Multicomponent Molecular Systems Based on Porphyrins, 1,3,5-Triazine and Carboranes: Synthesis and Characterization

2,4,6-Trichloro-1,3,5-triazine (cyanuric chloride) is an excellent coupling reagent for the preparation of highly structured multifunctional molecules. Three component systems based on porphyrin, cyanuric chloride and carborane clusters were prepared by a one-pot stepwise amination of cyanuric chloride with 5-(4-aminophenyl)-10,15,20-triphenylporphyrin, followed by replacement of the remaining chlorine atoms with carborane S- or N-nucleophiles. Some variants of 1,3,5-triazine derivatives containing porphyrin, carborane and residues of biologically active compounds such as maleimide, glycine methyl ester as well as thioglycolic acid, mercaptoethanol and hexafluoroisopropanol were also prepared. A careful control of the reaction temperature during the substitution reactions will allow the synthesis of desired compounds in a good to high yields. The structures of synthesized compounds were determined with UV-vis, IR, 1H NMR, 11B NMR, MALDI-TOF or LC-MS spectroscopic data. The dark and photocytotoxicity as well as intracellular localization and photoinduced cell death for compounds 8, 9, 17, 18 and 24 were evaluated.


Introduction
Natural and synthetic porphyrins due to their unique photophysical and photochemical properties have a great potential as systems for the design of molecular electronics and photonics devices [1,2], sensors [3,4], efficient catalysts [5,6], dyes for photovoltaic solar cells [7][8][9] and as photosensitisers (PS) for photodynamic therapy (PDT) against malignant and non-malignant diseases [10][11][12]. The intensive use of porphyrin derivatives in PDT is based on their special physico-chemical and structural properties, such as intense absorption and emission in the visible region where the biological tissues absorb only weakly, high triplet state quantum yield, selectivity for tumor cells and low in vivo toxicity. PDT is currently used as an alternative treatment for the control of malignant diseases [13][14][15] and relies on the combination of a non-toxic PS with harmless visible light to produce reactive oxygen species (ROS), mainly singlet oxygen ( 1 O 2 ) in target tissues, which can selectively kill cancer cells [16]. Due to that, PDT treatment is safe and has few side effects. Now, PDT is used in the clinic to treat various cancers including tumors of the skin, head, neck and lung [17]. In addition to PDT, other promising modalities for biomedical porphyrins applications as PS have been developed over recent years, such as photodynamic antimicrobial chemotherapy [18][19][20], photothermal therapy [21,22] and sonotherapy [23]. Recently, potential of their application as PS was extended for the treatment of other diseases, such as age-related macular degeneration, skin disorders [24][25][26] and for the inactivation of microorganisms such as fungi or viruses [27][28][29]. The creation of new porphyrin compounds with particular structure and promising biological activity based on affordable starting materials is of considerable interest and substantially extends the potential of porphyrin applications.
In this context the functionalization of porphyrin macrocycles with biologically active nitrogen-containing heterocycles represents an important approach for the improvement of their PDT efficiency [30]. Now, synthesis of nitrogen-containing heterocyclic compounds has attracted an increased interest because of their utility for various biological receptors with a high degree of binding affinity. Triazine derivatives are attractive compounds with biologically important properties, and they have found a number of applications in medicinal chemistry as anticancer [31,32], antimalarial [33][34][35][36][37][38][39], antiviral [40,41] and antimicrobial [42] agents. A convenient compound for the functionalization of the 1,3,5triazine core with a variety of pharmacaphore groups is 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride). Furthermore, the three symmetrically positioned electrophilic centers of trichlorotriazine enable an alternative method for the selective introduction of diversity of functionalities through the successive substitution of the chlorine atoms with nucleophiles via the addition-elimination mechanism [43]. In recent years, our studies have focused on the design of boronated porphyrin derivatives for PDT and BNCT containing covalently bound 1,2,3-triazole [30] and maleimide moieties [44] on the periphery of mesotetraarylporphyrins. There is available information on the improved antitumor properties of tetrapyrrole photosensitizers containing boron polyhedra in PDT as compared with their non-boronated analogues [45][46][47]. At the same time, the literature survey indicated that carboranyl-substituted 1,3,5-triazines also demonstrated biological activity [48] and are efficient compounds towards creation BNCT agents. One, two or three carboranyl residues were incorporated into cyanuric chloride [49][50][51][52].
In this study, we report the synthesis of carboranyl-substituted porphyrin conjugates containing 1,3,5-triazine (s-triazine) heterocycle as a rigid spacer group by replacing one or two chlorine atoms of cyanuric chloride with different Sand N-carborane nucleophiles. Therefore, the combining within one molecule porphyrin, s-triazine heterocycle and carborane cluster may result in the molecules with new biological profile.

Synthesis
For the synthesis of porphyrin-triazine conjugates containing carborane clusters, readily available 5-(4-aminophenyl)-10,15,20-triphenylporphyrin 1 [53] and cyanuric chloride 2 were used as starting compounds. Cyanuric chloride 2 is an electron-deficient heterocycle and behaves as a heterocyclic acid chloride analog, in which three of its reactive chlorine atoms are capable of easily being substituted in S N Ar reactions. Therefore, it can serve as starting material for the preparation of substituted s-triazines, where the substituents are linked via nucleophilic heteroatoms such as oxygen, nitrogen or sulfur [54,55]. This process is temperature-dependent since the reactivity decreases with increasing number of substituents linked to the heterocycle. The structure of the nucleophile, its basic strength and steric factors as well as solvent used for the reaction affected the course of the replacement [56]. Moreover, some examples have been already reported dealing with the preparation of conjugates between the compound 2 and tetrapyrrolic macrocycles such as porphyrins and phthalocyanines [57][58][59][60][61].
Porphyrin 1 as N-nucleophile was reacted with compound 2 (molar ratio 1:1) at 0 • C in THF in the presence of N,N-diisopropylethylamine (DIPEA) to afford conjugate 3, in which one s-triazine chlorine atom was substituted with porphyrin substituent [62]. Its formation was confirmed by disappearance of 1 by TLC analysis in hexane-CHCl 3 (2:3) system. The reaction was completed in 10 min and, after the separation, conjugate 3 was isolated in excellent (97%) yield. The conjugate 3 was used as key compound in step-wise substitution of the s-triazine chlorine atoms with carborane nucleophiles. The conjugate 3 prepared as describe above but without isolation from the reaction mixture was further reacted with an excess of 9-mercapto-m-carborane 4 [63] at 65 • C in THF in the presence of DIPEA for 10 h (Scheme 1) to give compound 5 in 82% yield. Treatment of 5 with Zn(OAc) 2 . 2H 2 O in CHCl 3 -MeOH mixture afforded corresponding zinc complex 6 in 95% yield (Scheme 1). substitution of the s-triazine chlorine atoms with carborane nucleophiles. The conjugate 3 prepared as describe above but without isolation from the reaction mixture was further reacted with an excess of 9-mercapto-m-carborane 4 [63] at 65 °C in THF in the presence of DIPEA for 10 h (Scheme 1) to give compound 5 in 82% yield. Treatment of 5 with Zn(OAc)2 . 2H2O in CHCl3-MeOH mixture afforded corresponding zinc complex 6 in 95% yield (Scheme 1). The reaction of conjugate 3 with 1-mercapto-o-carborane 7 [64] containing functional SH-group bound to the carbon atom of carborane polyhedron proceeded smoothly at ambient temperature in THF in the presence of DIPEA for 2 h to afford conjugate 8 in 82% yield. Treatment of 8 with Zn(OAc)2 . 2H2O in CHCl3-MeOH mixture afforded corresponding zinc complex 9 in 94% yield (Scheme 2). In addition, our attempts to substitute chlorine atoms in 3 with 3-amino-o-carborane (10) [65] in boiling THF in the presence of DIPEA were unsuccessful. We explain that by the decrease of reactivity of the remaining chlorine atoms in 3 [56] and the lowered nucleophilicity [66] of amino group in amine 10 in comparison to mercaptocarboranes. At the same time, we have found that reaction of amine 10 with compound 2 was readily proceeded in the presence of DIPEA in THF at 0 °C for 40 min to give carboranylamino dichlorotriazine derivative 11 in 71% yield (Scheme 3). Scheme 1. Synthesis of conjugates 5 and 6.
The reaction of conjugate 3 with 1-mercapto-o-carborane 7 [64] containing functional SH-group bound to the carbon atom of carborane polyhedron proceeded smoothly at ambient temperature in THF in the presence of DIPEA for 2 h to afford conjugate 8 in 82% yield. Treatment of 8 with Zn(OAc) 2 . 2H 2 O in CHCl 3 -MeOH mixture afforded corresponding zinc complex 9 in 94% yield (Scheme 2). prepared as describe above but without isolation from the reaction mixture was further reacted with an excess of 9-mercapto-m-carborane 4 [63] at 65 °C in THF in the presence of DIPEA for 10 h (Scheme 1) to give compound 5 in 82% yield. Treatment of 5 with Zn(OAc)2 . 2H2O in CHCl3-MeOH mixture afforded corresponding zinc complex 6 in 95% yield (Scheme 1). The reaction of conjugate 3 with 1-mercapto-o-carborane 7 [64] containing functional SH-group bound to the carbon atom of carborane polyhedron proceeded smoothly at ambient temperature in THF in the presence of DIPEA for 2 h to afford conjugate 8 in 82% yield. Treatment of 8 with Zn(OAc)2 . 2H2O in CHCl3-MeOH mixture afforded corresponding zinc complex 9 in 94% yield (Scheme 2). In addition, our attempts to substitute chlorine atoms in 3 with 3-amino-o-carborane (10) [65] in boiling THF in the presence of DIPEA were unsuccessful. We explain that by the decrease of reactivity of the remaining chlorine atoms in 3 [56] and the lowered nucleophilicity [66] of amino group in amine 10 in comparison to mercaptocarboranes. At the same time, we have found that reaction of amine 10 with compound 2 was readily proceeded in the presence of DIPEA in THF at 0 °C for 40 min to give carboranylamino dichlorotriazine derivative 11 in 71% yield (Scheme 3). In addition, our attempts to substitute chlorine atoms in 3 with 3-amino-o-carborane (10) [65] in boiling THF in the presence of DIPEA were unsuccessful. We explain that by the decrease of reactivity of the remaining chlorine atoms in 3 [56] and the lowered nucleophilicity [66] of amino group in amine 10 in comparison to mercaptocarboranes. At the same time, we have found that reaction of amine 10 with compound 2 was readily proceeded in the presence of DIPEA in THF at 0 • C for 40 min to give carboranylamino dichlorotriazine derivative 11 in 71% yield (Scheme 3). The introduction of porphyrin entity into compound 11 was achieved through the condensation of porphyrin 1 amino group with chlorine atom in compound 11 at ambient temperature for 3 h in THF using DIPEA as an acid scavenger to afford conjugate 12 in 72% yield after the purification by column chromatography (Scheme 4). The introduction of porphyrin entity into compound 11 was achieved through the condensation of porphyrin 1 amino group with chlorine atom in compound 11 at ambient temperature for 3 h in THF using DIPEA as an acid scavenger to afford conjugate 12 in 72% yield after the purification by column chromatography (Scheme 4). The introduction of porphyrin entity into compound 11 was achieved through the condensation of porphyrin 1 amino group with chlorine atom in compound 11 at ambient temperature for 3 h in THF using DIPEA as an acid scavenger to afford conjugate 12 in 72% yield after the purification by column chromatography (Scheme 4). It turned out that we failed to replace the remaining chlorine atom in conjugate 12 with carborane 10, but its substitution proceeded easily when reacting with thioglycolic acid (13), methyl ester of glycine hydrochloride (14), mercaptoethanol (15) and hexafluoro-2-propanol (HFIP) (16) in THF in the presence of DIPEA or DIPEA/DMAP system in THF to give corresponding derivatives 17-20 in rather good yields (Scheme 5). It can be also assumed that simultaneous introduction of substituents with COOH, COOMe, OH and CF3 functional groups along with porphyrin and carborane ones in s-triazine system may improve the biological characteristics of resulting compounds.  It turned out that we failed to replace the remaining chlorine atom in conjugate 12 with carborane 10, but its substitution proceeded easily when reacting with thioglycolic acid (13), methyl ester of glycine hydrochloride (14), mercaptoethanol (15) and hexafluoro-2-propanol (HFIP) (16) in THF in the presence of DIPEA or DIPEA/DMAP system in THF to give corresponding derivatives 17-20 in rather good yields (Scheme 5). It can be also assumed that simultaneous introduction of substituents with COOH, COOMe, OH and CF 3 functional groups along with porphyrin and carborane ones in s-triazine system may improve the biological characteristics of resulting compounds. The introduction of porphyrin entity into compound 11 was achieved through the condensation of porphyrin 1 amino group with chlorine atom in compound 11 at ambient temperature for 3 h in THF using DIPEA as an acid scavenger to afford conjugate 12 in 72% yield after the purification by column chromatography (Scheme 4).

Scheme 4. Synthesis of conjugate 12.
It turned out that we failed to replace the remaining chlorine atom in conjugate 12 with carborane 10, but its substitution proceeded easily when reacting with thioglycolic acid (13), methyl ester of glycine hydrochloride (14), mercaptoethanol (15) and hexafluoro-2-propanol (HFIP) (16) in THF in the presence of DIPEA or DIPEA/DMAP system in THF to give corresponding derivatives 17-20 in rather good yields (Scheme 5). It can be also assumed that simultaneous introduction of substituents with COOH, COOMe, OH and CF3 functional groups along with porphyrin and carborane ones in s-triazine system may improve the biological characteristics of resulting compounds. A similar synthetic approach was used for the preparation of trisubstituted s-triazine conjugate 21 starting from the disubstituted triazine conjugate 22, which was formed via the treatment of THF solution of conjugate 3 with 9-mercapto-m-carborane (4) (1 equiv, DIPEA, 8h). Compound 22 was not isolated but was further converted into the target compound 21 (85%) by refluxing with an excess of 2-aminoethanethiol hydrochloride (23) and DIPEA for 15 h (Scheme 6). A similar synthetic approach was used for the preparation of trisubstituted s-triazine conjugate 21 starting from the disubstituted triazine conjugate 22, which was formed via the treatment of THF solution of conjugate 3 with 9-mercapto-m-carborane (4) (1 equiv, DIPEA, 8h). Compound 22 was not isolated but was further converted into the target compound 21 (85%) by refluxing with an excess of 2-aminoethanethiol hydrochloride (23) and DIPEA for 15 h (Scheme 6). In light of maleimides importance in medicinal chemistry, conjugates 24, 25 containing a carboranylmaleimide fragments have been obtained by the reaction of phenylenediamine-substituted porphyrin 26 (prepared from conjugate 3 and . This reaction resulted in the formation of two products. The major product 24 Scheme 6. Synthesis of conjugate 21. In light of maleimides importance in medicinal chemistry, conjugates 24, 25 containing a carboranylmaleimide fragments have been obtained by the reaction of phenylenediaminesubstituted porphyrin 26 (prepared from conjugate 3 and 1,4-phenylenediamine 27) with 3bromo-1-(N-(o-carborane-3 -yl)maleimide (28) [67] (Scheme 7). This reaction resulted in the formation of two products. The major product 24 was zwitter-ionic nido-carboranemaleimidesubstituted conjugate (89% yield); the minor product was closo-carborane conjugate 25 (8% yield). Scheme 6. Synthesis of conjugate 21.

Spectroscopic Data
All synthesized compounds were isolated by column chromatography and their structures were fully characterized by UV-vis, IR and 1 H, 11 B, 11 B{ 1 H} NMR spectroscopies and MALDI or ESI mass spectrometry. IR spectra of boronated porphyrins 5, 6, 8, 9, 12, 17-21 and 25 contain strong bands for the B-H stretching vibrations in the region of 2587 to 2608 cm -1 supporting the carborane closo-structure, strong band for the B-H stretching vibrations at 2521 cm -1 for porphyrin 24 supporting the carborane nido-structure and for N-H stretching vibrations in the region 3269-3418 cm -1 . The band at 3054-3075 cm -1 indicated the presence of the carborane CH groups. Additionally, the IR spectra of all prepared porphyrins 5, 6, 8, 9, 12, 17-21, 24 and 25 showed the characteristic band in the regions 1521-1596 cm -1 corresponding to the C = N bonds of the s-triazine heterocycle. In 1 H NMR eight β-protons of porphyrin macrocycle for all compounds were found between 8.80 and 9.00 ppm and nineteen meso-phenyl protons of porphyrins were observed between 7.68 and 8.31 ppm.
The expected signals for functionalities linked at the s-triazine spacer were also observed confirming the structure of compounds 5, 6, 8, 9, 12, 17-21, 24 and 25 (See Materials and Methods and Supplementary Materials Figures S1-S13 for details).

UV-Visible Absorption and Fluorescence Spectra
The UV-vis spectra of newly synthesized compounds 5, 6, 8, 9, 17-21 recorded in CH 2 Cl 2 and compound 24 recorded in (CH 3 ) 2 CO are shown in Figure 1. Most of them demonstrate an intense Soret band between 419 and 421 nm, only for a maleimidesubstituted conjugate 24 the corresponding band was found at 416 nm likely due to an increase of HOMO-LUMO energy gap. between 7.68 and 8.31 ppm.
The expected signals for functionalities linked at the s-triazine spacer were also observed confirming the structure of compounds 5, 6, 8, 9, 12, 17-21, 24 and 25 (See Materials and Methods and Supplementary Materials Figures S1-S13 for details).

UV-Visible Absorption and Fluorescence Spectra
The UV-vis spectra of newly synthesized compounds 5, 6, 8, 9, 17-21 recorded in CH2Cl2 and compound 24 recorded in (CH3)2CO are shown in Figure 1. Most of them demonstrate an intense Soret band between 419 and 421 nm, only for a maleimide-substituted conjugate 24 the corresponding band was found at 416 nm likely due to an increase of HOMO-LUMO energy gap.
Spectra of all the porphyrins exhibit four Q bands between 514 and 647 nm. The intensity pattern of Q bands is of etio-type (IV > III > II > I) in spite of asymmetry of substitution. It reflects the absence of distortion of the π-conjugation of porphyrin core probably due to buffer effect of phenyl groups. In the case of metalloporphyrins (6 and 9), there are only two bands of Q type as a result of symmetry enhancement from D2h in porphyrins to D4h in metalloporphyrins. Spectra of all the porphyrins exhibit four Q bands between 514 and 647 nm. The intensity pattern of Q bands is of etio-type (IV > III > II > I) in spite of asymmetry of substitution. It reflects the absence of distortion of the π-conjugation of porphyrin core probably due to buffer effect of phenyl groups. In the case of metalloporphyrins (6 and 9), there are only two bands of Q type as a result of symmetry enhancement from D 2h in porphyrins to D 4h in metalloporphyrins.
Fluorescence spectra of synthesized compounds 8, 17, 18, 24 ( Figure 2) were similar, showing two maxima at 655 nm and 715 nm. The spectrum of porphyrin 9 also showed two fluorescence peaks, with maxima at 610 and 660 nm, which can be explained by the presence of zinc in the molecule.

Cytotoxicity
We evaluated the cytotoxicity of carboranylporphyrins 8, 9, 17, 18 and 24 for tumor cell lines in the dark and after light activation. To determine the dark cytotoxicity, the HCT116 human colon carcinoma cells were exposed to the compounds for 72 h followed

Cytotoxicity
We evaluated the cytotoxicity of carboranylporphyrins 8, 9, 17, 18 and 24 for tumor cell lines in the dark and after light activation. To determine the dark cytotoxicity, the HCT116 human colon carcinoma cells were exposed to the compounds for 72 h followed by MTT tests. Under these conditions, the cytotoxicity was insignificant: cell survival in the studied range of concentrations was > 75% (For details see Supplementary Materials Figure  S14). After photoexcitation with a diode laser at 400 nm, 2 J/cm 2 , cell viability markedly decreased. Upon photoactivation, carboranylporphyrins 9 and 24 were the most potent and caused a dose-dependent cell death after illumination. IC 50 values were 20 ± 1.8 and 16 ± 0.9 µM, respectively (see Supplementary Materials Figure S15). As positive controls, the disodium salt of chlorin e6 (Photolon) and the fluorinated carboranylchlorin FBCh with the reported PS characteristics were used [68].
Photoexcitation of porphyrins and chlorins in a biological experiment made it possible to compare the obtained results. Photoinduced cytotoxicity data ( Figure S15) indicated that the activity dosage form of the clinical PS was somewhat higher, probably due to the fact that the time of intracellular accumulation of prepared carboranylporphyrins 9, 24 is higher than of Photolon (24 h versus 1.5 h [69], respectively). The activity of compounds 9 and 24 was comparable with FBCh.
Finally, compared to original tetraphenylporphyrin (TPP), the carboranylporphyrins 9 and 24 exhibited an improved phototoxicity in HCT116 cells whereas the dark cytotoxicity was low. Indeed, TPP in the dark did not show cytotoxicity at 0.1-50 µM for 48 h. Upon intracellular accumulation for 24 h, photoexcitation after a higher light exposure (33 J/cm2) followed by 24 h incubation (to allow cell death), TPP showed no photodynamic activity [30].

Intracellular Localization and Photoinduced Cell Death
Colocalization of carboranylporphyrins 9 and 24 with mitochondrial and lysosomal trackers after 24h of accumulation is shown in Figure 3.  Both 9 and 24 were distributed in the cytoplasm but not in nuclei. Both compounds colocalized with mitochondria and lysosomes. Compound 9 was distributed as granules. In addition to mitochondria, its colocalization with the lysosomal dye was clearly detectable. For compound 24, accumulation in lysosomes was less pronounced. During activation with the microscope laser at 405 nm (excitation of Hoechst 33342) an increase in the ROS probe dihydrorhodamine 123 (DHR 123) (Figure 4). Both 9 and 24 were distributed in the cytoplasm but not in nuclei. Both compounds colocalized with mitochondria and lysosomes. Compound 9 was distributed as granules. In addition to mitochondria, its colocalization with the lysosomal dye was clearly detectable. For compound 24, accumulation in lysosomes was less pronounced. During activation with the microscope laser at 405 nm (excitation of Hoechst 33342) an increase in the ROS probe dihydrorhodamine 123 (DHR 123) (Figure 4). Both 9 and 24 were distributed in the cytoplasm but not in nuclei. Both compounds colocalized with mitochondria and lysosomes. Compound 9 was distributed as granules. In addition to mitochondria, its colocalization with the lysosomal dye was clearly detectable. For compound 24, accumulation in lysosomes was less pronounced. During activation with the microscope laser at 405 nm (excitation of Hoechst 33342) an increase in the ROS probe dihydrorhodamine 123 (DHR 123) (Figure 4).  ROS generation occurred 5 minutes after photoexcitation of porphyrins 9, 24 accumulated by the cell, which is an event preceding cell death. To analyze necrotic non-repairable membrane damage under light excitation of photosensitizers, propidium iodide was added to the cell buffer during shooting as a marker of the plasma membrane integrity, and it's binding to nucleic acids in nucleus made it possible to detect necrotic membrane damage. Compounds 9, 24 were accumulated cells for 24 h, nuclei were stained with Hoechst 33342 and photosensitizers were photoexcited with a confocal microscope laser at a wavelength of 405 nm in the presence of propidium iodide in the cell buffer ( Figure 5).
Molecules 2022, 27, 6200 9 of 18 ROS generation occurred 5 minutes after photoexcitation of porphyrins 9, 24 accumulated by the cell, which is an event preceding cell death. To analyze necrotic non-repairable membrane damage under light excitation of photosensitizers, propidium iodide was added to the cell buffer during shooting as a marker of the plasma membrane integrity, and it's binding to nucleic acids in nucleus made it possible to detect necrotic membrane damage. Compounds 9, 24 were accumulated cells for 24 h, nuclei were stained with Hoechst 33342 and photosensitizers were photoexcited with a confocal microscope laser at a wavelength of 405 nm in the presence of propidium iodide in the cell buffer ( Figure 5). Propidium iodide (PI) entered the cells (a hallmark of the plasma membrane damage) within the initial minutes of illumination. Both 9 and 24 demonstrated the photodynamic potency similar to the reference photosensitizer FBCh [61]. Thus, the mechanism of cell death upon photoexcitation of lead photosensitizers 9 and 24 is rapid necrosis.

General Information
Reagents were from Sigma-Aldrich unless specified otherwise. All reactions were performed in an atmosphere of dry argon. All solvents were dried as recommended in standard protocols. Propidium iodide (PI) entered the cells (a hallmark of the plasma membrane damage) within the initial minutes of illumination. Both 9 and 24 demonstrated the photodynamic potency similar to the reference photosensitizer FBCh [61]. Thus, the mechanism of cell death upon photoexcitation of lead photosensitizers 9 and 24 is rapid necrosis.

General Information
Reagents were from Sigma-Aldrich unless specified otherwise. All reactions were performed in an atmosphere of dry argon. All solvents were dried as recommended in standard protocols. 1  MALDI mass spectra for porphyrins were recorded on a Brucer autoflex speed timeof-flight (TOF) mass obtained mass spectrometer (Bruker Daltonics Inc., Bremen, Germany) equipped with a solid-state ultraviolet (UV) laser of 355 nm (1 kHz repetition rate, 1000 shots for each spectrum) and operated in positive reflectron mode. MALDI mass spectra were recorded by using stainless-steel targets (MTP 384 ground steel; Bruker Daltonics Inc., Germany) containing 384 cells for the deposition of the analyte mixed with matrix; the most intense peaks were given for each compound.
LC-MS (liquid chromatography-mass spectrometry) analysis of the reaction products was performed on Shimadzu LCMS-2020 High Performance Liquid Chromatograph Mass Spectrometer with electrospray ionization (ESI) method and single quadrupole detector (negative and positive ions). Desolvation line/heat block temperature were 250/400 • C. Nitrogen (99.5%) was used like nebulizer and drying gas. Acetonitrile (99.9 + % HPLC gradient grade, Chem-Lab) was used as mobile phase with flow rate 0.8 ml/min without any pretreatment. Compounds were dissolved in acetone, injection volume 30 µL. The mass range between 50 and 2000 was scanned.

Cell Culture
The human colon adenocarcinoma cell line HCT116 was obtained from American Type Culture Collection (Manassas, VA, USA). Cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (PanEco, Russia) at 37 • C, 5% CO 2 in a humidified atmosphere. Cells in logarithmic phase of growth were used in the experiments.

Cytotoxicity Studies
The cytotoxicity of compounds for HCT116 human colon carcinoma cells was assessed in MTT assays. Cells were plated in 96 well plates (NUNC, USA, 5·10 3 cells in 190 µl culture medium per well). After 24 h at 37 • C, 5% CO 2 in a humidified atmosphere, cells were treated with compounds dissolved from 10 mM stocks in DMSO. Dark cytotoxicity was assessed in a formazan conversion assay (MTT-test) after a 72 h exposure. For photoinduced cytotoxicity, cells were loaded with tested compounds for 24 h, the medium was replaced with a fresh one and cells were illuminated with a 400 nm laser, 2 J/cm 2 (AFC Polironics, Russia). After the completion of drug exposure in the dark or drug accumulation/illumination, 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent) was added to cells for 2 h, the culture medium was removed, cells were resuspended in 100 µL DMSO and the optical densities were measured on a Multiscan FC plate spectrophotometer (Thermo Scientific, Waltham, MA, USA) at a wavelength of 571 nm. The percentage of survived cells for each dose was calculated as the quotient of the average optical density in the wells after incubation with this dose to the average optical density of the control wells (the values of the latter are taken as 100%). Four independent experiments were performed for each concentration. Standard deviations did not exceed 10%.

Conclusions
A convenient synthetic approach for the preparation of s-triazines containing porphyrin and carborane clusters was developed by nucleophilic substitution of the chlorine atom in cyanuric chloride with 5-(4-aminophenyl)-10,15,20-triphenylporphyrin, followed by the substitution of remaining chlorine atoms with carborane clusters or other S, N, Onucleophile functionalities. The prepared compounds were fully characterized by UV-vis, IR, 1 H and 11 B NMR spectroscopy and mass-spectrometry. As a result, it was shown that cyanuric chloride is a convenient scaffold for the creation of multicomponent systems efficient for biomedical and other application.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27196200/s1, Figure S1. The 1 H, 11 B NMR spectra of 5 in CDCl 3 ; Figure S2. The 1 H, 11 B NMR spectra of 6 in (CD 3 ) 2 CO; Figure S3. The 1 H and 11 B NMR spectra of 8 in CDCl 3 ; Figure S4. The 1 H, 11 B NMR spectra of 9 in CDCl 3 ; Figure S5. The 1 H and 11 B NMR spectra of 11 in CDCl 3 ; Figure S6. The 1 H and 11 B NMR spectra of 12 in CDCl 3 ; Figure S7. The 1 H and 11 B NMR spectra of 17 in CDCl 3 ; Figure S8. The 1 H and 11 B NMR spectra of 18 in CDCl 3 ; Figure   S9. The 1 H and 11 B NMR spectra of 19 in CDCl 3 ; Figure S10. The 1 H, 11 B and 19 F spectra of 20 in CDCl 3 ; Figure S11. The 1 H, 11 B NMR spectra of 21 in CDCl 3 ; Figure S12. The 1 H and 11 B NMR spectra of 24 in (CD 3 ) 2 CO; Figure S13. The 1 H and 11