Adenine derivatives and their use as UV-photoprotective agents

Abstract

Adenine derivatives substituted at the C2, N6, and N9 purine positions having antisenescent and combined photoprotective UVA/UVB effects. These substances are particularly suitable as anti-senescent and UV-photoprotective component in cosmetic preparations, plant protection preparations and in preparations for the treatment/application of tissue cultures. ##STR00001##

Claims

1. Adenine derivatives of general formula I, ##STR00003## and pharmaceutically acceptable salts thereof with alkali metals, ammonia, amines, or addition salts with acids, wherein R2 is hydrogen or halogen; and R6 is selected from a group containing heteroaryl with 5- to 6-membered aromatic ring containing at least one heteroatom S whereas other ring atoms are carbon atoms, whereas the heteroaryl is unsubstituted or substituted by at least one substituent selected from the group consisting of C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogen, carboxyl, amino, di(C1-C4)alkylamino and amino(C1-C4)alkyl; heteroarylalkyl with 6-membered aromatic ring containing at least one heteroatom S whereas other atoms of the ring are carbon atoms, wherein the alkyl of the 6-membered heteroalkyl contains 1 to 4 carbon atoms, whereas the 6-membered heteroarylalkyl is unsubstituted or substituted by at least one substituent selected from the group C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogen, carboxyl, amino, di(C1-C4)alkylamino and amino(C1-C4)alkyl; heteroarylalkyl with 5-membered aromatic ring containing at least one heteroatom S whereas other atoms of the ring are carbon atoms, wherein the alkyl of the 5-membered heteroalkyl contains 1 to 4 carbon atoms, whereas the 5-membered heteroarylalkyl is unsubstituted or substituted by at least one substituent selected from the group C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogen, carboxyl, amino, di(C1-C4)alkylamino and amino(C1-C4)alkyl; heterocyclyl with 5- to 6-membered aliphatic ring containing at least one heteroatom selected from O and S whereas other atoms of the ring are carbon atoms, wherein the heterocycle is unsubstituted or substituted by at least one substituent selected from the group C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogen, carboxyl, amino, di(C1-C4)alkylamino and amino(C1-C4)alkyl; heterocyclylalkyl with 5- to 6-membered aliphatic ring containing at least one heteroatom selected from O and S whereas other atoms of the ring are carbon atoms, wherein the alkyl of the heterocyclylalkyl contains 1 to 4 carbon atoms, whereas the heterocyclylalkyl is unsubstituted or substituted by at least one substituent selected from the group C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogen, carboxyl, amino, di(C1-C4)alkylamino, amino(C1-C4)alkyl; and cycloalkylalkyl with ring containing 5 to 6 carbon atoms, wherein the alkyl of the cycloalkylalkyl contains 1 to 4 carbon atoms, whereas the cycloalkylalkyl is unsubstituted or substituted by at least one substituent selected from the group containing C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogen, carboxyl, amino, di(C1-C4)alkylamino and amino(C1-C4)alkyl.

2. Adenine derivatives according to claim 1, selected from the group consisting of: 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 6-(5-methyltetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 6-(1-tetrahydrofuran-2-ylethylamino)-9-(tetrahydrofuran-2-yl)purine; 6-(cyclopentylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 6-(thiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 6-(3-methylthiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 6-(5-methylthiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 6-(5-chlorothiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 6-(5-bromothiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 6-(1-thiophen-2-ylethylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(5-methyltetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(1-tetrahydrofuran-2-ylethylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(thiophen-2-ylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(thiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(3-methylthiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(5-methylthiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(5-chlorothiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(5-bromothiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine; 2-chloro-6-(1-thiophen-2-ylethylamino)-9-(tetrahydrofuran-2-yl)purine and 2-chloro-6-(cyclopentylmethylamino)-9-(tetrahydrofuran-2-yl)purine.

3. Adenine derivatives according to claim 1, wherein the substituent R6 is heteroaryl consisting of a 5-membered ring containing one heteroatom S, whereas the heteroaryl is unsubstituted or substituted by at least one substituent selected from the group containing C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogen, carboxyl, amino, di(C1-C4)alkylamino and amino(C1-C4)alkyl.

4. Adenine derivatives according to claim 1, wherein the substituent R6 is heteroarylalkyl consisting of a 5-membered ring and C1-C2 alkyl, whereas the 5-membered ring contains one heteroatom S, while the heteroarylalkyl is unsubstituted or substituted by at least one substituent selected from the group containing C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogen, carboxyl, amino, di(C1-C4)alkylamino and amino(C1-C4) alkyl.

5. Adenine derivatives according to claim 1, wherein the R6 substituent is heterocyclyl consisting of a 5-membered ring and the said 5-membered ring contains one heteroatom selected from O and S, while the heterocyclyl is unsubstituted or substituted by at least one substituent selected from the group comprising C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogene, carboxyl, amino, di (C1-C4)alkylamino and amino(C1-C4) alkyl.

6. Adenine derivatives according to claim 1, wherein the substituent R6 is heterocyclylalkyl consisting of a 5-membered ring and C1-C2 alkyl, and the said 5-membered ring contains one heteroatom selected from O and S, whereas the heterocyclylalkyl is unsubstituted or substituted by at least one substituent selected from the group consisting of C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogen, carboxyl, amino, di(C1-C4)alkylamino and amino(C1-C4)alkyl.

7. Adenine derivatives according to claim 1, wherein the substituent R6 is cyclopentylmethyl, which is unsubstituted or substituted by at least one substituent selected from the group comprising C1-C4 alkyl, mercapto(C1-C4)alkyl, formyl, acetyl, halogene, carboxyl, amino and di(C1-C4)alkylamino, amino(C1-C4)alkyl.

8. Cosmetic and/or therapeutic preparations, preparations for the protection of plants and/or preparations for the application to tissue cultures, which contain at least one compound of the general formula I according to claim 1.

Description

EXAMPLES OF CARRYING OUT THE INVENTION

Example 1

Preparation of 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine (1)

(1) 6-Chloro-9-(tetrahydrofuran-2-yl)purine (1 g; 4.46 mmol), tetrahydrofurfurylamine (554 l; 5.36 mmol) and triethylamine (Et.sub.3N) (3.2 ml; 22.3 mmol) were sequentially dissolved in propanol (50 ml). The mixture was stirred under reflux for 4 hours and then concentrated in vacuo. The residue was dissolved in water and extracted into EtOAC using liquid-liquid continuous extractor (24 h). Organic fraction was dried (Na2SO4) and evaporated in vacuo. The product was obtained after purification via column chromatography using (EtOAc:MeOH:NH.sub.3; 34:1:1; v:v) as eluent. Yield: 76%. .sup.1H NMR (500 MHz, DMSO-d.sub.6), ppm: 1.53-1.64 (m, 1H); 1.69-1.89 (m, 3H); 1.95-2.02 (m, 1H); 2.11-2.23 (m, 1H); 2.30-2.44 (m, 2H); 3.37-3.48 (m, 1H); 3.48-3.53 (m, 1H); 3.53-3.60 (m, 1H); 3.70-3.76 (m, 1H); 3.81-3.90 (m, 1H); 3.98 (q, J=7.03 Hz, 1H); 4.09 (q, J=7.44 Hz, 1H); 6.21 (dd, J=6.88, 3.82 Hz, 1H); 7.63 (br. s., 1H); 8.17 (br. s., 1H); 8.21 (s, 1H).

(2) TABLE-US-00001 TABLE 1 Compounds prepared according to example 1 Elemental analysis calculated/found ES MS N. R.sub.6 R.sub.2 % C % H % N [M + H].sup.+ 1 tetrahydrofuran-2-ylmethyl H 58.1/58.0 6.6/6.6 24.2/24.3 290.3 2 5-methylfuran-2-ylmethyl H 60.2/60.1 5.7/5.7 23.4/23.5 300.3 3 5-hydroxymethylfuran-2- H 57.1/57.2 5.4/5.4 22.2/22.5 316.3 ylmethyl 4 5-formylfuran-2-ylmethyl H 57.5/57.6 4.8/4.8 22.4/22.2 314.3 5 1-furan-2-ylethyl H 60.2/60.1 5.7/5.8 23.4/23.5 300.3 6 5-methyltetrahydrofuran-2- H 59.4/59.3 7.0/7.0 23.1/23.2 304.4 ylmethyl 7 1-tetrahydrofuran-2-ylethyl H 59.4/59.2 7.0/7.1 23.1/23.2 304.4 8 cyklopentylmethyl H 62.7/62.9 7.4/7.5 24.4/24.5 288.4

Example 2: Preparation of 6-(thiophen-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine (9)

(3) 6-Chloro-9-(tetrahydrofuran-2-yl)purine (0.5 g; 2.23 mmol), 2-thiophenemethylamine (275 l; 2.68 mmol) and triethylamine (Et.sub.3N) (1.6 ml; 11.15 mmol) were sequentially dissolved in propanol (25 ml) The mixture was stirred under reflux for 3 hours then 2-thiophenemethylamine (23 l; 0.23 mmol) was added and reaction mixture was stirred under reflux for an additional 1.5 hours. The mixture was concentrated in vacuo. The residue was dissolved in water and extracted into EtOAc. Organic fraction was dried (Na.sub.2SO.sub.4) and evaporated in vacuo. Product was obtained after precipitation in diethylether. Yield: 61%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 1.92-2.00 (m, 1H); 2.12-2.22 (m, 1H); 2.31-2.38 (m, 1H); 2.38-2.43 (m, 1H); 3.85 (td, J=7.68, 6.34 Hz, 1H); 4.08 (td, J=7.68, 6.50 Hz, 1H); 4.79 (br. s., 2H); 6.21 (dd, J=6.88, 3.82 Hz, 1H); 6.88 (dd, J=5.04, 3.44 Hz, 1H); 6.97 (dd, J=3.40, 1.03 Hz, 1H); 7.27 (dd, J=5.12, 1.22 Hz, 1H); 8.23 (s, 2H); 8.36 (br. s., 1H).

(4) TABLE-US-00002 TABLE 2 Compounds prepared according to example 2 Elemental analysis calculated/found ES MS N. R.sub.6 R.sub.2 % C % H % N [M + H].sup.+ 9 thiophen-2-ylmethyl H 55.8/55.6 5.0/5.4 23.2/23.3 302.4 10 3-methylthiophen-2-ylmethyl H 57.1/57.2 5.4/5.5 22.2/22.3 316.4 11 5-methylthiophen-2-ylmethyl H 57.1/57.0 5.4/5.3 22.2/22.1 316.4 12 5-chlorothiophen-2-ylmethyl H 50.0/50.1 4.2/4.3 20.9/20.8 336.8 13 5-bromothiophen-2-ylmethyl H 44.2/44.1 3.7/3.8 18.4/18.5 381.3 14 1-thiophen-2-ylethyl H 57.1/57.0 5.4/5.5 22.2/22.0 316.4

Example 3: Preparation of 2-chloro-6-furfurylamino-9-(tetrahydrofuran-2-yl)purine (15)

(5) 2,6-Dichloro-9-(tetrahydrofuran-2-yl)purine (0.5 g; 1.93 mmol), furfurylamine (204 l; 2.31 mmol) and triethylamine (Et.sub.3N) (1.32 ml; 9.65 mmol) were sequentially dissolved in propanol (25 ml). The mixture was stirred under reflux for 5 hours and then concentrated in vacuo. If product did not cristallize from reaction mixture it was evaporated in vacuo. Crude reaction mixture was precipitated from (CHCl.sub.3:EtOH; 1:8; v:v) or (CHCl.sub.3:Ether; 1:7; v:v) and filtrated. Solid product was washed with cold water and recrystallized from EtOH

(6) TABLE-US-00003 TABLE 3 Compounds prepared according to example 3 Elemental analysis calculated/found ES MS N. R.sub.6 R.sub.2 % C % H % N [M + H].sup.+ 15 furfuryl Cl 52.6/52.5 4.4/4.3 21.9/21.7 320.7 16 5-methylfuran-2-ylmethyl Cl 54.0/54.1 4.8/4.7 21.0/20.9 334.8 17 5-hydroxymethylfuran-2- Cl 51.5/51.6 4.6/4.7 20.0/20.1 350.8 ylmethyl 18 5-formylfuran-2-ylmethyl Cl 51.8/51.9 4.1/4.2 20.1/20.3 348.8 19 1-furan-2-ylethyl Cl 54.0/54.1 4.8/4.9 21.0/21.3 334.8 20 tetrahydrofuran-2-ylmethyl Cl 51.9/51.8 5.6/5.7 21.6/21.5 323.8 21 5-methyltetrahydrofuran-2- Cl 53.3/53.4 6.0/6.0 20.7/20.5 338.8 ylmethyl 22 1-tetrahydrofuran-2-ylethyl Cl 53.3/53.4 6.0/6.1 20.7/20.6 338.8 23 thiophen-2-ylmethyl Cl 50.1/50.1 4.2/4.3 20.9/20.8 336.8 24 3-methylthiophen-2- Cl 51.5/51.6 4.6/4.7 20.0/20.2 350.8 ylmethyl 25 5-methylthiophen-2- Cl 51.5/51.6 4.6/4.7 20.0/20.1 350.8 ylmethyl 26 5-chlorthiophen-2-ylmethyl Cl 45.4/45.6 3.5/3.7 18.9/18.8 371.3 27 5-bromthiophen-2- Cl 40.6/40.5 3.2/3.1 16.9/16.8 415.7 ylmethyl 28 1-thiophen-2-ylethyl Cl 51.5/51.6 4.6/4.7 20.0/20.1 350.8 29 cyklopentylmethyl Cl 56.0/56.1 6.3/6.4 21.8/21.9 322.8

Example 4: Evaluation of Cytotoxicity of Novel Derivatives for Skin Cell by MTT In Vitro Test

(7) MTT assay is a standard test of toxicity based on photometric measurement of the ability of metabolically active cells to reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Using the assay, the effects of 72 hour treatments with several concentrations of the compounds (sixfold dilution, maximal concentration=50 microM) on viability of skin fibroblasts BJ and keratinocytes HaCaT were evaluated. About 5,000 cells were seeded per well of a 96-well plate 24 hours before the treatment. DMSO vehiculum was used as a negative control. After 72 hour treatment, new medium with MTT (Sigma, M2128) was added to a final concentration of 0.5 mg/ml. After 3 hours, medium was removed and resulting formazan in the cells was dissolved in DMSO. The absorbance was measured at 570 nm (640 nm reference wavelength). The IC50 values were calculated from the dose-response curves. 6-furfurylaminopurine riboside was used as positive controls. The following results were obtained.

(8) TABLE-US-00004 TABLE 4 Cytotoxicity of prepared compounds in MTT in vitro assay. Compound IC.sub.50 (M) dimethylsulfoxid >100 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran- >100 2-yl)purine 6-(thiophen-2-ylmethylamino)-9-(tetrahydrofuran- >100 2-yl)aminopurine 2-chloro-6-furfurylamino-9-(tetrahydrofuran-2-yl)purine >100 6-furfurylamino-9-ribosylpurine (comperative example) 3

Example 5: Anti-Senescent Activity of Novel Compounds Tested in Senescent Bioassay on Wheat Leaf Segments

(9) 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 the growth chamber at 25 C. with a 16/8 h light period at 50 m.sup.2.Math.s.sup.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 L of the tested derivative solution each. 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. The results are means of five replicates and the entire test was repeated twice. In each experiment activities of the novel compounds were tested and compared with activity of BAP, which is known to be highly active cytokinin.

(10) The compounds to be tested were dissolved in dimethylsulfoxide (DMSO) and the solution brought up to 10.sup.3 M with distilled water. This stock solution was further diluted with the respective media used for the biotest to a concentration ranging from 10.sup.8 M to 10.sup.4 M. The final concentration of DMSO did not exceed 0.2% and therefore did not affect the biological activity in the assay system used. The activity obtained for 10.sup.4 M of BAP was postulated as 100%. Kinetin was used as the second standard. Newly prepared compounds generally exceeded the efficiency of standard (BAP) by 10% of its activity (Tab. 5).

(11) TABLE-US-00005 TABLE 5 Relative biological activity in detached wheat leaf senescence (chlorophyll retention) biotest compared with activity of 6-benzylaminopurine (BAP) standard (100% means activity of BAP in concentration 10.sup.4 mol .Math. l.sup.1) maximum effective activity (%) concentration [10.sup.4 mol .Math. l.sup.1 BAP = Compound (mol .Math. l.sup.1) 100%] BAP (comperative example) 10.sup.4 100 1 kinetin (comperative example) 10.sup.4 98 4 6-furfurylamino-9-(tetrahydro- 10.sup.4 114 3 furan-2-yl)purin 6-(3-methylthiophen-2-ylmethyl- 10.sup.4 110 7 amino)-9-(tetrahydrofuran- 2-yl)purine 6-(tetrahydrofuran-2-ylmethyl- 10.sup.4 125 9 amino)-9-(tetrahydrofuran- 2-yl)purine 6-(thiophen-2-ylmethylamino)-9- 10.sup.4 112 5 (tetra-hydrofuran-2-yl)purine 2-chloro-6-(tetrahydrofuran-2-yl- 10.sup.4 127 5 methylamino)-9-(tetrahydro- furan-2-yl)purine

Example 6: In Vitro Cytotoxic Activity of New Derivatives on Cancer Cell Lines

(12) One of the parameters used as the base for cytotoxic analysis is metabolic activity of viable cells. Microtiter assay, which uses the Calcein AM, is now widely used to quantify cell proliferation and cytotoxicity. The quantity of reduced Calcein AM corresponds to the number of viable cells in culture. The cell lines of breast cancer (MCF-7), human erythromleukemia (K562), BJ human fibroblast cells (BJ) and human keratinocyte cell line (HaCaT) were used for routine screening of cytotoxicity of the compounds. The cells were maintened in Nunc/Corning 80 cm.sup.2 plastic bottles and grown in media for cell culture (DMEM containing 5 g/l of glucose, 2 mM of glutamin, 100 U/ml of penicilin, 100 g/ml of streptomycin, 10% of fetal bovine serum and sodium hydrogencarbonate). Cell suspensions were diluted according to cell types and according to expected final cell density (10.sup.4 of cells per well according to characteristics of cell growth), pippeted 80 l of cell suspension on 96-well microtiter plates. Innoculates were stabilized by 24 hrs preincubation at 37 C. in CO.sub.2. Particular concentrations of tested compounds were added in time zero as 20 l aliquotto wells of microtiter plates. Usually, the compounds were diluted into six concentrations in four-fold dilution series. In routine testing, the highest well concentration was 166.7 M, of change dependent on the substance. All drug concentrations were examined in duplicates. The incubation of cells with tested derivatives lasted 72 hrs at 37 C., 100% humidity and in the atmosphere of CO.sub.2. At the end of the incubation period, the cells were tested and analysed according to the addition of Calcein AM (Molecular probes) solution and the incubation lasted for next 1 hour. Fluorescence (FD) was measured using Labsystem FIA reader Fluorskan Ascent (Microsystems). The survival of tumor cells (The tumor cell survival-TCS) was counted according to equation: GI.sub.50=(FD.sub.well with derivative/FD.sub.control well)100%. The value of GI.sub.50, that is equal to the concentration of compound at which 50% of tumour cells are terminated. To evaluate the antitumor activity was tested toxicity of new derivatives on panel of cell lines of different histogenetic and species origin (Tab. 6, G150 concentration given in M). It turned out that new compounds showed to be non toxic for neither of all tested tumor lines nor for nonmalignant cell lines BJ and HaCaT. Effective derivatives killed tumor cells in concentrations close to 0.1 to 50. None of the newly prepared compounds only reached the value.

(13) TABLE-US-00006 TABLE 6 Cytotoxicity of newly prepared compouds for various cell lines Compound MCF-7 K562 BJ HaCaT 6-furfurylamino-9-(tetrahydrofuran-2- >100 >100 >100 >100 yl)purine 6-(tetrahydrofuran-2-ylmethylamino)-9- >100 >100 >100 >100 (tetrahydrofuran-2-yl)purine 6-(thiophen-2-ylmethylamino)-9- >100 >100 >100 >100 (tetrahydrofuran-2-yl)purine 2-chloro-6-(tetrahydrofuran-2-ylmeth- >100 >100 >100 >100 ylamino)-9-(tetrahydrofuran-2-yl)purine

Example 7: In Vitro Test of Phototoxic Effects of Test Compounds on Normal Human Dermal Fibroblasts

(14) Phototoxic potential of test compound was determined by modified in vitro test validated phototoxicity evaluation (Spielmann H, Balls M, Dupuis J, Pape W J, Pechovitch G, de Silva O, Holzhiitter H G, Clothier R, Desolle P, Gerberick F, Liebsch M, Lovell W W, Maurer T, Pfannenbecker U, Potthast J M, Csato M, Sladowski D, Steiling W, Brantom P. The International EU/COLIPA In Vitro Phototoxicity Validation Study: Results of Phase II (Blind Trial). Part 1: The 3T3 NRU Phototoxicity Test. Toxicol In Vitro. 1998; 12:305-27). Normal human dermal fibroblasts (NHDF) were used as an in vitro model. Cells were isolated from tissue specimens obtained from healthy patients undergoing plastic surgery at the Department of Plastic and Aesthetic Surgery (University Hospital Olomouc). The use of skin tissue was in accordance with the Ethics Committee of the University Hospital and Faculty of Medicine and Dentistry, Palacky University, Olomouc and all patients signed written informed consent. Fibroblasts were used between the 2nd and 4th passage. For all experiments the fibroblasts were seeded onto 96-well plates at a density of 0.810.sup.5 cells/ml (0.2 ml per well) of cultivation medium (DMEM supplemented with fetal calf serum (10%, v/v), penicillin (100 mg/ml) and streptomycin (100 U/ml)). Test substances included compounds number 1, 3, 9, 15 and 20. Compounds were dissolved in DMSO and then diluted in serum free medium (DMEM supplemented with penicillin (100 mg/ml) and streptomycin (100 U/ml)). After 24 h incubation was cultivation medium changed to serum free medium containing test compound or DMSO (negative control). The final applied concentrations range 3.9-125 mol/l. As a control, serum free medium supplemented with appropriate concentration of DMSO (0.5%, v/v) was used. In parallel with test compound, chlorpromazine (CPZ; 0.8-50 mol/l) was used as a known phototoxic compound. The test compound was in parallel applied on two 96-well plates with NHDF. After 60 minutes incubation with test compound medium was discarded, cells were washed two-times with PBS and PBS supplemented with glucose (1 mg/ml) was applied. A plate was then exposed to a non-cytotoxic dose of UVA radiation (5.0 J/cm.sup.2) using a solar simulator SOL 500 (Dr. Hoenle Technology, Germany) equipped with a H1 filter transmitting wavelengths of 320-400 nm. Intensity of UVA radiation was evaluated before each irradiation by UVA-meter. A control (non-irradiated) plate was for the period of irradiation incubated in dark. After UVA exposure PBS with glucose was discarded and serum free medium was applied. After 24 hours (37 C., 5% CO.sub.2) cell damage was evaluated by neutral red (NR) incorporation into viable cells. Medium was discarded and NR solution (0.03% w/v, PBS) was applied. After 60 minutes NR solution was discarded, cells were fixed with a mixture of formaldehyde (0.5%, v/v) and CaCl.sub.2 (1%, m/v) in ratio 1:1 and then NR was dissolved in methanol (50%, v/v) with acetic acid (1%, v/v). After 5 minutes of intensive shaking absorbance was measured at 540 nm. Experiments were performed in four independent repetitions with use of cells from four donors to minimize individual sensitivity of donor cells. Phototoxic effect was evaluated as % of viability of control cells that was calculated from experimental data (absorbance) according to the following equation:

(15) $\mathrm{Viability}\left(\%\mathrm{of}\mathrm{control}\right)=\left(\frac{\left(A_S-A_B\right)}{\left(A_C-A_B\right)}\right).Math.100$ A.sub.S . . . absorbance of sample (cells pre-incubated with test compound in serum free medium and irradiated) A.sub.C . . . absorbance of control (cells pre-incubated with DMSO in serum free medium and irradiated) A.sub.B . . . absorbance of background (extraction solution)

(16) Result: Treatment with test compounds and following exposure to non-toxic UVA dose did not cause decrease in cell viabilityincorporation of NR and thus test compound can be considered as non-phototoxic in the used concentration range (3.9-125 mol/l). Results are given in Tab. 7. A well-known phototoxic compound chlorpromazine, which can be used for comparison, decreases the viability of NHDF cells: on exposure to UVA radiation (UVA+), the viability decreases below 80% of control in the presence of 6.3 mol/l of chlorpromazine, while the viability of unirradiated cells (UVA) decreases below 80% in the presence of 25 mol/l of chlorpromazine. A Above data indicate that test compounds are safe for cosmetic and dermatological application including use with following exposure of treated skin with solar radiation.

(17) TABLE-US-00007 TABLE 7 UVA-induced effects of test compounds on NHDF viability. 3.9 7.8 15.6 31.3 62.5 125 1 (mol/l) UVA % control 96.6 106.5 99.6 102.4 102.3 109.7 SMODCH 7.4 9.2 3.7 2.1 5.8 10.5 +UVA % control 105.1 110.6 106.8 109.5 103.3 107.5 SMODCH 10.3 10.7 13.4 10 8.9 8.9 3 (mol/l) UVA % control 103 104.9 104.2 107.7 106.6 110.4 SMODCH 4.2 5.4 5 8 3.2 4 +UVA % control 101.04 106.46 102.28 107.4 110.49 111.8 SMODCH 1.9 7.8 2.9 8.2 8.3 7.1 9 (mol/l) UVA % control 100 103 100.5 106.7 107.9 108.4 SMODCH 0.8 3.4 2 0.3 3.2 0.2 +UVA % control 97.8 102.3 100.8 105.9 102.8 106.3 SMODCH 1.5 4.4 1.4 4.3 3.7 1.2 15 (mol/l) UVA % control 100.3 103.5 104.3 105.4 106.3 101.2 SMODCH 1.6 4.3 3.2 4.7 3.9 1.8 +UVA % control 100.1 101.5 102.6 102.9 103.3 97.3 SMODCH 0.2 2.1 0.8 2.7 2.1 4 20 (mol/l) UVA % control 103.1 102.9 100.6 105.6 102 104 SMODCH 1.7 5.2 1.3 5.4 1.1 1.8 +UVA % control 100.1 102.9 100.4 102.7 105.1 104.8 SMODCH 3.5 4 3.7 3.9 6.1 5.2

Example 8: In Vitro Test of Photoprotective Effects of 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine on Dermal Fibroblasts

(18) Normal human dermal fibroblasts (NHDF) were used as an in vitro model. Cells were isolated from tissue specimens obtained from healthy patients undergoing plastic surgery at the Department of Plastic and Aesthetic Surgery (University Hospital Olomouc). The use of skin tissue was in accordance with the Ethics Committee of the University Hospital and Faculty of Medicine and Dentistry, Palacky University, Olomouc and all patients signed written informed consent. Fibroblasts were used between the 2nd and 4th passage. For all experiments the fibroblasts were seeded onto 96-well plates at a density of 0.810.sup.5 cells/ml (0.2 ml per well) of cultivation medium (DMEM supplemented with foetal calf serum (10%, v/v), penicillin (100 mg/ml) and streptomycin (100 U/ml)). Test compounds included 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine (1). Compounds were dissolved in DMSO and then diluted in serum free medium (DMEM supplemented with penicillin (100 mg/ml) and streptomycin (100 U/ml)). After 24 h incubation was cultivation medium changed to serum free medium containing test compound or DMSO (negative control). The final applied concentrations range of 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine was 3.9-500 mol/ll. As a control serum free medium supplemented with appropriate concentration of DMSO (0.5%, v/v) was used. Each test compound was in parallel applied on two 96-well plates with NHDF. After 60 minutes incubation medium with test compound was discarded, cells were washed two-times with PBS and PBS supplemented with glucose (1 mg/ml) was applied. To study UVA photoprotection, a plate was exposed to a cytotoxic dose of UVA radiation (7.5 J/cm.sup.2) using a solar simulator SOL 500 (Dr. Hoenle Technology, Germany) equipped with a H1 filter transmitting wavelengths of 320-400 nm. To study UVB photoprotection, a plate was exposed to a cytotoxic dose of UVB radiation (400 mJ/cm.sup.2) using the solar simulator equipped with a H2 filter transmitting wavelengths of 295-320 nm. Intensity of UVA or UVB radiation was evaluated before each irradiation by UVA- or UVB-meter. Control (non-irradiated) plates were for the period of irradiation incubated in dark. After UVA or UVB exposure PBS with glucose was discarded and serum free medium was applied. After 24 hours (37 C., 5% CO.sub.2) cell damage was evaluated by neutral red (NR) incorporation into viable cells. Medium was discarded and NR solution (0.03% w/v, PBS) was applied. After 60 minutes NR solution was discarded, cells were fixed with a mixture of formaldehyde (0.5%, v/v) and CaCl.sub.2 (1%, m/v) in ratio 1:1 and then NR was dissolved in methanol (50%, v/v) with acetic acid (1%, v/v). After 5 minutes of intensive shaking absorbance was measured at 540 nm. Experiments were performed in four independent repetitions with use of cells from four donors to minimize individual sensitivity of donor cells. Photoprotective effect was evaluated by comparison of experimental data (absorbance) of test compounds with a positive control and a negative control (according to the following equation:

(19) $\mathrm{Protection}\left(\%\right)=100-.Math.\frac{\mathrm{As}-\mathrm{Anc}}{\mathrm{Apc}-\mathrm{Anc}}.Math..Math.100$ As . . . absorbance of sample (cells pre-incubated with test compounds in serum free medium and irradiated) Anc . . . absorbance of negative control (cells pre-incubated with s DMSO in serum free medium and non-irradiated=incubated in dark) Apc . . . absorbance of positive control (cells pre-incubated with s DMSO in serum free medium and irradiated)

(20) Results: Cells pre-incubated with test compound and exposed to UVA or UVB radiation showed higher viability (ability to incorporate NR) compared to those pre-incubated with DMSO (control) and UVA or UVB irradiated (Tab. 8 and 9).

(21) TABLE-US-00008 TABLE 8 Photoprotective effect of 6-(tetrahydrofuran-2-ylmethylamino)-9- (tetrahydrofuran-2-yl)purine (1) on UVA-induced damage to NHDF. UVA photoprotection Concentration 1 (mol/l) protection (%) 3.9 27.7 (6.9) 7.8 57.4 (14.4) 15.6 43.2 (10.8) 31.3 47.5 (11.9) 62.5 41.3 (10.3) 125 .sup.49 (12.3) 250 45.3 (11.3) 500 18.5 (4.6)

(22) TABLE-US-00009 TABLE 9 Photoprotective effect of 6-(tetrahydrofuran-2-ylmethylamino)-9- (tetrahydrofuran-2-yl)purine (1) on UVB-induced damage to NHDF. UVB photoprotection Concentration 1 (mol/l) protection (%) 3.9 4.9 (1.2) 7.8 44.1 (11).sup. 15.6 35 (8.7) 31.3 38.3 (9.6) 62.5 44.4 (11.1) 125 42.7 (10.7) 250 46.6 (11.6) 500 26.3 (6.6)

Example 9: In Vitro Test of Photoprotective Effects of Test Compounds on Dermal Fibroblasts

(23) Normal human dermal fibroblasts (NHDF) were used as an in vitro model. Cells were isolated from tissue specimens obtained from healthy patients undergoing plastic surgery at the Department of Plastic and Aesthetic Surgery (University Hospital Olomouc). The use of skin tissue was in accordance with the Ethics Committee of the University Hospital and Faculty of Medicine and Dentistry, Palacky University, Olomouc and all patients signed written informed consent. Fibroblasts were used between the 2nd and 4th passage. For all experiments the fibroblasts were seeded onto 96-well plates at a density of 0.810.sup.5 cells/ml (0.2 ml per well) of cultivation medium (DMEM supplemented with fetal calf serum (10%, v/v), penicillin (100 mg/ml) and streptomycin (100 U/ml)). Test substances included compounds number 1, 3, 9, 15 and 20. Compounds were dissolved in DMSO and then diluted in serum free medium (DMEM supplemented with penicillin (100 mg/ml) and streptomycin (100 U/ml)). After 24 h incubation was cultivation medium changed to serum free medium containing test compound or DMSO (negative control). The final applied concentrations range was 3.9-31.3 mol/ll. As a control serum free medium supplemented with appropriate concentration of DMSO (0.5%, v/v) was used. Rosmarinic acid was used as positive control. Each test compound was in parallel applied on two 96-well plates with NHDF. After 60 minutes incubation medium with test compound was discarded, cells were washed two-times with PBS and PBS supplemented with glucose (1 mg/ml) was applied. To study UVA photoprotection, a plate was exposed to a cytotoxic dose of UVA radiation (7.5 J/cm.sup.2) using a solar simulator SOL 500 (Dr. Hoenle Technology, Germany) equipped with a H1 filter transmitting wavelengths of 320-400 nm. To study UVB photoprotection, a plate was exposed to a cytotoxic dose of UVB radiation (150 mJ/cm.sup.2) using the solar simulator equipped with a H2 filter transmitting wavelengths of 295-320 nm. Intensity of UVA or UVB radiation was evaluated before each irradiation by UVA- or UVB-meter. Control (non-irradiated) plates were for the period of irradiation incubated in dark. After UVA or UVB exposure PBS with glucose was discarded and serum free medium was applied. After 24 hours (37 C., 5% CO.sub.2) cell damage was evaluated by neutral red (NR) incorporation into viable cells. Medium was discarded and NR solution (0.03% w/v, PBS) was applied. After 60 minutes NR solution was discarded, cells were fixed with a mixture of formaldehyde (0.5%, v/v) and CaCl.sub.2 (1%, m/v) in ratio 1:1 and then NR was dissolved in methanol (50%, v/v) with acetic acid (1%, v/v). After 5 minutes of intensive shaking absorbance was measured at 540 nm. Experiments were performed in four independent repetitions with use of cells from four donors to minimize individual sensitivity of donor cells. Photoprotective effect was evaluated by comparison of experimental data (absorbance) of test compounds with a positive control and a negative control (according to the following equation:

(24) $\mathrm{Protection}\left(\%\right)=100-.Math.\frac{\mathrm{As}-\mathrm{Anc}}{\mathrm{Apc}-\mathrm{Anc}}.Math..Math.100$ As . . . absorbance of sample (cells pre-incubated with test compounds in serum free medium and irradiated) Anc . . . absorbance of negative control (cells pre-incubated with s DMSO in serum free medium and non-irradiated=incubated in dark) Apc . . . absorbance of positive control (cells pre-incubated with s DMSO in serum free medium and irradiated)

(25) Results: Cells pre-incubated with test compounds and exposed to UVA or UVB radiation showed higher viability (ability to incorporate NR) compared to those pre-incubated with DMSO (control) and UVA or UVB irradiated (Tab. 10 and 11). All test compounds showed higher or comparable photoprotective activity with rosmarinic acid used as positive control. Therefore test compounds has high photoprotective potential.

(26) TABLE-US-00010 TABLE 10 Photoprotective effect of test compounds and rosmarinic acid (RA, positive control) on UVA-induced damage to NHDF. 3.9 7.8 15.6 31.3 3 (mol/l) Protection (%) 14.7 16.3 24.6 25.8 SMODCH 3.2 3.3 5.8 4.5 9 (mol/l) Protection (%) 41.3 44.4 45.7 52.0 SMODCH 18.0 12.6 9.5 10.8 15 (mol/l) Protection (%) 24.5 29.9 33.0 35.3 SMODCH 11.9 10.8 12.1 11.5 20 (mol/l) Protection (%) 5.9 10.7 11.5 16.2 SMODCH 3.2 3.5 5.4 4.1 RA Protection (%) 6.1 14.7 17.0 19.3 SMODCH 3.0 3.5 3.8 3.4

(27) TABLE-US-00011 TABLE 11 Photoprotective effect of test compounds and rosmarinic acid (RA, positive control) on UVB-induced damage to NHDF. 3.9 7.8 15.6 31.3 3 (mol/l) Protection (%) 43.6 53.1 47.4 48.4 SMODCH 10.7 9.1 16.9 13.9 9 (mol/l) Protection (%) 34.9 62.6 64.4 68.7 SMODCH 23.7 21.4 14.6 6.2 15 (mol/l) Protection (%) 39.9 55.2 53 60.2 SMODCH 15.1 14.2 17.1 21 20(mol/l) Protection (%) 39.8 58.3 58.2 60 SMODCH 18.3 19.3 23.2 18.8 RA Protection (%) 36.8 50.9 52.9 67.7 SMODCH 5.9 5.8 11.8 5.1

Example 10: In Vitro Test of Phototoxic Effects of 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine on Normal Human Epidermal Keratinocytes

(28) Normal Human Epidermal Keratinocytes (NHEK) were used as an in vitro model. Cells were isolated from tissue specimens obtained from healthy patients undergoing plastic surgery at the Department of Plastic and Aesthetic Surgery (University Hospital Olomouc). The use of skin tissue was in accordance with the Ethics Committee of the University Hospital and Faculty of Medicine and Dentistry, Palacky University, Olomouc and all patients signed written informed consent. NHEK were used between the 3rd and 4th passage. For all experiments the keratinocytes were seeded onto 96-well plates at a density of 1104 cells/ml (0.2 ml per well) of growth medium for keratinocytes (EpiLife) supplemented with Human Keratinocyte Growth Supplement Kit and antibiotics (penicillin (100 mg/ml), streptomycin (100 mg/ml) and ampicillin (250 g/ml)). Test compounds included 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine (1). Compound was dissolved in DMSO and then diluted in serum free medium (EpiLife supplemented with penicillin (100 mg/ml) and streptomycin (100 U/ml) and ampicillin (250 g/ml)). After 24 h incubation was growth medium changed to serum free medium containing test compound or DMSO (negative control). The final applied concentrations range was 3.9-500 mol/l. As a control, serum free medium supplemented with appropriate concentration of DMSO (0.5%, v/v) was used. In parallel with test compound, chlorpromazine (CPZ; 0.8-50 mol/l) was used as a known phototoxic compound. The test compound was in parallel applied on two 96-well plates with NHEK. After 60 minutes incubation with test compound medium was discarded, cells were washed two-times with PBS and PBS supplemented with glucose (1 mg/ml) was applied. A plate was then exposed to a non-cytotoxic dose of UVA radiation (5.0 J/cm2) using a solar simulator SOL 500 (Dr. Hoenle Technology, Germany) equipped with a H1 filter transmitting wavelengths of 320-400 nm. Intensity of UVA radiation was evaluated before each irradiation by UVA-meter. A control (non-irradiated) plate was for the period of irradiation incubated in dark.

(29) After UVA exposure PBS with glucose was discarded and serum free medium was applied. After 24 hours (37 C., 5% CO2) cell damage was evaluated by neutral red (NR) incorporation into viable cells. Medium was discarded and NR solution (0.03% w/v, PBS) was applied. After 60 minutes NR solution was discarded, cells were fixed with a mixture of formaldehyde (0.5%, v/v) and CaCl2 (1%, m/v) in ratio 1:1 and then NR was dissolved in methanol (50%, v/v) with acetic acid (1%, v/v). After 5 minutes of intensive shaking absorbance was measured at 540 nm. Experiments were performed in four independent repetition with use of cells from four donors to minimize individual sensitivity of donor cells. Phototoxic effect was evaluated as % of viability of control cells that was calculated from experimental data (absorbance) according to the following equation:

(30) $\mathrm{Viability}\left(\%\mathrm{of}\mathrm{control}\right)=\left(\frac{\left(A_S-A_B\right)}{\left(A_C-A_B\right)}\right).Math.100$

(31) AS . . . absorbance of sample (cells pre-incubated with test compound in serum free medium and irradiated)

(32) AC . . . absorbance of control (cells pre-incubated with DMSO in serum free medium and irradiated)

(33) AB . . . absorbance of background (extraction solution)

(34) Result: Treatment with test compound and following exposure to non-toxic UVA dose did not cause decrease in cell viabilityincorporation of NR and thus test compound can be considered as non-phototoxic in the used concentration range (3.9-500 mol/l). Results are given in Tab. 12. A well-known phototoxic compound chlorpromazine, which can be used for comparison, decreases the viability of NHEK cells: on exposure to UVA radiation (UVA+), the viability decreases below 80% of control in the presence of 0.6 mol/l of chlorpromazine, while the viability of unirradiated cells (UVA) decreases below 50% in the presence of 12.5 mol/l of chlorpromazine. A. Above data indicate that test compounds are safe for cosmetic and dermatological application including use with following exposure of treated skin with solar radiation.

(35) TABLE-US-00012 TABLE 12 UVA-induced effects of 6-(tetrahydrofuran-2-ylmethylamino)-9- (tetrahydrofuran-2-yl)purine on NHEK viability 1 Concentration % of control (mol/l) UVA +UVA 3.9 112.2 (11) 109.1 (2.3) 7.8 135.5 (8.2) 120.6 (1.6) 15.6 116.5 (2.8) .sup.110 (2.9) 31.3 119.1 (8.7) 115.1 (2.8) 62.5 .sup.121 (8.7) 105.9 (8.7) 125 .sup.119 (2.8) 111.7 (6.8) 250 110.8 (12.4) 112.1 (3.7) 500 105.7 (7.1) 101.2 (3.3)

Example 11: Example 8: In Vitro Test of Photoprotective Effects of 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine on normal human epidermal keratinocytes

(36) Normal Human Epidermal Keratinocytes (NHEK) were used as an in vitro model. Cells were isolated from tissue specimens obtained from healthy patients undergoing plastic surgery at the Department of Plastic and Aesthetic Surgery (University Hospital Olomouc). The use of skin tissue was in accordance with the Ethics Committee of the University Hospital and Faculty of Medicine and Dentistry, Palacky University, Olomouc and all patients signed written informed consent. NHEK were used between the 3rd and 4th passage. For all experiments the keratinocytes were seeded onto 96-well plates at a density of 110.sup.4 cells/ml (0.2 ml per well) of growth medium for keratinocytes (EpiLife) supplemented with Human Keratinocyte Growth Supplement Kit and antibiotics (penicillin (100 mg/ml), streptomycin (100 mg/ml) and ampicillin (250 g/ml)). Test compounds included 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine (1). Compound was dissolved in DMSO and then diluted in serum free medium (EpiLife supplemented with penicillin (100 mg/ml) and streptomycin (100 U/ml) and ampicillin (250 g/ml)). After 24 h incubation was growth medium changed to serum free medium containing test compound. The final applied concentration range was 3.9-500 mol/l. As a control, serum free medium supplemented with appropriate concentration of DMSO (0.5%, v/v) was used. After 60 minutes incubation with test compound medium was discarded, cells were washed two-times with PBS and PBS supplemented with glucose (1 mg/ml) was applied. To study UVA photoprotection, a plate was exposed to a cytotoxic dose of UVA radiation (7.5 J/cm2) using a solar simulator SOL 500 (Dr. Hoenle Technology, Germany) equipped with a H1 filter transmitting wavelengths of 320-400 nm. To study UVB photoprotection, a plate was exposed to a cytotoxic dose of UVB radiation (200 mJ/cm2) using the solar simulator equipped with a H2 filter transmitting wavelengths of 295-320 nm. Intensity of UVA or UVB radiation was evaluated before each irradiation by UVA- or UVB-meter. Control (non-irradiated) plates were for the period of irradiation incubated in dark. After UVA or UVB exposure PBS with glucose was discarded and serum free medium was applied.

(37) After 24 hours (37 C., 5% CO2) cell damage was evaluated by neutral red (NR) incorporation into viable cells. Medium was discarded and NR solution (0.03% w/v, PBS) was applied. After 60 minutes NR solution was discarded, cells were fixed with a mixture of formaldehyde (0.5%, v/v) and CaCl2 (1%, m/v) in ratio 1:1 and then NR was dissolved in methanol (50%, v/v) with acetic acid (1%, v/v). After 5 minutes of intensive shaking absorbance was measured at 540 nm.

(38) Experiments were performed in four independent repetitions with use of cells from four donors to minimize individual sensitivity of donor cells. Photoprotective effect was evaluated by comparison of experimental data (absorbance) of test compounds with a positive control and a negative control (according to the following equation:

(39) $\mathrm{Protection}\left(\%\right)=100-.Math.\frac{\mathrm{As}-\mathrm{Anc}}{\mathrm{Apc}-\mathrm{Anc}}.Math..Math.100$

(40) As . . . absorbance of sample (cells pre-incubated with test compounds in serum free medium and irradiated)

(41) Anc . . . absorbance of negative control (cells pre-incubated with s DMSO in serum free medium and non-irradiated=incubated in dark)

(42) Apc . . . absorbance of positive control (cells pre-incubated with s DMSO in serum free medium and irradiated)

(43) Results: Cells pre-incubated with test compound and exposed to UVA or UVB radiation showed higher viability (ability to incorporate NR) compared to those pre-incubated with DMSO (control) and UVA or UVB irradiated (Tab. 13 and 14). Therefore test compound has high photoprotective potential.

(44) TABLE-US-00013 TABLE 13 Photoprotective effect of 6-(tetrahydrofuran-2-ylmethylamino)-9- (tetrahydrofuran-2-yl)purine (1) on UVA-induced damage to NHEK UVA photoprotection Concentration 1 (mol/l) protection (%) 3.9 39.7 (12.9) 7.8 60.2 (16.1) 15.6 53.5 (12.4) 31.3 46.8 (11.8) 62.5 47.9 (8.9) 125 51.7 (4.8) 250 48 (3.2) 500 .sup.30 (13.5)

(45) TABLE-US-00014 TABLE 14 Photoprotective effect of 6-(tetrahydrofuran-2-ylmethylamino)-9- (tetrahydrofuran-2-yl)purine (1) on UVb-induced damage to NHEK UVB photoprotection Concentration 1 (mol/l) % protection (%) 3.9 53.5 (7.6) 7.8 72 (6) 15.6 69.8 (8.6) 31.3 65.9 (9).sup. 62.5 65.9 (5).sup. 125 72.9 (6.9) 250 65.7 (3.6) 500 .sup.28 (3.2)

Example 12: The Effect of Compounds on Lifespan of Caenorhabditis elegans

(46) Caenorhabditis elegans is a model organism used for identification of compounds with possible beneficial effect on human aging and age-related diseases. The list of compounds that prolong the lifespan of C. elegans and are known to have beneficial effect on human health includes resveratrol, curcumin and many others. Some substances that prolong the lifespan of worms are also used in skin rejuvenating and anti-aging cosmetics, for example vitamin E, coenzyme Q10, green tea or pomegranade extracts and cytokinin kinetin. The Caenorhabditis elegans strain used in this experiment was fem-1/HT17. This strain has a heat-inducible mutation which causes all worms to develop into females when cultivated in 25 C. (13). That prevents further reproduction and contamination of the experiment with progeny. Compounds dissolved in DMSO (100 mM stock solutions) were added into fresh NGM (nematode growth medium) to the final concentration of 10 and 50 or 100 M and pipetted onto a Petri dish. Medium with DMSO vehiculum alone and non-treated medium were used as negative control. After solidification of NGM, the plates were seeded with 100 l of 20 concentrated overnight suspension of Escherichia coli strain OP50 in LB medium. Bacteria on plates were allowed to grow overnight in 37 C. Age synchronized young adults (obtained by hypochlorite treatment) were then pipetted onto plates. Plates were kept in 25 C. In regular time intervals (1-3 days), the plates were scanned on an Epson perfection V700 photo flatbed scanner. The number of surviving worms was established by image analysis based on comparison of several subsequent photographs and identification of moving objects. Pictures were analyzed in Fiji similarly as described here. Scripts from the original publication were slightly modified and the parameters adjusted to better suit our photo resolution and lighting. Three subsequent pictures of a plate were compared with each other. The average of the 3 resulting numbers was used to reduce the possibility of error. The overall results were then analyzed in programs OASIS and ED50v10. The statistical significance was evaluated by Log-Rank test and P-values were corrected by Bonferroni correction. The results are shown in table 15. Compounds 1, 15 and 20 significantly prolonged the lifespan of worms.

(47) TABLE-US-00015 TABLE 15 The effect of compounds on lifespan of Caenorhabditis elegans (in days) 15 15 20 20 1 1 DMSO 10 M 100 M 10 M 100 M 10 M 100 M medi- 8.7 9.4 10.3 11.2 10.2 10.3 10.2 an aver- 10.9 11.3 13.8 12.4 11.2 11.9 12.6 age

Example 13: In Vitro Test of Phototoxic Effects of Test Compounds on HaCaT

(48) Spontaneously Immortalized Aneuploid Human Keratinocyte Cell Line (HaCaT) was bought from CLS (Eppeheim, Germany) and used as an in vitro model. For all experiments the keratinocytes were seeded onto 96-well plates at a density of 1.610.sup.5 cells/ml (0.2 ml per well) of growth medium (DMEM supplemented with fetal bovine serum (10%), penicillin (100 mg/ml) and streptomycin (100 U/ml)).

(49) Test substances included compounds number 3, 9, 15 and 20. Compounds were dissolved in DMSO and then diluted in serum free medium (DMEM supplemented penicillin (100 mg/ml) and streptomycin (100 U/ml)). After 24 h incubation was growth medium changed to serum free medium containing test compound or DMSO (negative control). The final applied concentrations range was 3.9-125 mol/l. As a control, serum free medium supplemented with appropriate concentration of DMSO (0.5%, v/v) was used. In parallel with test compound, chlorpromazine (CPZ; 0.8-50 mol/l) was used as a known phototoxic compound. The test compound was in parallel applied on two 96-well plates with HaCaT. After 60 minutes incubation with test compound medium was discarded, cells were washed two-times with PBS and PBS supplemented with glucose (1 mg/ml) was applied. A plate was then exposed to a non-cytotoxic dose of UVA radiation (5.0 J/cm2) using a solar simulator SOL 500 (Dr. Hoenle Technology, Germany) equipped with a H1 filter transmitting wavelengths of 320-400 nm. Intensity of UVA radiation was evaluated before each irradiation by UVA-meter. A control (non-irradiated) plate was for the period of irradiation incubated in dark. After UVA exposure PBS with glucose was discarded and serum free medium was applied. After 24 hours (37 C., 5% CO2) cell damage was evaluated by neutral red (NR) incorporation into viable cells. Medium was discarded and NR solution (0.03% w/v, PBS) was applied. After 60 minutes NR solution was discarded, cells were fixed with a mixture of formaldehyde (0.5%, v/v) and CaCl2 (1%, m/v) in ratio 1:1 and then NR was dissolved in methanol (50%, v/v) with acetic acid (1%, v/v). After 5 minutes of intensive shaking absorbance was measured at 540 nm. Experiments were performed in four independent repetition with use of cells from four donors to minimize individual sensitivity of donor cells. Phototoxic effect was evaluated as % of viability of control cells that was calculated from experimental data (absorbance) according to the following equation:

(50) $\mathrm{Viability}\left(\%\mathrm{of}\mathrm{control}\right)=\left(\frac{\left(A_S-A_B\right)}{\left(A_C-A_B\right)}\right).Math.100$

(51) AS . . . absorbance of sample (cells pre-incubated with test compound in serum free medium and irradiated)

(52) AC . . . absorbance of control (cells pre-incubated with DMSO in serum free medium and irradiated)

(53) AB . . . absorbance of background (extraction solution)

(54) Result: Treatment with test compounds and following exposure to non-toxic UVA dose did not cause decrease in cell viabilityincorporation of NR and thus test compound can be considered as non-phototoxic in the used concentration range (3.9-125 mol/l). Results are given in Tab. 16. A well-known phototoxic compound chlorpromazine, which can be used for comparison, decreases the viability of HaCaT cells: on exposure to UVA radiation (UVA+), the viability decreases below 80% of control in the presence of 3.1 mol/l of chlorpromazine, while the viability of unirradiated cells (UVA) decreases below 70% in the presence of 25 mol/l of chlorpromazine. Above data indicate that test compounds are safe for cosmetic and dermatological application including use with following exposure of treated skin with solar radiation.

(55) TABLE-US-00016 TABLE 16 UVA-induced effects of test compounds on HaCaT viability 3.9 7.8 15.6 31.3 62.5 125 3 (mol/l) UVA % control 100.1 100.4 102.1 107.8 106.4 109.5 SMODCH 1.2 1.1 2.7 9.3 9.5 7.2 +UVA % control 103.88 111.08 107.93 118.43 120.78 124.66 SMODCH 1.7 1.6 1.4 3.7 3.2 4.2 9 (mol/l) UVA % control 101 101.1 100.4 101.8 103.9 103.4 SMODCH 2.3 1.8 1 3.8 4.8 5.1 +UVA % control 122.7 124.2 120.5 128.1 129.9 126.6 SMODCH 3.5 4.6 5.3 4.4 3.2 3.1 15 (mol/l) UVA % control 106.5 107.4 108.6 108.6 109.2 105 SMODCH 9.7 9.5 10.6 9.5 12.6 8.9 +UVA % control 116.4 119.9 119.9 118.8 115.7 104 SMODCH 0.7 4.9 6.3 7.7 9 8.8 20 (mol/l) UVA % control 103.5 102.9 104.1 108.6 110.7 110.5 SMODCH 6.1 3.1 5.3 2.4 1.7 1.2 +UVA % control 104.6 112.1 110.1 121.4 125.8 125.9 SMODCH 0.7 3.3 5.6 2.2 5.9 4.2

Example 14: In Vitro Test of Photoprotective Effects of Test Compounds on HaCaT

(56) Spontaneously Immortalized Aneuploid Human Keratinocyte Cell Line (HaCaT) was bought from CLS (Eppeheim, Germany) and used as an in vitro model. For all experiments the keratinocytes were seeded onto 96-well plates at a density of 1.610.sup.5 cells/ml (0.2 ml per well) of growth medium (DMEM supplemented with fetal bovine serum (10%), penicillin (100 mg/ml) and streptomycin (100 U/ml)).

(57) Test substances included compounds number 3, 9, 15 and 20. Compounds were dissolved in DMSO and then diluted in serum free medium (DMEM supplemented penicillin (100 mg/ml) and streptomycin (100 U/ml)). After 24 h incubation was growth medium changed to serum free medium containing test compounds. The final applied concentration range was 3.9-31.25 mol/l. As a control, serum free medium supplemented with appropriate concentration of DMSO (0.5%, v/v) was used. Rosmarinic acid was used as positive control. After 60 minutes incubation with test compounds medium was discarded, cells were washed two-times with PBS and PBS supplemented with glucose (1 mg/ml) was applied. To study UVA photoprotection, a plate was exposed to a cytotoxic dose of UVA radiation (10 J/cm2) using a solar simulator SOL 500 (Dr. Hoenle Technology, Germany) equipped with a H1 filter transmitting wavelengths of 320-400 nm. To study UVB photoprotection, a plate was exposed to a cytotoxic dose of UVB radiation (150 mJ/cm2) using the solar simulator equipped with a H2 filter transmitting wavelengths of 295-320 nm. Intensity of UVA or UVB radiation was evaluated before each irradiation by UVA- or UVB-meter. Control (non-irradiated) plates were for the period of irradiation incubated in dark. After UVA or UVB exposure PBS with glucose was discarded and serum free medium was applied. After 24 hours (37 C., 5% CO2) cell damage was evaluated by neutral red (NR) incorporation into viable cells. Medium was discarded and NR solution (0.03% w/v, PBS) was applied. After 60 minutes NR solution was discarded, cells were fixed with a mixture of formaldehyde (0.5%, v/v) and CaCl2 (1%, m/v) in ratio 1:1 and then NR was dissolved in methanol (50%, v/v) with acetic acid (1%, v/v).

(58) After 5 minutes of intensive shaking absorbance was measured at 540 nm. Experiments were performed in four independent repetitions with use of cells from four donors to minimize individual sensitivity of donor cells. Photoprotective effect was evaluated by comparison of experimental data (absorbance) of test compounds with a positive control and a negative control (according to the following equation:

(59) $\mathrm{Protection}\left(\%\right)=100-.Math.\frac{\mathrm{As}-\mathrm{Anc}}{\mathrm{Apc}-\mathrm{Anc}}.Math..Math.100$

(60) As . . . absorbance of sample (cells pre-incubated with test compounds in serum free medium and irradiated)

(61) Anc . . . absorbance of negative control (cells pre-incubated with s DMSO in serum free medium and non-irradiated=incubated in dark)

(62) Apc . . . absorbance of positive control (cells pre-incubated with s DMSO in serum free medium and irradiated)

(63) Results: Cells pre-incubated with test compound and exposed to UVA or UVB radiation showed higher viability (ability to incorporate NR) compared to those pre-incubated with DMSO (control) and UVA or UVB irradiated (Tab. 17 and 18). All test compounds showed higher or comparable photoprotective activity with rosmarinic acid used as positive control. Therefore test compound has high photoprotective potential.

(64) TABLE-US-00017 TABLE 17 Photoprotective effects of test compounds and rozmarinic acid (positive control) on UVA-induced damage to HaCaT 3.9 7.8 15.6 31.3 3 (mol/l) Protection (%) 26.8 34.3 46.0 50.7 SMODCH 6.9 9.7 6.9 6.1 9 (mol/l) Protection (%) 72.1 70.3 66.8 61.4 SMODCH 4.4 4.9 8.1 10.0 15 (mol/l) Protection (%) 41.2 56.9 55.3 76.7 SMODCH 5.5 7.8 1.7 9.7 20 (mol/l) Protection (%) 12.3 25.0 28.9 31.6 SMODCH 5.2 7.7 10.2 9.1 RA Protection (%) 23.6 42.3 36.2 44.0 SMODCH 7.2 7.4 12.0 16.0

(65) TABLE-US-00018 TABLE 18 Photoprotective effects of test compounds and rozmarinic acid (positive control) on UVA-induced damage to HaCaT 3.9 7.8 15.6 31.3 3 (mol/l) Protection (%) 27.3 46.5 47.8 53.8 SMODCH 4.4 11.6 14.4 14.4 9 (mol/l) Protection (%) 48.8 56 55.7 56.5 SMODCH 1.9 12.6 7.7 6.3 15 (mol/l) Protection (%) 12.3 35.3 37.8 51.3 SMODCH 7.7 12.6 7.5 3.5 20 (mol/l) Protection (%) 45.7 66.2 64 75.2 SMODCH 7.1 1.6 3 10.3 RA Protection (%) 22 48.5 55.7 54.3 SMODCH 0.4 1.2 2.2 13.1

Example 15: Markers of UVA Protection

(66) Normal human dermal fibroblasts (NHDF) were used as an in vitro model. Cells were isolated from tissue specimens obtained from healthy patients undergoing plastic surgery at the Department of Plastic and Aesthetic Surgery (University Hospital Olomouc). The use of skin tissue was in accordance with the Ethics Committee of the University Hospital and Faculty of Medicine and Dentistry, Palacky University, Olomouc and all patients signed written informed consent. Fibroblasts were used between the 2nd and 4th passage. For all experiments the fibroblasts were seeded onto 6-well plates at a density of 0.510.sup.5 cells/cm.sup.2 of cultivation medium (DMEM supplemented with fetal calf serum (10%, v/v), penicillin (100 mg/ml) and streptomycin (100 U/ml)).

(67) Test compounds included 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine and positive control (rosmarinic acid). Compounds were dissolved in DMSO and then diluted in serum free medium (DMEM supplemented with penicillin (100 mg/ml) and streptomycin (100 U/ml)). The final applied concentrations range was 2.5-20 mol/l. As a control serum free medium supplemented with appropriate concentration of DMSO (0.5%, v/v) was used. Each test compound was in parallel applied on two 6-well plates with NHDF (3.1510.sup.5 cells/cm.sup.2). After 60 minutes incubation medium with test compound was discarded, cells were washed two-times with PBS and PBS supplemented with glucose (1 mg/ml) was applied. To study UVA photoprotection, a plate was exposed to a cytotoxic dose of UVA radiation (10 J/cm2) using a solar simulator SOL 500 (Dr. Hoenle Technology, Germany) equipped with a H1 filter transmitting wavelengths of 320-400 nm. Intensity of UVA radiation was evaluated before each irradiation by UVA-meter. Control (non-irradiated) plates were for the period of irradiation incubated in dark. After UVA exposure PBS with glucose was discarded and serum free medium was applied. After 1 hour (37 C., 5% CO2) cell damage was evaluated by analysis of reactive oxygen species (ROS) production. In parallel, intracellular levels of glutathion (GSH) were measured 4 hours (37 C., 5% CO2) after UVA application.

(68) ROS Production

(69) ROS production was evaluated by 2,7-Dichlorodihydrofluorescein diacetate (H.sub.2DCFDA). NHDF were incubated with (H.sub.2DCFDA) (5 nmol/l, 20 min) 1 hour after UVA exposure. Subsequently, cells were washed two-times with PBS, scraped into PBS a sonicated. Samples were applied on 96-well plate and fluorescence was measured (488/525 nm) (INFINITE M200, Tecan, Switzerland) after centrifugation (10 000 rpm, 4 C., 10 min). Protein content was analyzed spectrophotometrically by bicinchoninic acid at 562 nm (INFINITE M200, Tecan, Switzerland).

(70) GSH Depletion

(71) GSH levels in NHDF were evaluated by reaction with 5,5-Dithiobis(2-nitrobenzoic acid) (DTNB). Cells were washed two-times with PBS, scraped into acetic acid (1%, v/v) and sonicated. Samples were applied on 96-well plate after centrifugation (10 000 rpm, 4 C., 10 min) and reaction mixture was added (0.8 mol/l Tris/HCl, 20 mmol/l EDTA, pH 8.2; 20 mg/ml DTNB). Absorbance was measured at 412 nm. Protein content was analyzed spectrophotometrically by Lowry's method at 680 nm.

(72) Activity of test compounds (not irradiated) in analyzed parameters were evaluated by comparison of experimental data according to the following equation:

(73) $\%\mathrm{of}\mathrm{control}=100.Math.\left(\frac{\left(A_V-A_P\right)}{\left(A_K-A_P\right)}\right)$

(74) A.sub.P . . . background value

(75) A.sub.V . . . sample value (cells pre-incubated with test compounds in serum free medium)

(76) A.sub.K . . . control value (cells pre-incubated with s DMSO in serum free medium)

(77) Photoprotective effect was evaluated by comparison of experimental data (absorbance, fluorescence) of test compounds with a positive control (cells pre-incubated with DMSO in serum free medium and irradiated) and a negative control (cells pre-incubated with DMSO in serum free medium and non-irradiated=incubated in dark) according to the following equation:

(78) $\mathrm{Protection}\left(\%\right)=100-\left\{\left(\frac{\left(A_V-A_K\right)}{\left(A_{\mathrm{UV}}-A_K\right)}\right)*100\right\}$

(79) A.sub.K . . . negative control value (DMSO in serum free medium and non-irradiated)

(80) A.sub.UV . . . positive control value (DMSO in serum free medium and irradiated)

(81) A.sub.V . . . sample value (cells pre-incubated with test compounds in serum free medium and irradiated)

(82) Results: 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine did not showed negative effect on tested parameters (on non-irradiated cells) in test concentrations (2.5-20 mol/l). Test compound protected cells against UVA-induced production of ROS as well as depletion of GSH (endogenous antioxidant) (Tab. 19 and 20) in a concentration-dependent manner. Compound 1 is more effective in protection against production of ROS but less effective in protection against GSH depletion in comparison with rosmarinic acid.

(83) TABLE-US-00019 TABLE 19 Protective effect of 6-(tetrahydrofuran-2-ylmethylamino)-9- (tetrahydrofuran-2-yl)purine and rozmarinic acid (positive control) on UVA-induced ROS production ROS production Concentration 1 RA (mol/l) Protection (%) 2.5 21.3 (6.9) 20.6 (8.4) 5 21.3 (5.2) 24.1 (1).sup. 10 29.6 (3.3) 24.1 (2.4) 20 48.3 (0.5) 30.3 (6.8)

(84) TABLE-US-00020 TABLE 20 Protective effect of 6-(tetrahydrofuran-2-ylmethylamino)-9- (tetrahydrofuran-2-yl)purine and rozmarinic acid (positive control) on UVA-induced GSH depletion GSH depletion Concentration 1 RA (mol/l) Protection (%) 2.5 25.5 (6.8) 53.9 (7.1) 5 27.9 (7.2) 42.8 (2.4) 10 61.6 (15.7) 68.6 (4.3) 20 35.5 (14.1) 50.5 (9).sup.

(85) Normal human dermal fibroblasts (NHDF) were used as an in vitro model. Cells were isolated from tissue specimens obtained from healthy patients undergoing plastic surgery at the Department of Plastic and Aesthetic Surgery (University Hospital Olomouc). The use of skin tissue was in accordance with the Ethics Committee of the University Hospital and Faculty of Medicine and Dentistry, Palacky University, Olomouc and all patients signed written informed consent. Fibroblasts were used between the 2nd and 4th passage. For all experiments the fibroblasts were seeded onto 6-well plates at a density of 0.510.sup.5 cells/cm.sup.2 of cultivation medium (DMEM supplemented with fetal calf serum (10%, v/v), penicillin (100 mg/ml) and streptomycin (100 U/ml)).

(86) Test compounds included 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine and positive control (rosmarinic acid). Compounds were dissolved in DMSO and then diluted in serum free medium (DMEM supplemented with penicillin (100 mg/ml) and streptomycin (100 U/ml)). The final applied concentrations range was 2.5-20 mol/l. As a control serum free medium supplemented with appropriate concentration of DMSO (0.5%, v/v) was used. Each test compound was in parallel applied on two 6-well plates with NHDF (3.1510.sup.5 cells/cm.sup.2). After 60 minutes incubation medium with test compound was discarded, cells were washed two-times with PBS and PBS supplemented with glucose (1 mg/ml) was applied. To study UVB photoprotection, a plate was exposed to a cytotoxic dose of UVB radiation (150 mJ/cm2) using a solar simulator SOL 500 (Dr. Hoenle Technology, Germany) equipped with a H2 filter transmitting wavelengths of 295-320 nm. Intensity of UVB radiation was evaluated before each irradiation by UVB-meter. Control (non-irradiated) plated were for the period of irradiation incubated in dark. After UVB exposure PBS with glucose was discarded and serum free medium was applied. After 4 hours (37 C., 5% CO2) cell damage was evaluated by analysis of caspase-3 activity.

(87) Caspase-3 Activity

(88) Caspase-3 activity was evaluated by specific substrate (Ac-DEVD-AMC). NHDF were washed two-times with PBS, scraped into lysis buffer (50 mmol/l HEPES, pH 7.4; TritonX-100 (0.5%; v/v), protease inhibitor, 5 mmol/l DDT). Samples were applied on 96-well plate after centrifugation (10 000 rpm, 4 C., 10 min) and reaction buffer was added (20 mmol/l HEPES, pH 7.1, 2 mmol/l EDTA, protease inhibitor, 5 mmol/l DDT) cantaining specific substrate or inhibitors. Fluorescence was measured at (400/505 nm) after 1 hour incubation (37 C., dark). Protein content was analyzed spectrophotometrically by Bradford protein assay (INFINITE M200, Tecan, Switzerland).

(89) 0$\%\mathrm{of}\mathrm{control}=100.Math.\left(\frac{\left(A_V-A_P\right)}{\left(A_K-A_P\right)}\right)$

(90) A.sub.P . . . background value

(91) A.sub.V . . . sample value (cells pre-incubated with test compounds in serum free medium)

(92) A.sub.K . . . control value (cells pre-incubated with s DMSO in serum free medium)

(93) Photoprotective effect was evaluated by comparison of experimental data (absorbance, fluorescence) of test compounds with a positive control (cells pre-incubated with DMSO in serum free medium and irradiated) and a negative control (cells pre-incubated with DMSO in serum free medium and non-irradiated=incubated in dark) according to the following equation:

(94) $\mathrm{Protection}\left(\%\right)=100-\left\{\left(\frac{\left(A_V-A_K\right)}{\left(A_{\mathrm{UV}}-A_K\right)}\right)*100\right\}$

(95) A.sub.K . . . negative control value (DMSO in serum free medium and non-irradiated)

(96) A.sub.UV . . . positive control value (DMSO in serum free medium and irradiated)

(97) A.sub.V . . . sample value (cells pre-incubated with test compounds in serum free medium and irradiated)

(98) Results: 6-(tetrahydrofuran-2-ylmethylamino)-9-(tetrahydrofuran-2-yl)purine did not showed negative effect on tested parameters (on non-irradiated cells) in test concentrations (2.5-20 mol/l). Compound 1 decreased UVB-induced activity of caspase-3 (Tab. 21).

(99) TABLE-US-00021 TABLE 21 Protective effect of 6-(tetrahydrofuran-2-ylmethylamino)-9- (tetrahydrofuran-2-yl)purine and rozmarinic acid (positive control) on UVB-induced caspase-3 activity Caspase-3 activity Concentration 1 RA (mol/l) Protection (%) 2.5 41.2 (8.2) 60.6 (3.1) 5 84.4 (27.2) 39.7 (13.1) 10 71.1 (30.3) 38.4 (5.9) 20 45.9 (4.7) 28.7 (3.8)

Example 15: Comparative Gene Expression Analysis

(100) Comparative gene expression analysis in human dermal fibroblast (HDF) was performed to gain insight into role of compound 1 in the photoprotection against UV-induced damage. Three independent HDF lines form three patients were treated with 5 M 1 for 24 h or mock-treated with DMSO. Alternatively, the above three biological samples (without 1) were irradiated with UV light (5 J/cm2). 1.5-3.010.sup.6 cells from each treatment or control was used for isolation of total RNA using trizol. cDNA sequencing libraries were prepared with the Illumina TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, Calif.) according to standard Illumina's protocols and sequenced on HiSeq 2500 apparatus (50 bp single-end reads).

(101) Data were subjected to differential transcriptomic analysis with the aim to characterize significantly regulated genes and their expression levels. To reveal the molecular mechanism of the action of 1, we compared data from mock (DMSO)-treated HDF or UV-treated HDF with those obtained after 24 h treatment with 1. For data analysis, we performed ab initio method where sequencing reads were mapped to the reference genome. Comparison of the control group vs samples treated with 1 did not show any significantly regulated genes indicating 1 had low effect on the gene expression under normal conditions. Interestingly, when 1-treated group of data was compared with UV-treated group we could detect 1306 differentially regulated genes (P<0.05). 865 of those were upregulated and 441 genes were downregulated. To limit the number of genes that respond most significantly to 1 treatment we set relatively stringent conditionswe selected genes which log.sub.2 FC>1.5, or those with log.sub.2 FC<1.5. Further inspection of the subgroups (41 upregulated genes and 41 downregulated, see Tab. 22 and Tab. 23, respectively) revealed major differences among those. In the group of the upregulated genes, we could observe a range of genes with regulatory, developmental or receptor/signaling function, such as the calcium sensing. These included calcium-activated potassium channel KCNN4, calcium sensor DYSF or calcium-dependent phospholipid-binding protein CPNE7. In addition, we noted increased expression of the negative regulator of reactive oxygen species NRROS and the scavenger cysteine-rich type 1 receptor CD163 that protects again oxidative damage suggesting that 1 facilitates a mechanism leading to the protection against damage caused by oxygen radicals. Thus, 1 seems to protect cells against oxidative damage caused primarily by UV-light or other stress conditions.

(102) In contrast, in the group of the downregulated genes we found regulatory genes with a large group of genes that may be related to immune response. We observed upregulation of two chemokines, CCL8 and CXCL9, and cytokines TNFSF13B and TNFSF10. In addition, several protein kinases, such as HCK and JAK3, and innate immune response-related proteins TLR2 and GBP2 were found to be downregulated by 1. Hence, the immunosuppression mediated by 1 may contribute to the in vivo function of the compound.

(103) TABLE-US-00022 TABLE 22 Genes upregulated in response to the treatment with the compound 1 (P 0.05 and log.sub.2FC > 1.5). Gene Description logFC KIF1A kinesin family member 1A 3.05 ANKRD33 ankyrin repeat domain-containing protein 33 2.69 HS3ST2 heparan sulfate-glucosamine 3-sulfotransfer- 2.50 ase 2 CHRNA9 neuronal acetylcholine receptor subunit 2.21 alpha-9 TMEM233 interferon-induced transmembrane domain- 2.12 containing protein D2 TM4SF1 transmembrane 4 L6 family member 1 2.08 SCG2 secretogranin-2 2.00 MYEOV myeloma-overexpressed gene protein 1.98 PPP2R2C serine/threonine-protein phosphatase 2A 1.95 55 kDa regulatory subunit B KCNN4 Intermediate conductance calcium-activated 1.85 potassium channel 4 RP11-184E9.1 non-coding RNA 1.78 ITGA10 integrin alpha-10 1.77 CCL26 C-C motif chemokine 26 1.75 NRROS negative regulator of reactive oxygen species 1.74 DYSF dysferlin 1.73 ANGPTL4 angiopoietin-related protein 4 1.73 FAM81A non-coding RNA 1.71 RP11-541M12.6 non-coding RNA 1.70 RP11-367F23.2 non-coding RNA 1.70 LINC00704 non-coding RNA 1.69 FOLR3 folate receptor gamma 1.68 CD163 scavenger receptor cysteine-rich type 1 1.67 protein M130 ARHGAP22 rho GTPase-activating protein 22 1.66 CYP51A1 lanosterol 14-alpha demethylase 1.65 LINC01204 non-coding RNA 1.64 CITED4 cbp/p300-interacting transactivator 4 1.63 RP11-54A9.1 non-coding RNA 1.63 MET MET proto-oncogene, receptor tyrosine 1.63 kinase EBF2 transcription factor COE2 1.62 CPNE7 copine-7 1.61 LINC00702 non-coding RNA 1.60 TGM2 protein-glutamine gamma-glutamyltransfer- 1.58 ase 2 AC002456.2 non-coding RNA 1.58 TMEM154 transmembrane protein 154 1.57 NUDT8 nucleoside diphosphate-linked moiety X 1.56 motif 8 SLC20A1 sodium-dependent phosphate transporter 1 1.56 B4GALNT1 beta-1,4-N-acetyl-galactosaminyltransfer- 1.55 ase 1 STX1A syntaxin-1A 1.55 CTD-2587H24.5 non-coding RNA 1.54 ADGRG1 adhesion G-protein coupled receptor G1 1.52 CCDC107 coiled-coil domain-containing protein 107 1.50

(104) TABLE-US-00023 TABLE 23 Genes downregulated in response to the treatment with the compound 1 (P 0.05 and log.sub.2FC <1.5). Gene Description logFC RP11-383F6.1 non-coding RNA 1.50 APOL6 apolipoprotein L6 1.51 RP11-400K9.4 non-coding RNA 1.51 TMEM178A transmembrane protein 178A 1.52 HAUS1P2 non-coding RNA 1.54 RORB nuclear receptor ROR-beta 1.57 RP11-363H12.1 non-coding RNA 1.58 SERPINB9 serpin B9 1.59 DHRS3 short-chain dehydrogenase/reductase 3 1.60 TP63 tumor protein 63 1.61 GBP2 guanylate-binding protein 2 1.61 TNFSF13B tumor necrosis factor ligand superfamily 1.61 member 13B ADAMTS9-AS2 non-coding RNA 1.63 TLR2 toll-like receptor 2 1.63 AC003986.6 non-coding RNA 1.65 HCK tyrosine-protein kinase HCK 1.66 GPR37L1 G protein-coupled receptor 37 like 1 1.66 JAK3 tyrosine-protein kinase JAK3 1.68 TNFRSF9 tumor necrosis factor receptor superfamily 1.68 member 9 RP11-1100L3.8 non-coding RNA 1.70 MOB3B MOB kinase activator 3B 1.70 RP11-379B8.1 non-coding RNA 1.72 BRINP2 BMP/retinoic acid-inducible neural-specific 1.77 protein 2 IFIT2 interferon-induced protein with 1.77 tetratricopeptide repeats 2 CXCL9 C-X-C motif chemokine 9 1.79 CLNS1AP1 non-coding RNA 1.80 RNF150 RING finger protein 150 1.80 BTC probetacellulin 1.84 FAM20A pseudokinase FAM20A 1.86 RPL34P31 non-coding RNA 1.89 BHLHE22 class E basic helix-loop-helix protein 22 1.90 FGD3 FYVE, RhoGEF and PH domain-containing 1.93 protein 3 RP11-21C4.1 non-coding RNA 1.95 CFB complement factor B 1.96 RARRES3 retinoic acid receptor responder protein 3 2.09 TNFSF10 tumor necrosis factor ligand superfamily 2.14 member 10 PSAT1P3 non-coding RNA 2.19 KCNT2 potassium channel subfamily T member 2 2.21 RP1-181J22.1 non-coding RNA 2.53 ABCG1 ATP-binding cassette sub-family G 2.63 member 1 CCL8 C-C motif chemokine 8 2.80