New Azido Coumarins as Potential Agents for Fluorescent Labeling and Their “Click” Chemistry Reactions for the Conjugation with closo-Dodecaborate Anion

Novel fluorescent 7-methoxy- and 7-(diethylamino)-coumarins modified with azido-group on the side chain have been synthesized. Their photophysical properties and single crystals structure characteristics have been studied. In order to demonstrate the possibilities of fluorescent labeling, obtained coumarins have been tested with closo-dodecaborate derivative bearing terminal alkynyl group. CuI catalyzed Huisgen 1,3-dipolar cycloaddition reaction has led to fluorescent conjugates formation. The absorption–emission spectra of the formed conjugates have been presented. The antiproliferative activity and uptake of compounds against several human cell lines were evaluated.

However, in some cases labelling requires the use of coumarin azides with a shorter spacer between the fluorescent part and the azide function. Herein, we report the synthesis, fluorescence properties, and crystal structures of two new azido derivatives of coumarin and their use for labelling of the closo-dodecaborate anion, including the evaluation of antiproliferative activity and uptake of the synthesized compounds against several human cell lines.
An effective approach to the functionalization of the closo-dodecaborate anion is the opening of its cyclic oxonium derivatives by nucleophiles [76]. This approach is widely used for the synthesis of its various functional derivatives and attachment to biomolecules Since the discovery by Sharpless the term of "click chemistry" for the [3+2] cycloaddition reactions of alkynes with organic azides, which are high-yielding, practical, operationally safe, avoid by-product formation, and proceed in environment friendly solvents at room temperature and generally under mild conditions [49,50], this approach is widely used for synthesis of various bioactive coumarin derivatives [51,52], as well as fluorescent labeling of nanoparticles and biomolecules using azido derivatives of coumarin, such as coumarin 343 azide ( Figure 1) [53][54][55].
However, in some cases labelling requires the use of coumarin azides with a shorter spacer between the fluorescent part and the azide function. Herein, we report the synthesis, fluorescence properties, and crystal structures of two new azido derivatives of coumarin and their use for labelling of the closo-dodecaborate anion, including the evaluation of antiproliferative activity and uptake of the synthesized compounds against several human cell lines.

Strategy
The sodium salt of the mercapto derivative of the closo-dodecaborate anion Na 2 [B 12 H 11 SH] (BSH) is one of two clinically approved agents for boron neutron capture therapy (BNCT) for cancer [56][57][58], which is an anti-cancer treatment based on the irradiation of 10 B-rich tumours with low energy neutrons. The neutron capture reaction produces high linear energy transfer particles, 4 He (α-particle) and 7 Li, which cause lethal damage to tumour cells through ionization process [59][60][61][62]. This causes increased interest in the synthesis of conjugates of the closo-dodecaborate anion with various biologically active molecules [57,58,[63][64][65][66].
An effective approach to the functionalization of the closo-dodecaborate anion is the opening of its cyclic oxonium derivatives by nucleophiles [76]. This approach is widely used for the synthesis of its various functional derivatives and attachment to biomolecules [64,77,78], and has also been used to modify other polyhedral boron hydrides such as the closo-decaborate anion [64], nido-carborane [79], and cobalt and iron bis(dicarbollides) [80]. In particular, the synthesis of the earlier described coumarin derivatives of the closo-dodecaborate anion is based on the opening of oxonium rings with hydroxy groups at various positions of the coumarin heterocycle [72][73][74]. This leads to a significant difference in the photophysical characteristics of the obtained compounds from the commonly used fluorescent labels (see above). Therefore, our goal was to preserve the structure of coumarins traditionally used for labeling. We considered it expedient to use the [3+2]cycloaddition reaction for the synthesis of coumarin derivatives of the closo-dodecaborate anion. The "click" chemistry reaction has found a wide synthetic application for the preparation of a wide range of conjugates based on the boron cluster [81][82][83][84][85][86][87][88][89][90]. However, the opening of the 1,4-dioxane derivative results in moieties where the boron cluster connected with functional fragment via a biologically compatible flexible di(ethylene glycol) spacer. Thus, in particular, derivatives of the closo-dodecaborate anion with a terminal alkynyl group were obtained [91,92]. Therefore, the use of commercially available coumarin 343 azide will result in a product with an unnecessarily long spacer between the boron cluster and the fluorescent label.

Synthesis
The target coumarins functionalized with azido group 2a,b were synthesized as shown in Scheme 1. [64,77,78], and has also been used to modify other polyhedral boron hydrides such as the closo-decaborate anion [64], nido-carborane [79], and cobalt and iron bis(dicarbollides) [80]. In particular, the synthesis of the earlier described coumarin derivatives of the closo-dodecaborate anion is based on the opening of oxonium rings with hydroxy groups at various positions of the coumarin heterocycle [72][73][74]. This leads to a significant difference in the photophysical characteristics of the obtained compounds from the commonly used fluorescent labels (see above). Therefore, our goal was to preserve the structure of coumarins traditionally used for labeling. We considered it expedient to use the [3+2]-cycloaddition reaction for the synthesis of coumarin derivatives of the closo-dodecaborate anion. The "click" chemistry reaction has found a wide synthetic application for the preparation of a wide range of conjugates based on the boron cluster [81][82][83][84][85][86][87][88][89][90]. However, the opening of the 1,4-dioxane derivative results in moieties where the boron cluster connected with functional fragment via a biologically compatible flexible di(ethylene glycol) spacer. Thus, in particular, derivatives of the closo-dodecaborate anion with a terminal alkynyl group were obtained [91,92]. Therefore, the use of commercially available coumarin 343 azide will result in a product with an unnecessarily long spacer between the boron cluster and the fluorescent label.

Synthesis
The target coumarins functionalized with azido group 2a,b were synthesized as shown in Scheme 1. The starting 7-methoxycoumarin-3-carboxylic acid 1a and 7-diethylaminocoumarin-3-carboxylic acid 1b were prepared according to the literature procedures by the Knoevenagel condensation reaction of 4-substituted salicylaldehyde with diethyl malonate [93], or with ethyl cyanoacetate [94], respectively, followed by hydrolysis reactions. Coumarins 1a,b undergo amidation reaction with 2-azidoethylamine hydrochloride in refluxing acetonitrile in the presence of Et3N as a base. The reaction progress was monitored by thin layer chromatography. It should be noted that, after the cooling of the reaction mixture, the products precipitate from the reaction mixture, and in the case of derivative 2а, in the form of crystals suitable for single crystal X-ray diffraction study. The structures of the obtained compounds 2a,b were established using IR, 1 H NMR, and 13 C NMR spectroscopy (see Supplementary Materials). The 1 H NMR spectra of each compound in CDCl3 contain, in addition to characteristic signals of the coumarin moiety, broad triplets of the NH-amide protons at 9.0 ppm and signals from the two side chain methylene groups at ~3.6 and 3.5 ppm. In the IR spectra, the characteristic absorption bands of the N3 group appear at 2125 and 2098 cm −1 for 2a and 2b, respectively.
To demonstrate the ability of novel azido coumarins 2a,b to form conjugates with fluorescent properties, we have tested them in reactions with a closo-dodecaborate derivative containing a terminal alkynyl group in the side chain 3[NBu4], under typical "click" reaction conditions. As a result, new 1,2,3-triazoles 4a[Cs] and 4b[Cs] were obtained in high yields (Scheme 2). The starting 7-methoxycoumarin-3-carboxylic acid 1a and 7-diethylaminocoumarin-3-carboxylic acid 1b were prepared according to the literature procedures by the Knoevenagel condensation reaction of 4-substituted salicylaldehyde with diethyl malonate [93], or with ethyl cyanoacetate [94], respectively, followed by hydrolysis reactions. Coumarins 1a,b undergo amidation reaction with 2-azidoethylamine hydrochloride in refluxing acetonitrile in the presence of Et 3 N as a base. The reaction progress was monitored by thin layer chromatography. It should be noted that, after the cooling of the reaction mixture, the products precipitate from the reaction mixture, and in the case of derivative 2a, in the form of crystals suitable for single crystal X-ray diffraction study. The structures of the obtained compounds 2a,b were established using IR, 1 H NMR, and 13 C NMR spectroscopy (see Supplementary Materials). The 1 H NMR spectra of each compound in CDCl 3 contain, in addition to characteristic signals of the coumarin moiety, broad triplets of the NH-amide protons at 9.0 ppm and signals from the two side chain methylene groups at~3.6 and 3.5 ppm. In the IR spectra, the characteristic absorption bands of the N 3 group appear at 2125 and 2098 cm −1 for 2a and 2b, respectively.
To demonstrate the ability of novel azido coumarins 2a,b to form conjugates with fluorescent properties, we have tested them in reactions with a closo-dodecaborate derivative containing a terminal alkynyl group in the side chain 3[NBu 4 ], under typical "click" reaction conditions. As a result, new 1,2,3-triazoles 4a[Cs] and 4b[Cs] were obtained in high yields (Scheme 2). The formation of conjugates 4a,b was monitored by the TLC method, and the full conversion was gained in 16 h. The conjugation products were isolated by precipitation in the form of the Cs + salts and characterized by 1 H, 11 B, 13 C NMR, and IR spectroscopy as well as high-resolution mass spectrometry (see Supplementary Materials). The formation of the 1,2,3-triazole heterocycle results in the appearance of the corresponding singlets at ~ 8.45 ppm in the 1 H NMR spectra. In the IR spectra of 4a,b, the absorption bands of the BH stretching (2487 cm −1 ) demonstrate the presence of the boron hydride cluster, and absorption bands corresponding to the 1,2,3-triazole heterocycle (1705 and 1710 cm −1 , respectively) were detected. In contrast to azido-coumarins 2a,b, fluorescent conjugates 4a,b possess a good solubility in water.

Single Crystal X-ray Diffraction Study of Azido Derivatives of Coumarins
The molecular structure of the obtained azido derivatives of coumarins 2a and 2b were determined by a single crystals X-ray diffraction study ( Figure 2). The formation of conjugates 4a,b was monitored by the TLC method, and the full conversion was gained in 16 h. The conjugation products were isolated by precipitation in the form of the Cs + salts and characterized by 1 H, 11 B, 13 C NMR, and IR spectroscopy as well as high-resolution mass spectrometry (see Supplementary Materials). The formation of the 1,2,3-triazole heterocycle results in the appearance of the corresponding singlets at~8.45 ppm in the 1 H NMR spectra. In the IR spectra of 4a,b, the absorption bands of the BH stretching (2487 cm −1 ) demonstrate the presence of the boron hydride cluster, and absorption bands corresponding to the 1,2,3-triazole heterocycle (1705 and 1710 cm −1 , respectively) were detected. In contrast to azido-coumarins 2a,b, fluorescent conjugates 4a,b possess a good solubility in water.

Single Crystal X-ray Diffraction Study of Azido Derivatives of Coumarins
The molecular structure of the obtained azido derivatives of coumarins 2a and 2b were determined by a single crystals X-ray diffraction study ( Figure 2). The formation of conjugates 4a,b was monitored by the TLC method, and the full conversion was gained in 16 h. The conjugation products were isolated by precipitation in the form of the Cs + salts and characterized by 1 H, 11 B, 13 C NMR, and IR spectroscopy as well as high-resolution mass spectrometry (see Supplementary Materials). The formation of the 1,2,3-triazole heterocycle results in the appearance of the corresponding singlets at ~ 8.45 ppm in the 1 H NMR spectra. In the IR spectra of 4a,b, the absorption bands of the BH stretching (2487 cm −1 ) demonstrate the presence of the boron hydride cluster, and absorption bands corresponding to the 1,2,3-triazole heterocycle (1705 and 1710 cm −1 , respectively) were detected. In contrast to azido-coumarins 2a,b, fluorescent conjugates 4a,b possess a good solubility in water.

Single Crystal X-ray Diffraction Study of Azido Derivatives of Coumarins
The molecular structure of the obtained azido derivatives of coumarins 2a and 2b were determined by a single crystals X-ray diffraction study ( Figure 2).    2a and 2b, respectively), which is typical for primary 3-arylamido-and 3-hydrazidocoumarins [95][96][97][98]. The conformations of the azidoethyl fragments are pronouncedly different in 2a and 2b: while the anti-conformation is observed for the substituents at C11-C12 bond in 2a (the N1-C11-C12-N2 torsion angle is 170.5(1)°), the gosh-conformation is observed in 2b (the corresponding torsion angle is 61.2(1)°). In its turn, the N3 group is antiperiplanar to the C11-C12 bond in 2a (175.0(1)°) and to the C12-H12A bond in 2b (173.8(1)°). This difference can be readily explained by the peculiarities of crystal packing ( Figure 3): in 2b, the C12(H) group participates in CH…O contacts with O2 oxygen atoms of two neighboring molecules, whereas the only meaningful interaction for the azidoethyl fragment in 2a is the stacking interaction between N3 moieties (N…N 3.07 Å), which were also observed in some other organic compounds with the azido group [99,100]. In general, the morphology of crystal packings of 2a and 2b are still similar (see Supplementary Materials, Figure S1): the layer-type arrangement of molecules is stabilized by stacking interactions between coumarin acylamide fragments and CH…O hydrogen bonds.

Photophysical Properties
The UV-vis spectra of compounds 2a and 2b were measured in acetonitrile. In contrast, the derivatives 4a and 4a are soluble in water, and their absorption spectra were measured in water correspondingly ( Figure 4). All compounds demonstrate intense bands in the short wavelength region of the π → π* character within the coumarin aromatic system. Differences at the absorption maxima suggest that the donor-acceptor properties of the 7-substituted group affect the excitation energy. Compounds 2a and 4a with -OMe substituent demonstrate bands at 350 and 343 nm respectively. Compounds 2b and 4b containing -NEt2 substituent demonstrate bands in the lower energy region (413 and 430 nm, respectively). Compounds 2a, 2b in CH3CN, and 4a, 4b in water demonstrate unstructured broad bands centered at ca. 400 and 470 nm for OMe and NEt2 substituted

Photophysical Properties
The UV-vis spectra of compounds 2a and 2b were measured in acetonitrile. In contrast, the derivatives 4a and 4a are soluble in water, and their absorption spectra were measured in water correspondingly ( Figure 4). All compounds demonstrate intense bands in the short wavelength region of the π → π* character within the coumarin aromatic system. Differences at the absorption maxima suggest that the donor-acceptor properties of the 7-substituted group affect the excitation energy. Compounds 2a and 4a with -OMe substituent demonstrate bands at 350 and 343 nm respectively. Compounds 2b and 4b containing -NEt 2 substituent demonstrate bands in the lower energy region (413 and 430 nm, respectively). Compounds 2a, 2b in CH 3 CN, and 4a, 4b in water demonstrate unstructured broad bands centered at ca. 400 and 470 nm for OMe and NEt 2 substituted analogues, respectively ( Figure 5, Table 1). The emission observed is a fluorescence, being typical for substituted coumarins [101,102]. Differences at the emission maxima demonstrate the effects of the donor properties of substituent on the emission. The 7-10 nm of the wavelength difference between 2a and 4a as well as between 2b and 4b may be explained by the solvent effect. analogues, respectively ( Figure 5, Table 1). The emission observed is a fluorescence, being typical for substituted coumarins [101,102]. Differences at the emission maxima demonstrate the effects of the donor properties of substituent on the emission. The 7-10 nm of the wavelength difference between 2a and 4a as well as between 2b and 4b may be explained by the solvent effect.     analogues, respectively ( Figure 5, Table 1). The emission observed is a fluorescence, being typical for substituted coumarins [101,102]. Differences at the emission maxima demonstrate the effects of the donor properties of substituent on the emission. The 7-10 nm of the wavelength difference between 2a and 4a as well as between 2b and 4b may be explained by the solvent effect.

Antiproliferative Activity and Cell Uptake of Boronated Coumarins
The antiproliferative activity of compounds 4a and 4b against the human HCT116 colorectal carcinoma, MCF7 breast adenocarcinoma, A549 non-small cell lung carcinoma and WI38 nonmalignant lung fibroblast cell lines was evaluated by means of a standard MTT colorimetric assay as the IC 50 value after 72 h of incubation. Compounds 4a and 4b were found to be not active up to 200 µM (See Supplementary Materials) and can be considered as non-toxic against cancerous and non-malignant cells. Therefore, they could be of potential interest for use in BNCT for cancer. Cellular accumulation plays a fundamental role in the antiproliferative activity of low-molecular-mass compounds [103,104]. One of the main requirements of effective BNCT is the selective accumulation of boron-containing compounds in the tumor with a fairly high concentration (approx. 10 9 10 B atoms/cell or 20-35 µg/gram [60]). Therefore, we measured uptake of compounds 4a and 4b at 250 µM concentration in A549 and WI38 cell lines for 1, 4, 6 and 24 h; the amount of boron was determined by ICP-MS ( Figure 6).

Antiproliferative Activity and Cell Uptake of Boronated Coumarins
The antiproliferative activity of compounds 4a and 4b against the human HCT116 colorectal carcinoma, MCF7 breast adenocarcinoma, A549 non-small cell lung carcinoma and WI38 nonmalignant lung fibroblast cell lines was evaluated by means of a standard MTT colorimetric assay as the IC50 value after 72 h of incubation. Compounds 4a and 4b were found to be not active up to 200 μM (See Supplementary Materials) and can be considered as non-toxic against cancerous and non-malignant cells. Therefore, they could be of potential interest for use in BNCT for cancer. Cellular accumulation plays a fundamental role in the antiproliferative activity of low-molecular-mass compounds [103,104]. One of the main requirements of effective BNCT is the selective accumulation of boron-containing compounds in the tumor with a fairly high concentration (approx. 10 9 10 B atoms/cell or 20-35 μg/gram [60]). Therefore, we measured uptake of compounds 4a and 4b at 250 μM concentration in A549 and WI38 cell lines for 1, 4, 6 and 24 h; the amount of boron was determined by ICP-MS ( Figure 6). The accumulation of compound 4b in both cancerous and normal cells occurs noticeably faster than compound 4a and reaches a maximum after 6 h for the WI38 cell line and after 24 h for the A549 cell line. At the same time, both compounds accumulated slightly better in the WI38 cell line. The cells of many tumours are known to be much larger than normal cells [105,106]; however, the close size of the A549 cells [107] and red blood cells [108] allows a rough estimate of the accumulation of the compound 4b in the cancer cells, which does not exceed 10 μg/g, which is apparently insufficient for effective BNCT.
All reactions were carried out in air. Analytical TLC was performed on Kieselgel 60 F245 (Merck, Darmstadt, Germany) plates. Boron compounds were visualized with PdCl2 stain solution, which upon heating gave dark brown spots. 1 H, 13 C and 11 B NMR spectra were recorded at 400.13, 100.61 and 128.38 MHz, respectively, on a BRUKER-Avance-400 spectrometer (Bruker, Karlsruhe-Zurich, Switzerland-Germany). Tetramethylsilane and BF3×Et2O were used as standards for 1 H and 13 C NMR and 11 B NMR, respectively. All The accumulation of compound 4b in both cancerous and normal cells occurs noticeably faster than compound 4a and reaches a maximum after 6 h for the WI38 cell line and after 24 h for the A549 cell line. At the same time, both compounds accumulated slightly better in the WI38 cell line. The cells of many tumours are known to be much larger than normal cells [105,106]; however, the close size of the A549 cells [107] and red blood cells [108] allows a rough estimate of the accumulation of the compound 4b in the cancer cells, which does not exceed 10 µg/g, which is apparently insufficient for effective BNCT.

Materials and Methods
Chemicals were reagent grade and received from commercial vendors. Acetonitrile was distilled from P 2 O 5 and then from CaH 2 . Compounds 1a [93], 1b [94], 2-azidoethylamine hydrochloride [109] and 3 [92] were prepared according to the literature procedures. All reactions were carried out in air. Analytical TLC was performed on Kieselgel 60 F245 (Merck, Darmstadt, Germany) plates. Boron compounds were visualized with PdCl 2 stain solution, which upon heating gave dark brown spots. 1 H, 13 C and 11 B NMR spectra were recorded at 400.13, 100.61 and 128.38 MHz, respectively, on a BRUKER-Avance-400 spectrometer (Bruker, Karlsruhe-Zurich, Switzerland-Germany). Tetramethylsilane and BF 3 × Et 2 O were used as standards for 1 H and 13 C NMR and 11 B NMR, respectively. All chemical shifts are reported in ppm (δ) relative to external standards. IR spectra were recorded on IR Prestige-21 (Shimadzu, Kyoto, Japan) instrument. The UV-vis spectra of solutions were measured on Cary 50 Uv-Vis Spectrophotometer (Varian, Palo Alto, CA, USA). The photoluminescence spectra were recorded on the Spectrofluorometer RF-6000 (Shimadzu). High resolution mass spectra (HRMS) were measured on a Bruker micrOTOF II instrument (Bruker Daltonic, Bremen, Germany) using electrospray ionization (ESI). For anionic boron containing compounds, the measurements were performed in a negative ion mode (3200 V) with a mass range from m/z 50 to m/z 3000; external and internal calibration was performed with ESI Tuning Mix (Agilent, Santa Clara, CA, USA). The boron content in cells was determined by inductively coupled plasma mass spectrometry (ICP-MS) using X-II spectrometer (Thermo Scientific, Waltham, MA, USA) and the following parameters: RF generator power 1400 W, nebulizer PolyCon, spray chamber cooling 3 • C, plasma gas flow rate 12 L/min, auxiliary flow rate 0.9 L/min, nebulizer flow rate 0.9 L/min, sample update 0.8 mL/min, resolution 0.8 M. The main parameters of MS measurements were: detector mode double (pulse counting and analogous) and scanning mode Survey Scan and Peak Jumping. The settings for the Survey Scan were: number of runs 10, dwell time 0.6 ms, channels per mass 10, acquisition 13.2 s. The settings for the Peak Jumping were: sweeps 25, dwell time 10 ms, channels per mass 1, acquisition 34 s.

Synthetic Procedures
To a suspension of 3-carboxy-7-methoxycoumarin 1a (0.5 g, 2.3 mmol) and 2-azidoethanamine hydrochloride (0.31 g, 2.5 mmol) in 50 mL of acetonitrile, NEt 3 was added in excess (approximately 0.5 mL). After the amine addition, the solution became clear. The reaction mixture was stirred under reflux for 16 h. Pale yellow crystals were formed after a slow cooling to room temperature; they were filtered washed with water (5 mL), cold CH 3 CN (5 mL) and air dried to give 0.32 g of 2. Another portion of 2a was purchased from the mother liquor by recrystallisation from EtOH to give while combining 0. To a suspension of 3-carboxy-7-diethylaminocoumarin 1b (0.5 g, 1.9 mmol) and 2azidoethanamine hydrochloride (0.26 g, 2.1 mmol) in 50 mL of acetonitrile, NEt 3 was added in excess (approximately 0.5 mL). After the amine addition, the solution became clear. The reaction mixture was stirred under reflux for 16 h. Then it was allowed to cool to room temperature. Yellow precipitate was formed after a cooling of the reaction mixture to 0 • C; it was filtered washed with water (5 mL), cold CH 3 CN (5 mL) and air dried. Mother liquor was evaporated to dryness and the oily residue was recrystallized from the EtOAc-EtOH mixture to give an additional portion of the title product. Orange solid. Yield: 63%. 0.4 g Crystals were obtained from the EtOAc solution while in slow evaporation.

Synthesis of Conjugate 4a
A mixture of alkyne 3 (NBu 4 ) (0.1 g, 0.2 mmol), 2a (0.052 g, 0.2 mmol), Et 3 N (0.5 mL) and CuI (0.01 g, 0.04 mmol) in 15 mL of CH 3 CN was stirred under reflux for 16 h. The reaction mixture was allowed to cool to room temperature and passed through a layer of silica gel (2-3 cm) on a Schott filter. The system was washed with CH 3 CN until the product ceased to be detected by thin layer chromatography. The solvent was removed on a rotary evaporator. The residue was dissolved in MeOH (10 mL) and an excess of CsF solution in MeOH was added. Formed precipitate was filtered, washed with MeOH (20 mL) and air dried to yield title 4a as a pale-yellow solid. 0.12 g. Yield: 91%. 1

Synthesis of Conjugates 4b
A mixture of alkyne 3 (NBu 4 ) (0.1 g, 0.2 mmol), 2b (0.06 g, 0.2 mmol), Et 3 N (0.5 mL) and CuI (0.01 g, 0.04 mmol) in 15 mL of CH 3 CN was stirred under reflux for 16 h. Then it was allowed to cool to room temperature and passed through a layer of silica gel (2-3 cm) on a Schott filter. The system was washed with CH 3 CN until the product ceased to be detected by thin layer chromatography. The solvent was removed on a rotary evaporator. The residue was dissolved in MeOH (10 mL) and an excess of CsF solution in MeOH was added. Formed precipitate was filtered (attention! very fine powder), washed with MeOH (20 mL) and air dried to yield title 4b as yellow solid. 0.09 g. Yield: 63%.

In Vitro Antiproliferative Assays and Cellular Uptake
The human HCT116 colorectal carcinoma, MCF7 breast adenocarcinoma, A549 nonsmall cell lung carcinoma, and WI38 nonmalignant lung fibroblast cell lines were obtained from the European collection of authenticated cell cultures (ECACC; Salisbury, UK). All cells were grown in DMEM medium (Gibco™, Irland) supplemented with 10% fetal bovine serum (Gibco™, Brazil). The cells were cultured in an incubator at 37 • C in a humidified 5% CO 2 atmosphere and subcultured 2 times a week. The effect of the investigated compounds on cell proliferation was evaluated using a common MTT assay. The cells were seeded in 96-well tissue culture plates («TPP», Switzerland) at a 1 × 10 4 cells/well in 100 µL of the medium. After overnight incubation at 37 • C, the cells were treated with the tested compounds in the concentration range of 0 to 200 µM. Cisplatin was used as a standard. After 72 h of treatment, solution was removed, a freshly diluted MTT solution (100 µL, 0.5 mg/mL in cell medium) was added to the wells, and the plates were further incubated for 50 min. Subsequently, the medium was removed, and the formazan product was dissolved in 100 µL of DMSO. The number of living cells in each well was evaluated by measuring the absorbance at 570 nm using the «Zenith 200 rt» microplate reader (Biochrom, Cambridge, UK). The cellular accumulation of the studied compounds was determined as described earlier [110]. Briefly, A549 and WI38 cells were seeded in 25 cm 2 cell flask (1 × 10 6 cells per flask). After 48 pre-incubations, the cells were exposed to compounds for 1, 4, 6, and 24 h. After the treatment, the cells were detached by trypsinization, exhaustively washed with ice-cold PBS (10mM, pH 7.4), counted by using cell counter, and collected by centrifugation. Finally, the cell pellets were digested, and the quantity of boron taken up by the cells was determined by ICP-MS.

Crystallography
X-ray diffraction data for crystals of 2a and 2b were measured using the Bruker APEX II diffractometer (Bruker AXC, Maddison, WI, USA) equipped with the Photon 2 detector (MoKα-radiation, graphite monochromator, ω-scans). The intensity data were integrated by the SAINT program [111] and were corrected for absorption and decay using SADABS [112]. Both structures were solved by direct methods using SHELXS [113] and refined using the full matrix least squares technique against F 2 using SHELXL-2018. Positions of hydrogen atoms were found from the difference Fourier synthesis of electron density. Non-hydrogen and hydrogen atoms were refined in the anisotropic and isotropic approximations, respectively. The main refinement details and parameters are given in the Table S1 in Supporting Information. CCDC 2173129-2173130 contain all additional supplementary data.

Conclusions
We have reported synthesis, X-ray structure study and photophysical properties of novel coumarins functionalized with azido group which are suitable for the fluorescent labeling of compounds containing terminal alkynyl group. Their representative application was performed on the "click" reaction with the closo-dodecaborate based alkynyl derivative. The antiproliferative activity and uptake of the synthesized boron compounds against several human cell lines were evaluated. Both compounds were found to be non-toxic against cancerous and non-malignant cells, however their cellar uptake is not sufficient for effective BNCT treatment.