New Triphenylphosphonium Salts of Spiropyrans: Synthesis and Photochromic Properties

The most important area of modern pharmacology is the targeted delivery of drugs, and one of the most promising classes of chemical compounds for creating drugs of this kind are the photochromic spiropyrans, capable of light-controlled biological activity. This work is devoted to the synthesis and study of the photochromic properties of new triphenylphosphonium salts of spiropyrans. It was found that all the synthesized cationic spiropyrans have high photosensitivity, increased resistance to photodegradation and the ability for photoluminescence.


Introduction
Reversible molecular rearrangements play an important role in the study of many biological, chemical and physicochemical processes, wherein photochromic transformations are of great interest-reversible changes in a molecule between two nonequivalent forms with different absorption spectra, induced by external influences.The development of new organic photochromic systems and materials based on them is one of the areas of modern organic synthesis.
Recently, interdisciplinary research aimed at studying controlled drug delivery systems has become increasingly popular.This is due to the fact that the targeted delivery of the drug to the affected area and in controlled quantities can reduce unwanted side effects caused by drug toxicity.Photochromic spiropyrans are promising agents for the creation of drugs of this kind, with a high selectivity and a high induced therapeutic effect, due to the ability to exist in two isomeric forms with different physicochemical properties.At the same time, in the global literature, there is only one work describing, using one example of water-soluble spiropyran, photoswitchable cytotoxicity towards Hek293 cancer cells [20].This approach will make it possible to hope that the closed form will not cause a toxic effect and will easily pass through the cell membrane, while the open form, on the contrary, will lead to an induced cytotoxic effect.
It is known that mitochondrial oxidative phosphorylation is the basis of cell life and death.Therefore, the introduction into the structure of spiropyrans of the triphenylphosphonium cation, which has a known ability to quickly penetrate through lipid membranes [22][23][24][25][26][27][28][29], and concentrate mainly in mitochondria, helps to manipulate mitochondrial functions, causing controlled apoptosis [30,31], which will not only enhance the cytotoxic effect, but also use it as a vector for the fast and targeted delivery of the active substance to affected target cells.Moreover, the presence of a positive photochromic effect and luminescent properties will contribute to the additional visualization of intracellular processes.
Previously [32], we synthesized ammonium salts of spiropyrans, which were subsequently tested on their cytotoxic activity against various cancer cell lines, including MCF7, A431, BT474, and HF (unpublished data).The results obtained to date cannot be called impressive.For example, when a culture of A431 human epidermoid carcinoma cells was exposed to ammonium salts, the proportion of living cells was in the range of 82 to 96%.Upon subsequent exposure to UV light, the proportion of living cells ranged from 66 to 91%.
In continuation of these studies, in order to develop the synthesis of new previously unstudied photochromic compounds that are promising for the creation of mitochondriatargeted antitumor drugs, in this work, we synthesized salts of indoline spiropyrans containing a lipophilic triphenylphosphonium cation in their structure, and also their photochromic and luminescent properties were studied.

Synthesis and Identification
The synthesis of indoline spiropyrans was carried out using methods described in the literature [32,33], according to Scheme 1: chondrial functions, causing controlled apoptosis [30,31], which will not only enhance the cytotoxic effect, but also use it as a vector for the fast and targeted delivery of the active substance to affected target cells.Moreover, the presence of a positive photochromic effect and luminescent properties will contribute to the additional visualization of intracellular processes.
Previously [32], we synthesized ammonium salts of spiropyrans, which were subsequently tested on their cytotoxic activity against various cancer cell lines, including MCF7, A431, BT474, and HF (unpublished data).The results obtained to date cannot be called impressive.For example, when a culture of A431 human epidermoid carcinoma cells was exposed to ammonium salts, the proportion of living cells was in the range of 82 to 96%.Upon subsequent exposure to UV light, the proportion of living cells ranged from 66 to 91%.
In continuation of these studies, in order to develop the synthesis of new previously unstudied photochromic compounds that are promising for the creation of mitochondriatargeted antitumor drugs, in this work, we synthesized salts of indoline spiropyrans containing a lipophilic triphenylphosphonium cation in their structure, and also their photochromic and luminescent properties were studied.

Synthesis and Identification
The synthesis of indoline spiropyrans was carried out using methods described in the literature [32,33], according to Scheme 1: Scheme 1. Synthesis of compounds 12-16.
All synthesized triphenylphosphonium salts 12-16 were isolated from the reaction mixture using column chromatography.The structures of compounds 12-16 were All synthesized triphenylphosphonium salts 12-16 were isolated from the reaction mixture using column chromatography.The structures of compounds 12-16 were determined using NMR ( 1 H, 13 C and 31 P) spectroscopy, as well as HRMS high-resolution mass spectrometry (see Supplementary Materials).
The structure of the synthesized compounds 12-16 was also confirmed by highresolution mass spectrometry.For example, the HRMS spectrum of compound 12 exhibits an intense peak of a molecular ion with the m/z C 39 H 36 N 2 O 3 P [M-Br − ] = 611.2459(calculated: for MBr = 690.1646m/z; for [M-Br − ] = 611.2458m/z) (Figure 1).

PEER REVIEW
3 of 11 determined using NMR ( 1 H, 13 C and 31 P) spectroscopy, as well as HRMS high-resolution mass spectrometry (see Supplementary Materials).The structure of the synthesized compounds 12-16 was also confirmed by high-resolution mass spectrometry.For example, the HRMS spectrum of compound 12 exhibits an intense peak of a molecular ion with the m/z С39H36N2O3P [M-Br − ] = 611.2459(calculated: for MBr = 690.1646m/z; for [M-Br − ] = 611.2458m/z) (Figure 1).The spectra of compounds 13-16 are presented in the Supplementary Materials.

UV-Vis and Photoluminescent Studies
Taking into account the sensitivity of spiropyrans to a wide range of external stimuli [1][2][3][4][5]9,[34][35][36][37][38][39][40], we studied the photoinduced transformations of synthesized salts 12-16 in ethanol using absorption and luminescence spectroscopy.The choice of the latter as a solvent was due to the excellent solubility of the synthesized compounds in ethanol, as well as plans to conduct tests for biological activity in aqueous-alcoholic solutions.
The measurement of the absorption spectra of salts 12-16 without and with irradiation with ultraviolet (UV) light showed that all the synthesized compounds have positive photochromism.The results of the measurements of the main spectral and kinetic characteristics of compounds 12-16 are summarized in Table 1.The spectra of compounds 13-16 are presented in the Supplementary Materials.

UV-Vis and Photoluminescent Studies
Taking into account the sensitivity of spiropyrans to a wide range of external stimuli [1][2][3][4][5]9,[34][35][36][37][38][39][40], we studied the photoinduced transformations of synthesized salts 12-16 in ethanol using absorption and luminescence spectroscopy.The choice of the latter as a solvent was due to the excellent solubility of the synthesized compounds in ethanol, as well as plans to conduct tests for biological activity in aqueous-alcoholic solutions.
The measurement of the absorption spectra of salts 12-16 without and with irradiation with ultraviolet (UV) light showed that all the synthesized compounds have positive photochromism.The results of the measurements of the main spectral and kinetic characteristics of compounds 12-16 are summarized in Table 1.
Figure 2, using 12 as an example, shows the typical absorption spectra of spiropyrans 12-16 in ethanol without and with UV irradiation.The absorption of spiropyran molecules 12-16 in the closed cyclic form is characterized by intense bands in the short wavelength region of the spectrum at 224-225, 267, and 335-339 nm (Figure 2, curve 1).
As can be seen from the data in Table 1, the position of the absorption bands of the spiropyrans slightly (∆ max = 4 nm) shifts to longer wavelengths with an increase in the number of methylene groups in the spacer.When This change observed in the absorption spectra clearly indicate that under the influence of activating UV radiation, photochromic transformations occur; the pyran ring of molecules 12-16 opens and the merocyanine form of the compounds is formed (Scheme 2).In this case, the initially colorless solution instantly acquires a pink color (Figure 2, inset), due to the absorption of merocyanines in the green region of the visible spectrum.The position of this long wavelength maximum (542-551 nm) in the absorption spectrum of merocyanines, as in the case of the closed forms of spiropyrans, depends on the length of the spacer.When going from compound 12 to 14, a small hypsochromic shift is observed (∆ max = 9 nm).Considering that the absorption bands of spiropyrans, both in open and closed forms, are caused by singlet transitions S 0 → S n between n-π* and π-π* orbitals localized on the indole and pyran fragments, these shifts of absorption bands with increasing spacer length can be associated with the effect of the triphenylphosphonium cation on the electron density of the indole fragment.  The most characteristic absorption bands are given. 2Rate constant for spontaneous dark decolorization of the merocyanine form of spiropyran at room temperature. 3Rate constant for photobleaching of the merocyanine form of spiropyran at room temperature. 4Efficiency of photodegradation (τ 1/2 ), time of decrease by a factor of 2 of the value of optical density at the maximum of the absorption band of the photoinduced merocyanine form under continuous irradiation with UV light. 5Photosensitivity of a photochromic compound (S), S = ∆D phot /D max , the ratio of the absorbance values at the maximum of the absorption band of the merocyanine form of spiropyran to the value of the optical density at the maximum of the absorption band of the spiropyran form.As can be seen from the data in Table 1, the position of the absorption bands of the spiropyrans slightly (Δmax = 4 nm) shifts to longer wavelengths with an increase in the number of methylene groups in the spacer.When This change observed in the absorption spectra clearly indicate that under the influence of activating UV radiation, photochromic transformations occur; the pyran ring of molecules 12-16 opens and the merocyanine form of the compounds is formed (Scheme 2).In this case, the initially colorless solution instantly acquires a pink color (Figure 2, inset), due to the absorption of merocyanines in the green region of the visible spectrum.However, these changes are reversible; therefore, in the dark (dark relaxation, dark bleaching) or upon irradiation with visible light (photorelaxation, photobleaching), molecules 12-16 cyclize again, forming the original closed form (Scheme 2).A comparison of the kinetic curves of the dark relaxation process of spiropyrans 12-16 (Figure 3) shows that compound 12 has the highest rate of dark bleaching, and compound 16, on the contrary, has the lowest.However, these changes are reversible; therefore, in the dark (dark relaxation, dark bleaching) or upon irradiation with visible light (photorelaxation, photobleaching), molecules 12-16 cyclize again, forming the original closed form (Scheme 2).A comparison of the kinetic curves of the dark relaxation process of spiropyrans 12-16 (Figure 3) shows that compound 12 has the highest rate of dark bleaching, and compound 16, on the contrary, has the lowest.This observation is confirmed by a quantitative assessment of the rate constants of spontaneous dark bleaching k1 (Table 1), which are obtained from the slope of linear dependences when constructing kinetic curves in the Ln(I)-t(c) coordinates (Figure 4).For all studied compounds 12-16, the initial section of anamorphoses has a linear appearance, i.e., dark relaxation is described by a first-order reaction equation.However, these changes are reversible; therefore, in the dark (dark relaxation, dark bleaching) or upon irradiation with visible light (photorelaxation, photobleaching), molecules 12-16 cyclize again, forming the original closed form (Scheme 2).A comparison of the kinetic curves of the dark relaxation process of spiropyrans 12-16 (Figure 3) shows that compound 12 has the highest rate of dark bleaching, and compound 16, on the contrary, has the lowest.This observation is confirmed by a quantitative assessment of the rate constants of spontaneous dark bleaching k1 (Table 1), which are obtained from the slope of linear dependences when constructing kinetic curves in the Ln(I)-t(c) coordinates (Figure 4).For all studied compounds 12-16, the initial section of anamorphoses has a linear appearance, i.e., dark relaxation is described by a first-order reaction equation.This observation is confirmed by a quantitative assessment of the rate constants of spontaneous dark bleaching k 1 (Table 1), which are obtained from the slope of linear dependences when constructing kinetic curves in the Ln(I)-t(c) coordinates (Figure 4).For all studied compounds 12-16, the initial section of anamorphoses has a linear appearance, i.e., dark relaxation is described by a first-order reaction equation.
The kinetics of death of merocyanines upon irradiation with visible light (SZS-9 light filter, maximum transmission wavelength 490 nm) is also described by a first-order equation for all spiropyrans 12-16 (Figure 4).Table 1 shows that the rate constants for photobleaching of compounds 12-16 are three orders of magnitude greater than the rate constant for dark bleaching.
The measurements of the kinetic curves of the alternating photocoloration and dark bleaching of solutions 12-16 showed that the switching cycle can be repeated many times (Figure 5).However, after each «irradiation-relaxation» cycle, a decrease in the optical density of compounds 12-16 is observed, which may be due to their photodestruction.The efficiency of the photodegradation reaction measured from the absorption spectra (Figure 6, Table 1) confirms this assumption.Based on Table 1, it can be stated that with the constant irradiation of the solutions of spiropyrans 12-16 with UV light, the optical density at the maximum of the absorption band of the merocyanine form decreases.Moreover, for these connections, this time ranges from 32 to 99 min.Moreover, spiropyran 12 is characterized by the most pronounced photodegradation.Although the photosensitivity, determined by the ∆D max /D max ratio, is almost the same for all spiropyrans, 12-16, studied (Table 1).The detected difference in the measured kinetic characteristics of compounds 12-16 confirms our assumption that in the case of a short spacer length (n = 3 for 12), the triphenylphosphonium cation is able to influence the spiropyran molecule.A detailed explanation of the influence of the spacer length on the spectral-kinetic characteristics of the spiropyrans under study requires special experiments and theoretical calculations, and will be the subject of our future research.The kinetics of death of merocyanines upon irradiation with visib filter, maximum transmission wavelength 490 nm) is also described by tion for all spiropyrans 12-16 (Figure 4).Table 1 shows that the rate c bleaching of compounds 12-16 are three orders of magnitude greate stant for dark bleaching.
The measurements of the kinetic curves of the alternating photoc bleaching of solutions 12-16 showed that the switching cycle can be re (Figure 5).However, after each «irradiation-relaxation» cycle, a dec density of compounds 12-16 is observed, which may be due to their ph efficiency of the photodegradation reaction measured from the absorp 6, Table 1) confirms this assumption.Based on Table 1, it can be stated stant irradiation of the solutions of spiropyrans 12-16 with UV light, at the maximum of the absorption band of the merocyanine form decre these connections, this time ranges from 32 to 99 min.Moreover, spiro terized by the most pronounced photodegradation.Although the pho mined by the ∆D max /D max ratio, is almost the same for all spiropyrans, 1 1).The detected difference in the measured kinetic characteristics of confirms our assumption that in the case of a short spacer length (n phenylphosphonium is able to influence the spiropyran mole The kinetics of death of merocyanines upon irradiation w filter, maximum transmission wavelength 490 nm) is also desc tion for all spiropyrans 12-16 (Figure 4).Table 1 shows that th bleaching of compounds 12-16 are three orders of magnitud stant for dark bleaching.
The measurements of the kinetic curves of the alternatin bleaching of solutions 12-16 showed that the switching cycle c (Figure 5).However, after each «irradiation-relaxation» cycl density of compounds 12-16 is observed, which may be due to efficiency of the photodegradation reaction measured from the 6, Table 1) confirms this assumption.Based on Table 1, it can stant irradiation of the solutions of spiropyrans 12-16 with U at the maximum of the absorption band of the merocyanine for these connections, this time ranges from 32 to 99 min.Moreov terized by the most pronounced photodegradation.Although mined by the ∆D max /D max ratio, is almost the same for all spirop 1).The detected difference in the measured kinetic character confirms our assumption that in the case of a short spacer le phenylphosphonium cation is able to influence the spiropyra planation of the influence of the spacer length on the spectralspiropyrans under study requires special experiments and th will be the subject of our future research.1).C = 10 −4 M, l = 0.1 cm, spectrophotometer Agil 60.
It is known that photochromic compounds capable of luminescence are of terest because they can be used as luminescent molecular switches [2][3][4].In addi minescence analysis is one of the main methods for studying the state of matter in ical systems, and the study of luminescence spectra is a necessary step in the complex photobiological systems.In this regard, we studied the spectral and lum properties of the synthesized triphenylphosphonium salts 12-16 in ethanol at ro perature (25 °С).
As expected, no photoluminescence (PL) was detected for the spirocyclic form synthesized compounds 12-16.These data are in good agreement with the know ture data [4,7] and our previous experiments on studying the PL of spiropyrans of chemical structures [32,40].However, upon irradiation of solutions of 12-16 in PL in the red region of the visible spectrum with a maximum at 636-640 nm was (Figure 7).As can be seen from this figure, the position of the maxima in the spe the PL intensity slightly depend on the number of methylene groups in the spi spacer.It is known that photochromic compounds capable of luminescence are of great interest because they can be used as luminescent molecular switches [2][3][4].In addition, luminescence analysis is one of the main methods for studying the state of matter in biological systems, and the study of luminescence spectra is a necessary step in the study of complex photobiological systems.In this regard, we studied the spectral and luminescent properties of the synthesized triphenylphosphonium salts 12-16 in ethanol at room temperature (25 • C).
As expected, no photoluminescence (PL) was detected for the spirocyclic forms of the synthesized compounds 12-16.These data are in good agreement with the known literature data [4,7] and our previous experiments on studying the PL of spiropyrans of various chemical structures [32,40].However, upon irradiation of solutions of 12-16 in ethanol, PL in the red region of the visible spectrum with a maximum at 636-640 nm was detected (Figure 7).As can be seen from this figure, the position of the maxima in the spectra and the PL intensity slightly depend on the number of methylene groups in the spiropyran spacer.1).C = 10 −4 M, l = 0.1 cm, spectrophotometer Agilent Cary-60.
It is known that photochromic compounds capable of luminescence are of great interest because they can be used as luminescent molecular switches [2][3][4].In addition, luminescence analysis is one of the main methods for studying the state of matter in biological systems, and the study of luminescence spectra is a necessary step in the study of complex photobiological systems.In this regard, we studied the spectral and luminescent properties of the synthesized triphenylphosphonium salts 12-16 in ethanol at room temperature (25 °С).
As expected, no photoluminescence (PL) was detected for the spirocyclic forms of the synthesized compounds 12-16.These data are in good agreement with the known literature data [4,7] and our previous experiments on studying the PL of spiropyrans of various chemical structures [32,40].However, upon irradiation of solutions of 12-16 in ethanol, PL in the red region of the visible spectrum with a maximum at 636-640 nm was detected (Figure 7).As can be seen from this figure, the position of the maxima in the spectra and the PL intensity slightly depend on the number of methylene groups in the spiropyran spacer.

Materials and Methods
The 1 H, 13 C and 31 P NMR spectra were run on a Bruker Avance-500 spectrometer (Bruker, Billerica, MA, USA) at 500.17 and 125.78 MHz or on a Bruker Avance-400.13 spectrometer (Bruker, Billerica, MA, USA) at 400.17 and 100.62 MHz, respectively.CDCl 3 was used as a solvent.High-resolution mass spectrometry (HRMS) was performed on a MaXis impact, Bruker.Thin-layer chromatography was performed on silica gel plates (Sorbfil TLC, Krasnodar, Russia) to monitor the reactions.Spots were made visible with UV light.Column chromatography was performed with silica gel (Sigma-Aldrich, Burlington, MA, USA, high-purity grade, 60 Å/63-200 µm).
The spectrophotometric measurements were performed on a Cary-60 UV-Vis Spectrophotometer in 1 and 10 mm thick quartz cells.For photochemical measurements, the solutions were irradiated by an L8253 xenon lamp included in an LC-8 radiation unit (Hamamatsu, Shizuoka, Japan) at a medium radiation power through ultraviolet UFS-1 (from 240 nm to 400 nm with maximum transmission at 330 nm) and blue-green SZS-9 (from 380 nm to 580 nm with maximum transmission at 480 nm) filters.
The photoluminescence (PL) spectra of test compounds were registered using Horiba Jobin Yvon Fluorolog-3 spectrofluorometer (model FL-3-22) equipped with doublegrating monochromators, dual lamp housing containing a 450 W Xenon lamp and photomultiplier tube detector Hamamatsu R928 (Shizuoka, Japan).The PL spectra were corrected in all cases for source intensity (lamp and grating) and emission spectral response (detector and grating) by standard instrument correction provided in the instrument software FluorEssence 3.5 [41].
The detailed procedure of the synthesis and characterization of the products are given in the Supplementary Materials.

Conclusions
Thus, new photochromic spiropyrans containing in their structure a triphenylphosphonium cation with different spacer lengths were synthesized, and their photochromic and luminescent properties were studied.It is shown that all the synthesized compounds exhibit positive photochromism, as evidenced by their reversible photoinduced transformations under the influence of ultraviolet and visible radiation.The influence of structural factors on the spectral properties and kinetic characteristics of the photochromic transformations of the synthesized spirophotochromes was established.First, the positions of the absorption bands of the cyclic and merocyanine forms 12-16 slightly depend on the spacer length between the spiropyran and triphenylphosphonium fragments.Thus, the hypsochromic shift of the spectral bands in the series 12-16 is 4 nm for the spirocyclic form and 9 nm for the merocyanine form.Second, the measured relaxation rate constants of spiropyrans 12-16 from the open to the closed form differ several times.Thus, the results of the study demonstrate the possibility of controlling the efficiency of the photoinduced transformations of spiropyrans by varying the spacer length.Moreover, the presence of a positive photochromic effect with photoswitchable luminescence will make it possible to monitor the progress of various intracellular processes.
Summarizing the above, we can conclude that the synthesized cationic spiropyrans 12-16, due to their high photosensitivity, displayed increased resistance to photodegradation and an ability to photoluminesce, determine the relevance of the development of research in the field of the synthesis of new structures and the need to continue further research into the study of their physicochemical properties and biological activity.

11 Figure 2 ,
Figure 2, using 12 as an example, shows the typical absorption spectra of spiropyrans 12-16 in ethanol without and with UV irradiation.The absorption of spiropyran molecules 12-16 in the closed cyclic form is characterized by intense bands in the short wavelength region of the spectrum at 224-225, 267, and 335-339 nm (Figure 2, curve 1).

Figure 5 .
Figure 5. Kinetics of alternating photocoloration of solutions 12 (a) a influence of UV light (through a UFS-1 filter (Elektrosteklo LLC

Table 1 .
Spectral-kinetic characteristics of the studied photochromic compounds in spiropyran (SP) and merocyanine (MC) forms.

Table 1 .
Spectral-kinetic characteristics of the studied photochromic compounds in spiropyran (SP) and merocyanine (MC) forms.