PLIHALOVA LUCIE (CZ)
ZATLOUKAL MAREK (CZ)
DOLEZAL KAREL (CZ)
KOPRNA RADOSLAV (CZ)
STRNAD MIROSLAV (CZ)
WALLA JAN (CZ)
BALONOVA JARMILA (CZ)
WO2016095881A1 | 2016-06-23 | |||
WO2009003428A2 | 2009-01-08 | |||
WO1997034485A1 | 1997-09-25 |
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CLAIMS 1. N6-substituted adenine mesylate represented by the general formula I wherein R is selected from the group comprising C3-C15 cycloalkyl, furfuryl, allyl, 4-hydroxy-3- methylbut-2-en-1-yl, 3-methylbut-2-en-1-yl, 4-hydroxy-3-methylbutyl, 4-hydroxy-1,3-dimethylbut-2- en-1-yl, 4-hydroxy-1,3-dimethylbutyl, benzyl, wherein the cycloalkyl, allyl, benzyl and furfuryl may be unsubstituted or optionally substituted with 1 to 3 substituents selected from the group comprising hydroxy, halogen, methyl, hydroxymethyl and methoxy, and salts, solvates and addition salts therof. 2. Compound of general formula I according to claim 1, selected from the group comprising 6- furfurylaminopurine mesylate, 6-(2-chlorobenzylamino)purine mesylate, 6-(3- chlorobenzylamino)purine mesylate, 6-(2-fluorobenzylamino)purine mesylate, 6-(3- fluorobenzylamino)purine mesylate, 6-(4-fluorobenzylamino)purine mesylate, 6-(2- hydroxybenzylamino)purine mesylate, 6-(3-hydroxybenzylamino)purine mesylate, 6-(2- methoxybenzylamino)purine mesylate, 6-(3-methoxybenzylamino)purine mesylate, 6-(3-methylbut-2- en-1-ylamino)purine mesylate, 6-benzylaminopurine mesylate, 6-(4-hydroxy-3-methylbut-2-en-1- ylamino)purine mesylate, 6-(Z)-(4-hydroxy-3-methylbut-2-en-1-ylamino)purine mesylate, 6-(E)-(4- hydroxy-3-methylbut-2-en-1-ylamino)purine mesylate, 6-(Z)-(4-hydroxy-1,3-dimethylbut-2-en-1- ylamino)purine mesylate, 6-(E)-(4-hydroxy-1,3-dimethylbut-2-en-1-ylamino)purine mesylate, 6-(4- hydroxy-3-methylbutylamino)purine mesylate, and 6-(4-hydroxy-1,3-dimethylbutylamino)purine mesylate. 3. Compound of general formula I according to claim 1, selected from the group containing: - crystalline form of benzylaminopurine mesylate having characteristic peaks in XRPD obtained using CuKα radiation: 6.2; 10.0; 15.2; 15.7; 18.6; 18.8; 19.3; 20.4; 23.5; 23.8; 24.4; 27.6 ± 0.2° 2-theta; - crystalline meta-topolin mesylate having characteristic peaks in XRPD obtained using CuKα radiation: 8.1; 9.7; 13.1; 15.6; 16.8; 17.6; 19.1; 19.5; 22.2; 24.6; 24.8; 24.9; 25.7 ± 0.2° 2-theta; - crystalline ortho-topolin mesylate having characteristic peaks in XRPD obtained using CuKα radiation: 9.8; 12.1; 16.8; 17.8; 18.3; 18.3; 20.7; 23.3; 24.1; 25.1; 26.8 ± 0.2° 2-theta; - crystalline kinetin mesylate having characteristic peaksin XRPD obtained using CuKα radiation: 6.2; 18.9; 19.8; 20.4; 22.6; 23.9; 27.7; 27.8 ± 0.2° 2-theta. 4. Use of compounds of general formula I according to any one of claims 1 to 3 for protection of plant cells in vivo and in vitro against oxidative and/or electrophilic stress, and/or as antioxidants for the inhibition of lipid, protein and DNA peroxidation in plants in vivo or in vitro. 5. Use of compounds of general formula I according to any one of claims 1 to 3 for increasing the yield of crops, for improving rooting, for increasing the number of tillers, for increasing multiplication, and/or for delaying plant senescence, especially in the production of agricultural crops, preferably selected from cereals, beets, malvaceae, stone fruits, small fruits, legumes, oilseeds, pumpkins, fibrous plants, citruses, vegetables and plants such as tobacco, nuts, eggplant, sugar cane, tea, vines, hops, bananas, natural rubber, medicinal and ornamental plants. 6. Use of compounds of general formula I according to any one of claims 1 to 3 in tissue cultures as growth-regulatory and prodifferentiation factors, for stimulating cell proliferation and morphogenesis, and/or for suppressing undesirable physiological disorders such as hyperhydricity, necrosis of growth vesicles, chimera formation, inhibition of root formation during acclimatization, and early senescence. 7. Use of compounds of general formula I according to any one of claims 1 to 3 as growth regulators for in vitro cloning of plant or animal germ cells and embryos, preferably oocytes, and for propagation and differentiation of embryos, excluding human embryos. 8. Use of compounds of general formula I according to any one of claims 1 to 3 for inhibiting or delaying the senescence of plant cells and whole plants; and/or to improve the overall appearance and condition of plants, in particular plant epidermal and mesophyll cells, and/or also for inhibition, delaying or reduction of yellowing, chloroplast loss and chlorophyll loss; and/or for rejuvenation of plant cells, and/or for stimulation of cell proliferation and/or for cell differentiation in plant organism. 9. Agricultural and/or biotechnological preparations, characterized in that they contain at least one compound of general formula I according to any one of claims 1 to 3, and at least one solvent, carrier and/or excipient. 10. Method for increasing the yield of crops, for improving rooting, for increasing the number of tillers, for increasing multiplication, and/or for delaying plant senescence, said method comprising the step of applying at least one compound of general formula I according to claim 1 on the crops or plants in need of such treatment. 11. Method for stimulating cell proliferation and morphogenesis, and/or for suppressing undesirable physiological disorders selected from hyperhydricity, necrosis of growth vesicles, chimera formation, inhibition of root formation during acclimatization, and early senescence, said method comprising the step of applying at least one compound of general formula I according to claim 1 on the crops or plants in need of such treatment. 12. Method for inhibiting or delaying the senescence of plant cells and whole plants; and/or to improve the overall appearance and condition of plants, in particular plant epidermal and mesophyll cells, and/or also for inhibition, delaying or reduction of yellowing, chloroplast loss and chlorophyll loss; and/or for rejuvenation of plant cells, and/or for stimulation of cell proliferation and/or for cell differentiation in plant organism, said method comprising the step of applying at least one compound of general formula I according to claim 1 on the crops or plants in need of such treatment. |
The comparison of XRPD patterns for BAP mesylate and BAP is shown in Fig. 1 while the peaks detected for BAP mesylate are given in Table 1. The peaks for free base BAP are given in Table 2. The comparison of meta-topolin mesylate crystalline salt and meta-topolin XRPD patterns is shown in Fig. 2. The peaks detected for meta-topolin mesylate are given in Table 3 while the peaks for meta-topolin free base are given in Table 4. The comparison of XRPD patterns of ortho-topolin mesylate crystalline salt and ortho-topolin is shown in Fig. 3 and the comparison of XRPD pattern of kinetin mesylate crystalline salt and kinetin is shown in Fig.4. The XRPD peaks for ortho-topolin mesylate are given in Table 5, while for ortho-topolin in Table 6. The XRPD peaks for kinetin mesylate are given in Table 7 and for kinetin free base in Table 8. As apparent, the patterns of crystalline mesylate salts at Figs 1, 2, 3 and 4 significantly differ from the XRPD patterns of original bases, and all the materials including free bases used for the comparison are crystalline. Table 1: Relative intenstities of diffracted radiation, measured angles, d-spacing obtained by XRPD for BAP mesylate crystalline salt Table 2: Relative intenstities of diffracted radiation, measured angles, d-spacing obtained by XRPD for BAP Table 3: Relative intenstities of diffracted radiation, measured angles, d-spacing obtained by XRPD for meta-topolin mesylate (m-topolin mes)
Table 4: Relative intenstities of diffracted radiation, measured angles, d-spacing obtained by XRPD for meta-topolin Table 5: Relative intenstities of diffracted radiation, measured angles, d-spacing obtained by XRPD for ortho-topolin-mesylate
Table 6: Relative intenstities of diffracted radiation, measured angles, d-spacing obtained by XRPD for ortho-topolin
Table 7: Relative intenstities of diffracted radiation, measured angles, d-spacing obtained by XRPD for kinetin mesylate
Table 8: Relative intenstities of diffracted radiation, measured angles, d-spacing obtained by XRPD for kinetin Example 10: Characterization of crystalline cytokinin mesylate salts by 1 H Nuclear Magnetic Resonance (NMR) 1 H Nuclear Magnetic Resonance (NMR): 1 H NMR was performed on a JEOL 500 SS operating at the temperature of 300 K and a frequency of 500.13 MHz. The samples were prepared by dissolving the compounds in DMSO-d6. Tetramethylsilane (TMS) was used as an internal standard. Calibration of chemical shift related to shift of residual solvent peak in 1 H DMSO-d 6 , 2.50 ppm. Each sample was prepared in ca. 5 mg concentration. Chemical shifts identified in the spectra of selected cytokininmesylate crystalline salt respond to described chemical compound and are as follows: meta-topolin mesylate (6-(3-hydroxybenzylamino)purine mesylate): 1 H NMR (500 MHz, DMSO-D 6 ) δ 9.55 (s, 1H), 8.64 (s, 1H), 8.53 (s, 1H), 7.28 (t, J = 7.9 Hz, 1H), 7.00 – 6.94 (m, 2H), 6.87 (d, J = 8.2 Hz, 1H), 4.81 (s, 2H), 2.35 (s, 3H). BAP mesylate: 1 H NMR (500 MHz, DMSO-D6) δ 9.66 (s, 1H), 8.64 (s, 1H), 8.54 (s, 1H), 7.40 – 7.29 (m, 5H), 7.26 (t, J = 7.1 Hz, 1H), 4.80 (d, J = 5.9 Hz, 2H), 2.35 (s, 3H). kinetin mesylát: 1 H NMR (500 MHz, DMSO-D6) δ 9.53 (s, 1H), 8.69 (s, 1H), 8.56 (s, 1H), 7.65 (s, 1H), 6.45 (s, 2H), 4.85 (s, 2H), 2.40 – 2.34 (m, 3H). ortho-topolin mesylate: 1 H NMR (500 MHz, DMSO-D 6 ) δ 9.27 (s, 1H), 8.60 (s, 1H), 8.46 (s, 1H), 7.18 (d, J = 7.2 Hz, 1H), 7.14 – 7.07 (m, 1H), 6.83 (d, J = 7.9 Hz, 1H), 6.74 (t, J = 7.3 Hz, 1H), 4.69 (d, J = 5.6 Hz, 2H), 2.29 (s, 3H). (Z)-zeatin (cis-zeatin) mesylate: 1 H NMR (DMSO-d6): δ 1.67 (s, 3H); 3.81 (d, 2H, J = 5.2); 4.15 (m, 2H); 4.72 (t,1H, J = 5.55); 5.55 (t, 1H, J = 6.8); 7.67 (bs, 1H); 8.07 (s, 1H); 8.19 (s, 1H) (E)-zeatin (trans-zeatin) mesylate: 1 H NMR (DMSO-d6): 1.69 (s, 3H); 3.80 (d, 2H, J = 7.0); 4.15 (bs, 2H); 4.75 (t, 1H); 5.53 (t, 1H, J = 7.0); 8.09 (s, 1H); 8.19 (s, 1H) Dihydrozeatin (racemic) mesylate: 1 H NMR (DMSO-d6): 1.00 (d, 3H, J = 6.8); 1.51 (m, 1H); 1.75 (m, 1H); 1.85 (m, 1H); 3.44 (dd, 1H, J = 11.0, J = 6.0); 3.48 (dd, 1H, J = 11.0); 3.66 (m, 2H); 8.06 (s, 1H), 8.22 (s, 1H) Isopentenyladenin mesylate: 1 H NMR (DMSO-d6): 1.67 (s, 3H), 1.70 (s, 3H), 4.08 (br s, 1H), 5.31(s,1H), 7.55 (bs,1H), 8.04 (s,1H), 8.16 (s.1H) When compared to chemical shifts of their respective bases (BAP, meta-topolin, ortho-topolin, kinetin, Z-zeatin, E-zeatin, dihydrozeatin, isopentenyladenine) that are known, described compounds, significant differences are apparent, especially of those chemical shifts that are in the vicinity of protonated hydrogens. Example 11: Single crystal X-ray diffraction of crystalline meta-topolin mesylate salt and description of molecular structure Single Crystal Preparation: Crystals were grown from solutions of crystalline meta-topolin mesylate salt (ca.100 mg) dissolved in metanol (40 cm 3 ). The solution was then allowed to slowly evaporate through pierced parafilm. Translucent crystals were visible after ca.14 days of evaporation. Single Crystal X-Ray Diffraction: Suitable crystal of the sample was selected for data collection. Diffraction data were collected using four/circle diffractometer Supernova with mirror-collimated Cu/Kα radioation from a micro-focus sealed X-ray tube, equipped with the CCD areal detector Atlas S2 operation at 94.94 (13) K. On indexing the data set, the crystal structure was determined to be a centrosymmetric dimer linked by below discussed hydrogen bonding. Crystal system was identified as monoclinic with Pna21 space group and with the following cell dimensions: a=14.9313 (1), b=7.8302 (3), c=25.1594(1) Å, α=90°, β=90°, γ=90° and cell volume V=2941.51 Å 3 . Data collection, reduction and absorption corrections for all compounds were carried out using CrysAlisPro software (Rigaku Oxford Diffraction, 2018) CrysAlis version 1.171.40.53). The asymmetric unit of the title compound is formed by a 6-(3- hydroxybenzylamino)purinium cation and a mesylate anion. Hydrogen atoms attached to carbon were placed in calculated positions. Some hydrogen atoms attached to oxygen could be located in difference map. Intermolecular hydrogen bonds between N-H atom of purine and oxygen atom of mesylate anion connect two molecules as centrosymmetric dimers. These hydrogen bonds stabilize the system and connect N6a..H6b of purine structure with O1a of mesylate anion, N7a..H7a of purine structure with O5b of mesylate anion, O4b..H4b of hydroxyl attached to benzyl ring of meta-topolin with O5b of mesylate anion and also O4a…H4a of meta/topolin with O1b of mesylate (Fig 4). The final ‘conventional’ R-factor [based on F and 5945 reflections with [F 2 > 2σ (F 2 )] was 0.0257. The single crystal structure of crystalline meta-topolin mesylate crystalline salt shows that the compound has general formula of C 13 H 15 N 5 O 4 S based on 6-(3-hydroxybenzylamino)purinium cation and mesylate anion. Centrosymmetric dimers are bond together by a number of above listed intermolecular hydrogen bonds that are mainly realized between protonized cation and mesylate anion that contributes to excellent solubility of the compound in water. Important torsion angles, that also have impact on physical properties and solubility of the compounds, are stongly influenced by the formation of such thick network of intermolecular hydrogen bonding. Example 12: Single crystal X-ray diffraction and structural analysis of ortho-topolin mesylate crystalline salt Single Crystal Preparation: Crystals were grown from solutions of crystalline ortho-topolin mesylate salt (ca. 100 mg) dissolved in metanol (30 cm 3 ). The solution was then allowed to slowly evaporate through pierced parafilm. Translucent crystals were visible after ca.7 days of evaporation. Single Crystal X-Ray Diffraction: Suitable crystals of the sample were selected for data collection. Diffraction data were collected using four/circle diffractometer Xcalibur Gemini ultra with Cu/Kα radiation (λ = 1.54184 Å) with graphite monochromator equipped with the CCD area detector Atlas S2 operation at 120.01(10) K. Crystal system was identified as monoclinic with P2 1 /c space group and with the following cell dimensions: a=10.3822 (4), b=21.0265 (5), c=8.1025 (2) Å, α=90°, β=107.466°, γ=90° and cell volume V=1688.21 (9) Å 3 . The structure model was found using Superflip or SHELXT and refined by full- matrix least-squares by JANA2006 software. Data collection, reduction and absorption corrections for all compounds were carried out using CrysAlisPro Software (Rigaku, Oxford Diffraction, 2018, CrysAlis version 1.171.40.35a). The asymmetric unit of the title compound is formed by 6-(2- hydroxybenzylamino)purinium cation and mesylate anion and one molecule of methanol solvent. Hydrogen atoms attached to carbon were placed in calculated positions. Some hydrogen atoms attached to oxygen could be located in difference maps. Intermolecular hydrogen bonds between N-H atom of purine and oxygen atom of mesylate anion connectmesylate anions with 6-(2- hydroxybenzylamino)purinium cations and the cation is also connected via hydrogen bonding with methanol solvent. Hydrogen bonds network stabilizes the system and connect mainly N6..H6 atom of purine structure with O2 of mesylate anion and also N7..H7 of purine structure with O2 of mesylate anion. Further, there are linked O5..H5 of methanol to O2 of mesylate anion and concurrently to N7..H7 of purine moiety as shown at Fig 5. O1…H1 of hydroxyl attached to benzene ring is linked with O3b..H.3b atom of the mesylate anion that compensates the next 6-(2-hydroxybenzylamino)purinium cation (Fig 5).The final ‘conventional’ R-factor [based on F and 3024 reflections with [F 2 > 2σ (F 2 )] was 0.0324. The single crystal structure of crystalline ortho-topolin mesylate crystalline salt shows that the compound has general formula of C 14 H 19 N 5 O 5 S, 2-hydroxybenzylaminopurinium cation and mesylate anion, accompanied with a molecule of methanol solvent. Centrosymmetric dimers of asymmetric unit are bonded together by a network of the above listed intermolecular hydrogen bonds that are mainly realized between protonized cation and mesylate anion and also include methanol solvent and that contribute to good solubility of the compound in water. Hydrogen atom placement inferred from X-ray data is usually regarded as tentative, but the position of particular molecules within the frame of the structure is persuasive. Besides, important torsion angles, that also have impact on physical properties and solubility of the compounds, are stongly influenced by the formation of such thick network of intermolecular hydrogen bonding. Example 13: Aqueous solubility of meta-topolin mesylate salt Aqueous solubility was measured using the following protocol. Supersaturated solution of meta-topolin mesylate salt was prepared. The solutions were filtered off from visible undissolved particles of the salt to obtain homogenic saturated solution. Saturated solution was diluted 1:10000 and measured using HPLC Alliance Waters 2690 with C18 Symmetry column with the diameter 2.1 mm and 150 mm length with a porosity of 5 µm. The sample was dissolved in the mobile phase (MeOH: HCOONH 4 - 1: 9). The sample was washed with a methanol gradient (10-90%, 35 min) at pH 4 and a flow rate of 0.3 ml/min. The absorbances of the components were detected in the UV region at 210-400 nm. The peak area of meta-topolin mesylate salt was 595027. The standard was measured as shown in the Table 9 below. Obtained peak area was compared to area peaks measured at certain concentration and the final concentration of saturated solution was determined Table 9: Peak areas for certain concentrations of meta-topolin salts in water. It means that the concentration of meta-topolin mesylate crystalline salt is approximately 24010,81 μM, it means 24,011 mM (8 mg/ml), which is one order of magnitude better than solubility defined for meta- topolin base. Meta-topolin is reported to exhibit an aqueous solubility of < 0.25 mg/ml, we determined the concentration of saturated solution of meta-topolin to be only 0.06 mg/ml. Solubility of other cytokinin (CK) mesylates was also determined by this method and the concentrations of saturated solutions are given below in Table 10: Example 14: Solubility of meta-topolin mesylate compared to meta-topolin in organic solvents Solubility in organic solvents was measured using the following protocol. Approximately 25 mg portions of meta-topolin and crystalline meta-topolin mesylate salt were placed in 48 different vials, separately. 5 volume aliquots of each solvent were added progressively (repeatedly) to the vials. After each addition, the mixture was checked for dissolution and if no dissolution was visible, the procedure was continued until dissolution was observed or until 50 volumes have been added. The results are shown in Table 11. While the solubility in polar solvents such as water or alcohols differ significantly, almost no changes are observed in other organic solvents. TABLE 11: Solubility of meta-topolin mesylate and comparison with meta-topolin in various organic solvents Example 15: Storage stability study of meta-topolin mesylate crystalline salt 50 mg sample of crystalline meta-topolin mesylate crystalline salt was placed in an eppendorf tube and sealed. The sample was kept at 25°C for 60 days. No color change was noted during the storage period. XRPD of samples were taken after 60 days to investigate any solid form change. FIG. 6 shows the XRPD patterns of the initial sample and samples of crystalline meta-topolin mesylate crystalline salt after 60 days. The test did not reveal any changes in XRPD spektra, or change in color after 60 days of the compound storage period in solid form. Example 16: Disproportionation stability study of meta-topolin mesylate crystalline salt A 50 mg sample of crystalline meta-topolin mesylate crystalline salt was slurried in 250 μL distilled water for ca.48 hours and then checked by XRPD for disproportionation. No signs of disproportionation were observed. Example 17: In vitro cytotoxic activity of crystalline cytokinin mesylate salts (metabolisation of calcein) Because toxic compounds adversely affect cell metabolic processes, many standard cytotoxicity assays are based on measuring the rate of metabolism of various artificial substrates. The resulting product is then quantified, for example, by spectrometry. Assays can be easily modified for use in 96-well plates. A microtiter assay based on the quantification of Calcein AM metabolism was used to evaluate the cytotoxicity of cytokinins, for example meta-topolin, ortho-topolin, kinetin, BAP or its mesylate salts, but also a number of other cytokinin mesylate derivatives according to the invention. The test is widely used in drug screening programs and in chemosensitivity testing. In living cells, Calcein AM is enzymatically hydrolyzed and the accumulation of the resulting calcein is manifested by green fluorescence. The following cell lines - RPMI 8226 (multiple myeloma), CEM (T-lymphoblastic leukemia), K562 (chronic myeloid leukemia), HL-60 (promyelocytic leukemia), MCF-7 (breast adenocarcinoma), HeLa (cervical cancer), G361 (malignant melanoma), HOS (human osteosarcoma) and BJ (human foreskin fibroblasts) - were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were maintained in standard DMEM or RPMI medium (Sigma, MO, USA) supplemented with heat-inactivated fetal bovine serum (10%) with 2 mM L-glutamine and penicillin-streptomycin (1%) under standard cell culture conditions (37 °C, 5% CO 2 in a humid environment) and subcultured two or three times a week using a standard trypsinization procedure. Approximately 10,000 cells in 80 μl of medium were seeded in a 96-well microtiter plate. After 12 hours of incubation, the compounds to be tested were added in 20 μl aliquots. Control cultures were treated with DMSO alone. The final concentration of DMSO in the medium did not exceed 0.5%. Serial, 3-fold dilutions (six in total, peak concentrations in 166 μM incubations) of each compound were tested. After 72 hours of incubation, Calcein AM solution (Molecular Probes) was added to a final concentration of 1 μg/ml and the cells were incubated for another hour. Free calcein fluorescence was then quantified using a Fluoroscan Ascent fluorometer (Microsystems), and the percentage of surviving cells in each well was calculated by dividing the OD obtained from each cell by the exposed cells by the mean OD obtained from control wells × 100%. Finally, IC50 values (concentration causing a 50% decrease in cellular esterase activity) were calculated for each compound generated from dose-response curves (Kryštof et al., 2005, Bioorg. Med. Chem. Lett. 12, 3283-3286). The IC50 values reported here are averages obtained from at least three independent experiments, where the individual replication values fell within 20% of the average. Growth inhibition was calculated using the following equation: IC50 = (mean FDwell exposed to drug - mean FDblank) / (mean FDcontrol well - mean FDblank) x 100%. Cytoxicity of compounds was tested on panel of cell lines of different histogenetic and species origin. As shown in Table 12. IC50 of cytokinin derivatives exceeded maximal concentration tested which shows that the compounds can be applied at concentrations causing desired effect without negative side effects. For comparison, mesylate salts exhibited similar or even lower cytotoxicity than free bases. Table 12. Cytotoxic activity of cytokinin bases and their mesylate salts expressed as IC 50 values in a 3-day Calcein-AM assay. Presented values are averages of at least 3 independent experiments, where individual replicates fall into 20 % interval around the average. Example 18: In vitro cytotoxicity of novel derivatives for non-cancer cells, skin fibroblasts (BJ) and keratinocytes (HaCaT, ARPE-19), evaluated using resazurine reduction assay Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a blue weakly fluorescent compound that is irreversibly reduced into red highly fluorescent resofurin by mitochondria. It is used for viability testing of both bacterial and eukaryotic cells. The effect of the novel compounds in several concentrations (maximum concentraton of 100 microM and 51.5-fold dilutions) on viability of skin fibroblasts BJ, keratinocytes HaCaT and retinal epitelium cells ARPE-19 was evaluated after 72 hour treatment and results are given in Table 13. The cells were maintained in standard cultivation conditions (5.5 % CO2, 37 `C, 100% humidity). Cultivation medium was DMEM with 10% FBS. Before the experiment, the cells were subcultured for 2 weeks. For the cytotoxicity evaluation, the cells were trypsinized and pipetted into 96 well plates (5000 cells per well in 80 microliters). After 24 hours, 5x concentrated solutions of the tested compounds in the medium were added. After 72 hours, 10x concentrated solution of resazurin in the medium (prepared from 1000x concentrated DMSO solution) was added to the cells into the final concentration of 0.0125 mg/ml. Fluorescence (ex = 570 nm, em= 610 nm) was measured after 1 hour (ARPE-19) or 3 hours (HaCaT and BJ) of incubation and the results are shown in Table 13. IC 50 values were calculated from dose response curves using drc library for R programming environment. Table 13: Cytotoxicity for Fibroblasts (GI 20 , µM) Example 19: Inhibition of senescence by cytokinin mesylate salts tested on winter wheat leaf segments Seeds of winter wheat, Triticum aestivum cv. Hereward, were washed under running water for 24 hours and then sown on vermiculite soaked with Knop´s solution. They were placed in a growth chamber at 25°C with a 16/8 h light/dark period at 50 µmol.m -2 .s -1 . After 7 days, the first leaf was fully developed and the second leaf had started to grow. A tip section of the first leaf, approximately 35 mm long, was removed from 5 seedlings and trimmed slightly to a combined weight of 100 mg. The basal ends of the five leaf tips were placed in the wells of a microtiter polystyrene plate containing 150 µl of each tested compound solution. The entire plate was inserted into a plastic box lined with paper tissues soaked in distilled water to prevent leaf sections from drying out. After 96 h incubation in the dark at 25°C, the leaves were removed and chlorophyll extracted by heating at 80°C for 10 min in 5 ml of 80% ethanol (v/v). The sample volume was then restored to 5 ml by the addition of 80% ethanol (v/v). The absorbance of the extract was recorded at 665 nm. In addition, chlorophyll extracts from fresh leaves and leaf tips incubated in deionised water were measured. From the obtained data, the concentration with highest activity was selected for each tested compound. Relative activity of the compound at this concentration was calculated (Tab.3). The activity obtained for 10 -4 M 6-benzylaminopurine (BAP) was postulated as 100%. The values shown are means of five replicates and the whole experiment was repeated twice. The tested cytokinin bases were dissolved in dimethylsulfoxide (DMSO) and the solution brought up to 10- 3 M with distilled water. Cytokinin mesylate salts which are more water-soluble (BAP mesylate, meta- topolin mesylate and kinetin mesylate) could be directly dissolved to form a 10 -3 M solution in distilled water. This stock was further diluted in distilled water to concentrations ranging from 10 -8 M to 10 -4 M. The final concentration of DMSO if used did not exceed 0.2% and therefore did not affect biological activity in the assay system used. The results in Table 14 show that the new cytokinin mesylate salts led to an increase of the cytokinin activity in the senescence bioassay in comparison to the classical cytokinin free bases. Table 14: Effect of new cytokinin mesylates on retention of chlorophyll in excised wheat leaf tips. Standard deviations are of the mean for 10 replicate determination. Example 20: Stimulation effect of the new cytokinin mesylates on plant cell division Stimulation effect of the cytokinin mesylates was tested in tobacco callus biotest using cytokinin dependent tobacco callus. Cytokinin-dependent tobacco callus Nicotiana tabacum L. cv. Wisconsin 38 was maintained at 25°C in darkness on modified MS medium, containing per 1 liter: 4 µmol nicotinic acid, 2.4 µmol pyridoxine hydrochloride, 1.2 µmol thiamine, 26.6 µmol glycine, 1.37 µmol glutamine, 1.8 µmol myo-inositol, 30 g of sucrose, 8 g of agar, 5.37 µmol NAA and 0.5 µmol BAP. Subcultivation was carried out every three weeks. Fourteen days before the bioassay, the callus tissue was transferred to the media without BAP. Biological activity was determined from the increase in fresh callus weight after four weeks of cultivation. Five replicates were prepared for each cytokinin concentration and the entire test was repeated twice. From the obtained data, the concentration with highest activity was selected for each compound tested. Relative activity of the compound at this concentration was calculated (Tab. 15). The activity obtained for 10 -5 M 6-benzylaminopurine (BAP) was postulated as 100%. The cytokinin bases to be tested were dissolved in dimethylsulfoxide (DMSO) and the solution brought up to 10 -3 M with distilled water. The mesylate salts were dissolved directly in distilled water. This stock was further diluted in the respective media used for the biotest to concentrations ranging from 10 -8 M to 10 -4 M. The final concentration of DMSO did not exceed 0.2% and therefore did not affect biological activity in the assay system used. The results in Table 15 show that the new cytokinin mesylate salts led to an increase of the cytokinin activity in the callus bioassay in comparison to the classical cytokinin free bases. Table 15: The effect of cytokinin bases and cytokinin mesylate salts on growth of cytokinin-dependent tobacco callus Nicotiana tabacum L. cv. Wisconsins 38 Example 21: The efect of new derivatives on in vitro and post vitro multiplication and rooting of rose (Rosa multiflora) The aim of this experiment was to test whether the new compounds are of practical use in tissue culture practice. The multiplication rate was investigated and the post vitro effects on rooting were examined. Rosa hybrida (pot rose cultivar) was cultured in 350 ml vessels with a screw on polycarbonate lid. Each culture vessel contained 100 ml autoclaved medium (120 °C, 20 min). The cultures were maintained at 23±2°C under a 16 h photoperiod at 40 µM.m -2 s -1 PAR. The basal medium (BMR) contained Murashige and Skoog (1962) macroelements, microelements and vitamins, 95 µM NaFeEDTA, 555 µM myo- inositol, 111 mM sucrose, 1.332 mM glycine, 684 µM L-glutamine and 7g/l agar. This medium was supplemented with 10 µM BAP, mT, or 3MeOBAP, or the corresponding mesylate salts. The control medium didn’t contain any cytokinin. After a culture period of 8 weeks, the number of induced shoots per explant was determined, as well as root number/explant and total root length/explant. The roots were removed and the explants (shoot clusters) were planted in unfertilised peat. After four weeks of acclimatising in a fog unit, root number and root length was determined. As expected, a cytokinin free medium yielded almost no new shoots. The original shoot explant grew out as a good quality single shoot that rooted very well. BAP gave a high shoot multiplication rate, but the shoots rooted badly (Table 16). Explants growing on cytokininin mesylates were characterized by a high multiplication index (a large number of new shoots) and were more rooted in comparison with free bases. The use of cytokinin mesylates led to an increase in all monitored parameters in plant culture compared to free cytokinin bases. Table 16: The influence of mesylates on in vitro and post vitro multiplication and rooting of rose Rosa multiflora. Example 22: Increase in grain yield and number of productive tillers of spring barley (var. Francin) Spring barley (variety Francin) was treated in 2019 at the locality in Olomouc, sprayed with substances - marked BAP (benzylaminopurine), meta-topolin mesylate and BAP mesylate. The seed was 3.5 million germinating seeds, the experiment was established in 5 randomized replicates for each variant, the control was an untreated variant. The experiment was performed according to the GEP (Good Experimental Practice) methodology, the size of individual plots was 10 m 2 . The application of the substances was carried out at a rate of 300 l / ha, in the growth phase BBCH 31-33 (beginning of peeling). The concentration of test substances in the spray bed was 5 µMol. The results are summarized in Table 17. Table 17: The influence of BAP, meta-topolin mesylate and BAP mesylate on spring barley
Foliar application of all new cytokinin derivatives increased number of strong (prodductive) tillers (from 36.11 to 55.56 % over control), while number of medium tillers decreased. Grain yield were increased only in case of meta-topolin mesylate and BAP mesylate (108.03 and 104.34 % at control). BAP used as possitive control did not affect grain yield. In all test variants, the density of plants increased, too (106.62 – 112.06 % compared at control).