The First Selenoanhydride in the Series of Chlorophyll a Derivatives, Its Stability and Photoinduced Cytotoxicity

In this work, we obtained the first selenium-containing chlorin with a chalcogen atom in exlocycle E. It was shown that the spectral properties were preserved in the target compound and the stability increased at two different pH values, in comparison with the starting purpurin-18. The derivatives have sufficiently high fluorescence and singlet oxygen quantum yields. The photoinduced cytotoxicity of sulfur- and selenium-anhydrides of chlorin p6 studied for the first time in vitro on the S37 cell line was found to be two times higher that of purpurin-18 and purpurinimide studied previously. Moreover, the dark cytotoxicity increased four-fold in comparison with the latter compounds. Apparently, the increase in the dark cytotoxicity is due to the interaction of the pigments studied with sulfur- and selenium-containing endogenous intracellular compounds. Intracellular distributions of thioanhydride and selenoanhydride chlorin p6 in S37 cells were shown in cytoplasm by diffusion distribution. The intracellular concentration of the sulfur derivative turned out to be higher and, as a consequence, its photoinduced cytotoxicity was higher as well.


Introduction
The physical and chemical properties of selenium, an element belonging to the chalcogen group, are like those of sulfur, which makes it possible to compare not only the elements themselves but also their compounds [1][2][3]. For example, an analogy may be drawn between cysteine and selenocysteine that are proteinogenic amino acids, with the difference that the sulfur atom in the thiol group of cysteine is replaced by a selenium atom in the selenol group of selenocysteine [4]. The latter is found in more than 25 types of protein molecules in mammal organisms [5][6][7]. Selenium is also a member of the group of glutathione peroxidase (GPx) enzymes that are involved in the reduction of hydrogen peroxide to water [8,9].
An alternative method for the preparation of cyclic chlorin p6 selenium anhydride (4) was developed. It was carried out by treatment with a mixture of elemental selenium and lithium aluminum hydride of compound (5). Compound (5) was obtained from purpurin-18 (1) by opening the anhydride ring with a 0.1 M alcoholic solution of potassium hydroxide to form tribasic acid (2), which was treated with thionyl chloride to give 13,15,17-trichloroanhydride (5) (Scheme 2). The absorption spectrum of selenium anhydride (4) is like that of thioanhydride (3) and contains a Qy absorption band that shows a slight hypsochromic shift, with respect to the position in the spectrum of the starting purpurin-18 (1). The absorption maxima for compounds (1), (3), and (4) in the long-wavelength region of the spectrum were recorded at 700, 698, and 696 nm, respectively ( Figure 1).  (1), (3) and (4). The absorption spectra were obtained at the same solution concentrations, with dichloromethane as a solvent.
The fluorescence spectra were recorded with excitation into the absorption band at 550 nm and the maximum fluorescence for compound (4) was observed at 722 nm ( Figure  2). Сhlorin p6 thioanhydride (3) or chlorin p6 selenoanhydride (4) have rather high quantum fluorescence yields and amounted to φ = 0.13 and 0.11, respectively. The fluorescence spectra were recorded with excitation into the absorption band at 550 nm and the maximum fluorescence for compound (4) was observed at 722 nm ( Figure 2). Chlorin p 6 thioanhydride (3) or chlorin p 6 selenoanhydride (4) have rather high quantum fluorescence yields and amounted to ϕ = 0.13 and 0.11, respectively.  3) and (4). The absorption spectra were obtained at the same solution concentrations, with dichloromethane as a solvent.
The photoinduced production of singlet oxygen and •OH radicals was investigated using chlorin p6 thioanhydride (3) or chlorin p6 selenoanhydride (4) in 1% micellar solutions of Kolliphor ELP ( Figure 4).  The photoinduced activity of compounds (3) and (4) was studied with a culture of tumor cells of mouse sarcoma S-37 (Table S1). The time of incubation with photosensitizers before exposure to light was 4 h, and the incubation time of cells after irradiation was 24 h. The cells were irradiated using a standard light dose of 10 J/cm 2 and an exposure time of 13 min. The starting compound 1 and purpurinimide were selected as the reference compounds. The tumor cell survival was estimated both visually using an inverted microscope and by the MTT test, whose results are shown in Figure 5.
The photoinduced activity of compounds (3) and (4) was studied with a culture of tumor cells of mouse sarcoma S-37 (Table S1). The time of incubation with photosensitizers before exposure to light was 4 h, and the incubation time of cells after irradiation was 24 h. The cells were irradiated using a standard light dose of 10 J/cm 2 and an exposure time of 13 min. The starting compound 1 and purpurinimide were selected as the reference compounds. The tumor cell survival was estimated both visually using an inverted microscope and by the MTT test, whose results are shown in Figure 5. It was found that chlorin p6 thioanhydride (3) and chlorin p6 selenoanhydride (4) accumulate efficiently in S37 cells in Figure 6. According to the extraction data, the average cytoplasmic concentrations (ACCs) in S37 cells are 59 ± 9 μmol L −1 for chlorin p6 thioanhydride (3) and 53 ± 8 μmol L −1 for chlorin p6 thioanhydride (4) after incubation with chlorin p6 thioanhydride (3) or chlorin p6 selenoanhydride (4) at 1 μmol L −1 for 2 h. microscope and by the MTT test, whose results are shown in Figure 5. It was found that chlorin p6 thioanhydride (3) and chlorin p6 selenoanhydride (4) accumulate efficiently in S37 cells in Figure 6. According to the extraction data, the average cytoplasmic concentrations (ACCs) in S37 cells are 59 ± 9 μmol L −1 for chlorin p6 thioanhydride (3) and 53 ± 8 μmol L −1 for chlorin p6 thioanhydride (4) after incubation with chlorin p6 thioanhydride (3) or chlorin p6 selenoanhydride (4) at 1 μmol L −1 for 2 h.

Discussion
Photodynamic therapy (PDT) is an efficient method of cancer treatment where the optimal choice of the photosensitizer (PS) is the major factor in successful tumor elimination [32][33][34]. The PS can be selectively accumulated in a tumor tissue. Some of them possess subcellularly oriented photoinduced cytotoxicity that damages vital cell organelles due to generation of reactive oxygen forms [35,36].
One of the promising approaches in the development of new PS involves their chemical design based on molecules of known pigments, namely, by replacing oxygen, nitrogen, and sulfur atoms in the exocycles of purpurin, cycloimide, and thioanhydride with a selenium atom. Since our research team has accumulated extensive experience in the chemical modification of porphyrins and their hydrogenated analogs [37][38][39][40], it seems interesting to create a selenoanhydride exocycle annulated with the main macrocycle to obtain new photosensitizers of chlorin series. As shown previously, a thioanhydride exocycle created in a chlorin molecule results in new physicochemical and spectral properties appearing in the pigment [41][42][43][44][45][46][47]. The improved properties of the sulfur-containing derivatives of porphyrins and chlorins thus obtained made it possible to use them as new complexing agents and potential photosensitizers in photodynamic therapy [48,49].
The creation of a six-membered ring annulated with the chlorin macrocycle increases the stability of the pigment molecule and causes a bathochromic shift of the Q y spectral band. For example, the formation of an imide exocycle results in a bathochromic shift of the main absorption band of purpurinimides to the region around 715 nm and increases the overall stability of the molecule. Moreover, their structure opens rich possibilities for chemical modification.
The possibility of synthesizing cyclic selenoanhydrides from derivatives of various dicarboxylic acids, including aliphatic, aromatic, and heterocyclic ones, was reported by a few researchers [23][24][25][26]. The synthesis of cyclic chlorin thioanhydride p 6 (2) was first performed by our team [50]. A specific feature of the suggested reaction, which determines the type of the resulting product, is that a particular molar ratio of the initial reagents, i.e., purpurin-18 (1) and sodium sulfide, was used. If their ratio was equimolar, no thioanhydride was formed, while the use of a two-fold excess of purpurin-18 (1) gave the target thioanhydride.
In this work, the previously identified regularities of this reaction were considered to obtain chlorin p 6 selenoanhydride (4). The reaction of equimolar amounts of purpurin-18 (1) and sodium selenide gives only chlorin p 6 (2) in the form of a triacid, whereas the addition of a double amount of purpurin-18 resulted in a mixture of the target selenoanhydride (4) and chlorin p 6 (2) (Scheme 1).
The previously suggested mechanism of thioanhydride formation can be extrapolated to the formation of the selenoanhydride. Apparently, the initial monoseleniumchlorin p 6 derivative (6) reacts with the second molecule of purpurin-18 (1) taken in two-fold excess to give acyclic anhydride (7), in which the intramolecular attack of the selenium atom on the adjacent carbonyl carbon atom gives selenoanhydride (4) and causes the elimination of a chlorin p 6 (2) molecule (Scheme 3).
The reaction product depends on the molecular ratios of purpurin-18 (1) and sodium selenide (Na 2 Se). If a two-fold excess of purpurin-18 is taken (1), then acyclic anhydride (7) is formed, while at a 1:1 ratio (condition iii, Scheme 1), the abovementioned anhydride is not formed, but hydrolysis of monoseleniumchlorin p 6 derivative (6) in an acidic medium leads to the formation of chlorin p 6 (2).
To confirm the structure of compound (4), an alternative synthesis was carried out: chlorin p 6 (2) was obtained as a triacid by opening the anhydride ring in purpurin-18 (1) with 0.1 M alcoholic solution of potassium hydroxide; tribasic acid (2) was converted with oxalyl chloride to 13,15,17-trichloroanhydride (5), and, finally, cyclic selenoanhydride (4) was obtained by treatment with a mixture of elemental selenium with lithium aluminum hydride (Scheme 2). The LiAlHSeH formed in the reaction is used for the synthesis of diacyl selenides from diacyl chlorides, including the synthesis of selenoanhydrides from various dicarboxylic acid chlorides, both aliphatic and aromatic [51]. The reaction, according to Scheme 2, gave product (4).
The target chlorin p 6 selenoanhydride (4) was developed according to Scheme 1 in preparative amounts for further research since it consists of only one-step and it is easy to purify the target compound.
The MALDI-TOF mass spectrum of compound (4) contained a signal corresponding to the calculated value for the molecular ion of cyclic anhydride selenium, while the 1 H NMR spectrum was identical to that of purpurin-18 (1). An elemental analysis of the compound (4) confirmed the qualitative and quantitative composition of the molecule corresponding to the proposed structure ( Figures S1-S3). To confirm the structure of compound (4), an alternative synthesis was carried out: chlorin p6 (2) was obtained as a triacid by opening the anhydride ring in purpurin-18 (1) with 0.1 M alcoholic solution of potassium hydroxide; tribasic acid (2) was converted with oxalyl chloride to 13,15,17-trichloroanhydride (5), and, finally, cyclic selenoanhydride (4) was obtained by treatment with a mixture of elemental selenium with lithium aluminum hydride (Scheme 2). The LiAlHSеH formed in the reaction is used for the synthesis of diacyl selenides from diacyl chlorides, including the synthesis of selenoanhydrides from various dicarboxylic acid chlorides, both aliphatic and aromatic [51]. The reaction, according to Scheme 2, gave product (4).
The target chlorin p6 selenoanhydride (4) was developed according to Scheme 1 in preparative amounts for further research since it consists of only one-step and it is easy to purify the target compound.
The MALDI-TOF mass spectrum of compound (4) contained a signal corresponding to the calculated value for the molecular ion of cyclic anhydride selenium, while the 1 H NMR spectrum was identical to that of purpurin-18 (1). An elemental analysis of the compound (4) confirmed the qualitative and quantitative composition of the molecule corresponding to the proposed structure ( Figures S1-S3).
In this work, we compared the chemical stability of chlorin p6 selenoanhydride (4), chlorin p6 thioanhydride (3), and purpurin-18 (1) at different pH values. The experiment involved monitoring the changes in the absorption spectra of the pigments dissolved in 4% aqueous micellar solutions of Kolliphor ELP with time at different pH values. Since In this work, we compared the chemical stability of chlorin p 6 selenoanhydride (4), chlorin p 6 thioanhydride (3), and purpurin-18 (1) at different pH values. The experiment involved monitoring the changes in the absorption spectra of the pigments dissolved in 4% aqueous micellar solutions of Kolliphor ELP with time at different pH values. Since the opening of O-, S-, and Se-containing exocycles is accompanied by a significant hypsochromic shift of the long-wavelength absorption band to the 660 nm region, an appearance and intensity increase of the latter would indicate that the structure changes with time ( Figure 4). Figure 4 shows that the stability at pH = 9 increases in the series: purpurin-18 (1), chlorin p 6 thioanhydride (3), chlorin p 6 selenoanhydride (4). In 24 h, compound (1) is completely converted to chlorin p 6 in the form of triacid (2) and the degree of conversion of chlorin p 6 thioanhydride (3) is no higher than 50%, whereas chlorin p 6 selenoanhydride (4) remains stable throughout the experiment.
The choice of a different value, pH = 4, was dictated by the need to simulate the environment of the tumor interstitial tissue, which is known to be acidic [52]. In these experiments, we used 4% micellar solutions of Kolliphor ELP solubilizer with purpurin-18 (1), chlorin p 6 thioanhydride (3), and chlorin p 6 selenoanhydride (4) at the same concentration of 1 mmol/L in the buffer solution with pH = 4. The stability results obtained for compounds (1), (3), and (4) are summarized in Table 1. As one can see from Table 1, the stability at pH = 4 increases in the series of compounds: purpurin-18 (1), chlorin p 6 thioanhydride (3), chlorin p 6 selenoanhydride (4). No complete exocycle opening occurred in the latter, even after 24 h. The more electronegative heteroatoms (O, N, S, Se) in the exocycle, the higher the reactivity of the adjacent carbonyl carbon atom in nucleophilic addition/substitution reactions. Apparently, this is the reason for the greater stability of the selenium anhydride ring.
The photoinduced activity of compounds (3) and (4) was studied with a culture of tumor cells of mouse sarcoma S-37 (Table S1). The values presented in Figure 5a show a two-fold increase in photoinduced cytotoxicity in Se-containing derivatives (4) compared to the pigments containing an anhydride or imide exocycle. Moreover, the dark cytotoxicity increased four-fold, in comparison with the latter compounds (Figure 5b). Apparently, the increase in the dark cytotoxicity is due to the interaction of the pigments studied with sulfur-and selenium-containing endogenous intracellular compounds. Such interactions can cause a violation of the redox balance in tumor cells, for example, by blocking the glutathione antioxidant system [19] or suppressing DNA repair [15]. The photoinduced cytotoxicity of compounds (3) and (4) is an order of magnitude higher than the dark cytotoxicity, which is comparable to the previously studied chlorophyll a derivatives and commercial PS [53].

Materials and Instruments
All the solvents used in this work were prepared according to common techniques as described elsewhere. Column chromatography was performed on silica gel 40/60 (Merck, Darmstadt, Germany). Preparative thin-layer chromatography (TLC) was performed on glass plates with silica gel 60 (Merck, Darmstadt, Germany). Analytical TLC was performed on aluminum plates with Silica gel 60 F 254 with a fluorescent probe (Merck, Darmstadt, Germany).
Absorption and fluorescence spectra were recorded on Shimadzu UV1800 UV/VIS (Shimadzu, Duisburg, Germany) and Shimadzu RF-5301 (Shimadzu, Duisburg, Germany) spectrophotometers in quartz cuvettes (0.4 × 1.0 cm) with an optical path length of 1 cm (spectral slit width 1 nm), with dichloromethane as a solvent. The absorption spectra were recorded in the range of 350-750 nm. Fluorescence spectra were recorded in the range 650-770 nm (excitation wavelength 520 nm), with dichloromethane as a solvent. The fluorescence quantum yield was determined by the relative method. A solution of purpurin-18 in toluene was used as a standard [54]. 1 H and 13 C NMR spectra were registered on Bruker DPX 300 (Bruker, Alzenau, Germany). NMR samples were prepared in CDCl 3 . Matrixassisted laser desorption/ionization (MALDI) mass spectra were recorded on a Bruker Ultraflex TOF/TOF spectrometer (Bruker, Alzenau, Germany), with 2,5-dihydroxybenzoic acid as the matrix. Elemental analysis was performed on EA 1112, Thermo Finnigan, Thermo Scientific, CE Instruments (Thermo Fisher Scientific, Waltham, MA, USA).
Extraction of chlorophyll a from Spirulina platensis, synthesis of purpurin-18, and synthesis of cyclic thioanhydride of chlorin p 6 were carried out as described previously [50].
Thionyl chloride (134 µmol, 26 µL) and triethylamine (10 µmol, 1.5 µL) were added to the solution of compound (2) (67 µmol) in diethyl ether (3 mL), and the reaction was performed at 22 • C for 30 min in argon atmosphere. After this, the LiAlHSeH reagent was added to product 7. The reagent was prepared beforehand by the addition of lithium aluminum hydride (2.6 mg) to a solution of selenium (2.2 mg) in tetrahydrofuran (250 µL). The resulting solution was incubated in an inert argon atmosphere for 24 h at 22 • C, and the compound (4) was extracted with chloroform (15 mL) and repeatedly washed with water (150 mL). The solution was concentrated in a vacuum, and the product was purified by preparative TLC (CH 2 Cl 2 /CH 3 OH, 60/1, v/v). The yield of the target compound (4) 13

A Study of Stability at Different pH Values
The stability was estimated by the variation in the intensity of the Q y absorption band in the electronic spectra of the samples.
A total of 4% Micellar solutions of Kolliphor ELP with purpurin-18 (1), chlorin thioanhydride p 6 (3), and chlorin p 6 selenoanhydride (4) were added to buffer solutions prepared according to standard methods, so that the final concentration of the pigments in the solution was 1 mmol/L and the pH was 4 or 9. The changes in the absorption spectra of the resulting solutions were monitored for 24 h of incubation.

A Study of Photoinduced Production of Singlet Oxygen and ·OH-Radicals
The ability of chlorin p 6 thioanhydride (3) and chlorin p 6 selenoanhydride (4) for the photoinduced generation of singlet oxygen was estimated using a 4-nitroso-N, Ndimethylaniline-histidine (RNO) assay [55], as in [56], with minor modifications. Rose Bengal was used as a reference compound with a known quantum yield of singlet oxygen generation (Φ( 1 O 2 ) = 0.75) [57]. The RNO-based assay, in the absence of histidine, was used under the same experimental conditions for the detection of ·OH radicals [58].

A Study of Photoinduced Activity In Vitro
The cells were cultivated under standard conditions at 37 • C in a humidified atmosphere with 5% CO 2, in a DMEM environment with the addition of L-glutamine (2 mM) and fetal calf serum (10%, PanEco, Moscow, Russia). The photoinduced efficiency was estimated as follows: S37 cells were seeded in a 96-well flat-bottomed plate for microtitration (Costar, New York, NY, USA). The tested PS was added after 24 h at concentrations ranging from 0.03 µg/mL to 20 µg/mL. The time of the incubation with photosensitizers before exposure to light was 4 h. After that, the cells were irradiated with a halogen lamp through a KS-19 broadband filter that passed light with wavelengths above 720 nm, and a water filter 5 cm thick, equipped with a liquid circulation system (λ ≥ 1000 nm). The power density was 21.0 ± 1.0 mW/cm 2 and the calculated light dose was 10 J/cm 2 . The starting compound 1 and purpurinimide were selected as the reference compounds. After irradiation, the cells were incubated for 24 h under standard conditions. To analyze the PS cytotoxicity and the cytotoxicity (without irradiation), cells were kept for 24 h in the dark. Survival was estimated by visual inspection and colorimetrically using the MTT test. Cell-growth inhibitions of more than 50% were considered biologically significant. This value was calculated as the average of three independent tests.

A Study Intracellular Accumulation
To study intracellular accumulation of chlorin p 6 thioanhydride (3) and chlorin p 6 selenoanhydride (4), cells were seeded on cover glasses in 24-well plates (seeding density of 5 × 10 4 cells per well) a day before the experiment. Confocal fluorescent images were recorded using a laser scanning confocal microscope Leica TCS SP2 (Leica, Wetzlar, Germany) with a 63× water-immersion lens (numerical aperture 1.2). Lateral and axial resolutions were 0.2 and 1 µm, respectively. Fluorescence of chlorin p 6 thioanhydride (3) and chlorin p 6 selenoanhydride (4) was excited at the 561 nm wavelength and recorded in the 680-750 nm range.

Conclusions
A selenium-containing derivative of purpurin-18, namely, chlorin p 6 selenoanhydride, was obtained in this work. The chemical synthesis was performed using two approaches. The first involved a direct reaction of purpurin-18 with sodium selenide, accompanied by a replacement of oxygen in exocycle E with a selenium atom. The second involved the reaction of chlorin p 6 13,15,17-trichloroanhydride with elemental selenium in the presence of lithium aluminum hydride.
The target compound was characterized by spectral and physicochemical methods. Stability studies showed that the selenoanhydride was more stable under acid (pH = 4) and basic conditions (pH = 9) than the thioanhydride and the starting purpurin-18. The derivatives have sufficiently high fluorescence and singlet oxygen quantum yields. In vitro biological studies of chlorin p 6 selenoanhydride on the S37 cell line showed a two-fold increase in the photoinduced cytotoxic effect, compared to the previously obtained purpurinimide and the original purpurin-18, while the dark cytotoxicity increased four-fold, in comparison with the latter. We have shown that the chlorin p 6 thioanhydride accumulates better in S37 cells and has a higher photoinduced cytotoxicity.