ppm): ‐145.82 – ‐146.46 (m).

HRMS: calc. for C₃₅H₃₅B₂F₄N₄O⁺ (3+H)⁺, 625.2933; found C₃₅H₃₅B₂F₄N₄O⁺ (3+H)⁺, 625.2934.

Dimer BODIPY‐Pt(IV) complex Pt‐4. Dimer BODIPY 3 (72 mg, 0.12 mmol) was dissolved in 4.3 ml of DMF, and Cu(CH₃CN)₄•BF₄ (8.8 mg, 28 μmol), TBTA (15 mg, 28 μmol) were added under argon atmosphere. The reaction mixture was stirred for 15 min at r.t., complex [Pt(OAc)(2‐azidoacetate)(Cl₂(NH₃)₂)] (43 mg, 0.094 mmol) was added, and the solution was stirred for another 3 h at r.t. The solvent was evaporated under reduced pressure. The residue was purified by column chromatography (CH₂Cl₂:MeOH = 10:1). Complex Pt‐4 was obtained as an orange powder. Yield: 33 mg (33 %).

¹H NMR (400 MHz, DMSO‐d₆, δ, ppm): 8.16 (s, 1H, H = CH), 7.37 (d, J = 8.6 Hz, 2H, H = 3,4), 7.24 (d, J = 8.5 Hz, 2H, H = 1,2), 6.51 (s, 6H, H = NH₃), 6.34 (s, 1H, H = 6‐BP), 6.21 (s, 2H, H = 2′, 6′‐BP), 5.22 (s, 2H, H = OCH₂), 5.19 (s, 2H, H = CH₂), 2.50 (s, H = DMSO + 5‐Me), 2.43 (s, 6H, H = 3′, 5′‐Me), 2.27 (s, 3H, H = 3‐Me), 1.92 (s, 3H, H = CH₃), 1.67 (s, 6H, H = 1′, 7′‐Me), 1.45 (s, 3H, H = 7‐Me), 1.23 (s, 3H, H = 1‐Me).

¹³C NMR (101 MHz, CDCl₃, δ, ppm): 178.07, 173.31, 159.00, 158.40, 155.34, 148.77, 145.50, 142.86, 142.13, 137.38, 133.29, 132.30, 131.07, 130.69, 129.22, 128.72, 127.71, 126.11, 124.56, 122.72, 121.53, 115.56, 79.17, 61.23, 52.71, 50.54, 22.63, 14.45, 14.29, 13.40, 12.32, 11.89.

¹⁹F NMR (376 MHz, DMSO‐d₆, δ, ppm): ‐143.22 – ‐143.73 (m).

¹⁹⁵Pt NMR (86 MHz, DMSO‐d₆, δ, ppm): 1229.57.

HRMS: calc. for C₃₉H₄₆B₂Cl₂F₄N₉O₅Pt⁺ (Pt‐4+H)⁺, 1084.2771; found C₃₉H₄₆B₂Cl₂F₄N₉O₅Pt⁺ (Pt‐4+H)⁺, 1084.2776.

Funding

This work was supported by the Russian Science Foundation, Grant No. 22‐15‐00182‐П.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.