Cyclodextrin-based transporter of nucleoside triphosphate transporter across the cell membrane, its preparation and use

Abstract

Compounds of general formulae 6 and 13 where X is —NH—C(NH.sub.2)═N+H.sub.2 or —N+H.sub.3, Y is a linear oligomer of arginine units terminated with an aminodimethylenamide unit (-Arg)n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca)n-NH.sub.2, where n=6 to 10, A-═CF.sub.3COO— or Cl— and m=1-2. Preparation and use of compounds of general formula 6 and 13 as carriers of nucleoside triphosphates across the cell membranes for the purpose of incorporation of modified nucleoside triphosphates into cellular DNA or RNA. Use of compounds of general formula 6 and 13 as carriers of nucleoside triphosphates across the cell membrane for determining the virostatic activities of modified nucleoside triphosphates. Use of compounds of general formula 6 and 13 as carriers of modified nucleoside triphosphates across the cell membrane for determining cell proliferation and S phase of the cell cycle.

Claims

1. Compounds of general formula 6 ##STR00017## where X is —NH—C(NH.sub.2)═N.sup.+H.sub.2 or —N.sup.+H.sub.3 and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, in a form of salt with pharmaceutically acceptable anion.

2. A method for the preparation of compounds of general formula 6 where X is —NH—C(NH.sub.2)=N.sup.+H.sub.2 CF.sub.3COO.sup.− and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, characterized in that the starting compound of formula 1 ##STR00018## is converted with allyl bromide and sodium hydride in DMF to a mixture of 2.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin and 3.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin; this mixture is then treated with acetic anhydride, N,N-diisopropylethylamine and N,N-dimethylaminopyridine in acetonitrile at room temperature to isolate compound of structural formula 2 ##STR00019## which is deacetylated in a further step, by treatment with sodium methoxide in anhydrous methanol to give the product 2.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin of formula 3 ##STR00020## from which 2.sup.I-O-allyl-heptakis(6-amino-6-deoxy)-β-cyclodextrin heptakis(trifluoro-acetate) of structural formula 4 ##STR00021## is prepared by treatment with triphenyl phosphine and aqueous ammonia in dimethylformamide, which is converted to 2.sup.I-O-allyl-heptakis(6-guanidino-6-deoxy)-β-cyclodextrin heptakis(trifluoroacetate) of formula 5 ##STR00022## by treatment with 1H-pyrazole-1-carboxamidine hydrochloride, and then the compound 5 is treated with the photoinitiator 2,2-dimethoxy-2-phenylacetophenone and light of wavelength 365 nm, or alternatively with a radical initiator azobisisobutyronitrile and heating, and thiols of general formulae SH—(CH.sub.2).sub.5—CO-(Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2 or SH—(CH.sub.2).sub.5—CO-(Arg-Aca).sub.n-NH.sub.2 and converted into compounds of the general formula 6, ##STR00023## where X is —NH—C(NH.sub.2)=N.sup.+H.sub.2 CF.sub.3COO.sup.− and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10.

3. Method of transporting nucleoside triphosphates across cell membranes, comprising the step of bringing into contact the compound of general formula 6 according to claim 1 and a nucleoside triphosphate and applying the resulting complex to a cell culture.

4. Method of incorporation of nucleoside triphosphates into the cellular nucleic acids, comprising the step of bringing into contact the compound of general formula 6 according to claim 1 and a nucleoside triphosphate and applying the resulting complex to a cell culture.

5. Method of determining virostatic or anticancer activity of nucleoside triphosphates, comprising the step of bringing into contact the compound of general formula 6 according to claim 1 and a nucleoside triphosphate and applying the resulting complex to a cell culture.

6. Method of determining cell proliferation and S phase of the cell cycle, comprising the step of bringing into contact the compound of general formula 6 according to claim 1 and a modified nucleoside triphosphate and applying the resulting complex to a cell culture.

7. A method for the preparation of compounds of general formula 6 according to claim 1, where X is —N.sup.+H.sub.3 CF.sub.3COO.sup.− and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10 characterized in that the starting compound of formula 1 ##STR00024## is treated with allyl bromide or allyl iodide and sodium hydride or potassium tert-butoxide in dimethylformamide to produce a mixture of 2′-(9-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin 3 and 3.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin 9 in ratio approximately 9:1, ##STR00025## which is then treated with ethyl trifluoroacetate and DIPEA in methanol to produce a mixture of isomeric compounds 10 and 11 ##STR00026## which are separated by means of reversed-phase HPLC; compound 10 is then treated with light of wavelength 365 nm, a photoinitiator 2,2-dimethoxy-2-phenylacetophenone (or azobisisobutyronitrile and heating) and thiols of general formulae SH—(CH.sub.2).sub.5—CO-(Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2 or SH—(CH.sub.2).sub.5—CO-(Arg-Aca).sub.n-NH.sub.2, where n=6-10, and, in this way, converted to compounds of general formula 12, ##STR00027## where Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, which are subsequently treated with aqueous ammonia to remove protective groups to obtain final products of general formula 6, ##STR00028## where X is —N.sup.+H.sub.3 CF.sub.3COO.sup.− and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10.

8. Compounds of general formula 13 ##STR00029## where X is —NH—C(NH.sub.2)=N.sup.+H.sub.2 or —N.sup.+H.sub.3 and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, A.sup.− is a pharmaceutically acceptable anion; the number of counteranions is from 10 to 16 per molecule.

9. A method for the preparation of compounds of general formula 13, where X is —NH—C(NH2)=N.sup.+H.sub.2 and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg)n-NH—(CH2)2-NH2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca)n-NH2, where n=6 to 10, characterized in that the starting compound octakis(6-azido-6-deoxy)-γ-cyclodextrin is converted by treatment with allyl bromide or allyl iodide and sodium hydride or potassium tert-butoxide in dimethylformamide to 2.sup.I-O-allyl-octakis(6-azido-6-deoxy)-γ-cyclodextrin 14 ##STR00030## reduction of which by action of triphenylphosphine and aqueous ammonia yields 2.sup.I-O-allyl-octakis(6-amino-6-deoxy)-γ-cyclodextrin 16, ##STR00031## which is treated with 1H-pyrazole-1-carboxamidine hydrochloride and converted to 2.sup.I-O-allyl-octakis(6-guanidino-6-deoxy)-γ-cyclodextrin octakis (trifluoroacetate) of structural formula 17; ##STR00032## compound 17 is treated with light of wavelength 365 nm, a photoinitiator 2,2-dimethoxy-2-phenylacetophenone, or alternatively by azobisisobutyronitrile and heating, and thiols of general formulae SH—(CH.sub.2).sub.5—CO-(Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2 or SH—(CH.sub.2).sub.5—CO-(Arg-Aca).sub.n-NH.sub.2 and, in this way, converted to compounds of general formula 13, ##STR00033## where X is —NH—C(NH.sub.2)═N.sup.+H.sub.2 and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, A.sup.− is CF.sub.3COO.sup.− or Cl.sup.−; the number of counteranions varies from 10 to 16 per molecule.

10. Method of transporting nucleoside triphosphates across cell membranes, comprising the step of bringing into contact the compound of general formula 13 according to claim 8 and a nucleoside triphosphate and applying the resulting complex to a cell culture.

11. Method of incorporation of nucleoside triphosphates into the cellular nucleic acids, comprising the step of bringing into contact the compound of general formula 13 according to claim 8 and a nucleoside triphosphate and applying the resulting complex to a cell culture.

12. Method of determining virostatic or anti cancer activity of nucleoside triphosphates, comprising the step of bringing into contact the compound of general formula 13 according to claim 8 and a nucleoside triphosphate and applying the resulting complex to a cell culture.

13. Method of determining cell proliferation and S phase of the cell cycle, comprising the step of bringing into contact the compound of general formula 13 according to claim 8 and a modified nucleoside triphosphate and applying the resulting complex to a cell culture.

14. Compounds of general formula 6 ##STR00034## where X is —NH—C(NH.sub.2)═N.sup.+H.sub.2 or —N.sup.+H.sub.3 and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg)n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca)n-NH.sub.2, where n=6 to 10, in a form of salt with pharmaceutically acceptable anion, or compounds of general formula 13 ##STR00035## where X is —NH—C(NH.sub.2)═N.sup.+H.sub.2 or —N.sup.+H.sub.3 and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, A.sup.− is a pharmaceutically acceptable anion; the number of counteranions is from 10 to 16 per molecule, where some or all arginine units in said general formula 6 or 13 are replaced with naturally occurred amino acids containing guanidine moiety or guanidino peptidomimetics chosen from norarginine, homoarginine and β-homoarginine.

15. Compounds of general formula 6 ##STR00036## where X is —NH—C(NH.sub.2)═N.sup.+H.sub.2 or —N.sup.+H.sub.3 and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, in a form of salt with pharmaceutically acceptable anion, or compounds of general formula 13 ##STR00037## where X is —NH—C(NH.sub.2)═N.sup.+H.sub.2 or —N.sup.+H.sub.3 and Y is a linear oligomer consisting of arginine units units terminated with an aminodimethylenamide unit (-Arg)n-NH—(CH2)2-NH2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH2, where n=6 to 10, A-is a pharmaceutically acceptable anion; the number of counteranions is from 10 to 16 per molecule, where some or all amido groups are replaced by different functional groups chosen from ester group, amine group, carbamate group or ether group.

16. Compounds of general formula 6 according to claim 1, wherein the pharmaceutically acceptable anion is CF.sub.3COO.sup.− or a chloride form.

17. Compounds of general formula 13 according to claim 8, wherein the pharmaceutically acceptable anion is CF.sub.3COO— or Cl.sup.−.

18. A method for the preparation of compounds of general formula 6 where X is —N.sup.+H.sub.3 CF.sub.3COO.sup.− and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, characterized in that the starting compound of formula 1 ##STR00038## is converted with allyl bromide and sodium hydride in DMF to a mixture of 2.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin and 3.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin; this mixture is then treated with acetic anhydride, N,N-diisopropylethylamine and N, X-dimethylaminopyridine in acetonitrile at room temperature to isolate compound of structural formula 2, ##STR00039## which is deacetylated in a further step, by treatment with sodium methoxide in anhydrous methanol to give the product 2.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin of formula 3, ##STR00040## from which 2.sup.I-O-allyl-heptakis(6-amino-6-deoxy)-β-cyclodextrin heptakis(trifluoro-acetate) of structural formula 4 ##STR00041## is prepared by treatment with triphenyl phosphine and aqueous ammonia in dimethylformamide, and then the compound 4 is treated with the photoinitiator 2,2-dimethoxy-2-phenylacetophenone and light of wavelength 365 nm, or alternatively with a radical initiator azobisisobutyronitrile and heating, and thiols of general formulae SH—(CH.sub.2).sub.5—CO-(Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2 or SH—(CH.sub.2).sub.5—CO-(Arg-Aca).sub.n-NH.sub.2 to obtain compounds of the formula 6, ##STR00042## where X is —N.sup.+H.sub.3 CF.sub.3COO.sup.− and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10.

19. A method for the preparation of compounds of general formula 13, where X is —N.sup.+H.sub.3 and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, A.sup.− is CF.sub.3COO.sup.− or Cl, characterized in that the starting compound octakis(6-azido-6-deoxy)-γ-cyclodextrin is converted by treatment with allyl bromide or allyl iodide and sodium hydride or potassium tert-butoxide in dimethylformamide to 2.sup.I-O-allyl-octakis(6-azido-6-deoxy)-γ-cyclodextrin 14 ##STR00043## reduction of which by action of triphenylphosphine and aqueous ammonia yields 2.sup.I-O-allyl-octakis(6-amino-6-deoxy)-γ-cyclodextrin 16; ##STR00044## compound 16 is treated with light of wavelength 365 nm, a photoinitiator 2,2-dimethoxy-2-phenylacetophenone, or alternatively with azobisisobutyronitrile and heating, and thiols of general formulae SH—(CH.sub.2).sub.5—CO-(Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2 or SH—(CH.sub.2).sub.5—CO-(Arg-Aca).sub.n-NH.sub.2 and, in this way, converted to compounds of general formula 13, ##STR00045## where X is-N.sup.+H.sub.3 and Y is a linear oligomer consisting of arginine units terminated with an aminodimethylenamide unit (-Arg).sub.n-NH—(CH.sub.2).sub.2—NH.sub.2, where n=6-10, or arginine-aminocaproic units (-Arg-Aca).sub.n-NH.sub.2, where n=6 to 10, A.sup.− is CF.sub.3COO.sup.− or Cl.sup.−; the number of counteranions varies from 10 to 16 per molecule.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1: Structural formula of Conjugate 6a

(2) FIG. 2: Structural formula of Conjugate 6b

(3) FIG. 3: Structural formula of Conjugate 6c

(4) FIG. 4: Structural formula of Conjugate 6d

(5) FIG. 5: a) Monitoring the penetration of a complex of compound 6a and fluorescently labelled NTP (ChromaTide® Alexa Fluor® 488-5-dUTP; ThermoFisher Scientific, Cat. No.: C11397) into U2-OS cells; b) control experiment performed by applying NTP to the cell culture (ChromaTide® Alexa Fluor® 488-5-dUTP; ThermoFisher Scientific, Cat. No.: C11397) without compound 6a.

(6) FIG. 6: Monitoring mitosis of U2-OS cells, which achieved incorporation of the fluorescent NTP into genomic DNA (light areas in the figures) with the help of compound 6b and NTP (Aminoallyl-dUTP-Cy3; Jena Bioscience, Cat. No. NU-803-S-Cy3).

(7) FIG. 7: Graph showing the dependence of the number of viral particles (corresponding to relative fluorescence) on the concentration of substances applied to the cell culture.

(8) FIG. 8: Structural formula of conjugate 13a

(9) FIG. 9: Dot chart representation of flow cytometry analysis described in Example 18.

(10) FIG. 10: Chemical shifts of .sup.1H NMR (ppm) in the spectra of compounds 2-5, 10 and 14. Spectra were taken at a frequency of 600 MHz.

(11) FIG. 11: Chemical shifts of .sup.13C NMR (ppm) in the spectra of compounds 2-5, 10 and 14. Spectra were taken at a frequency of 151 MHz.

(12) The invention will be further illustrated by the following examples, however, it is not restricted only to these.

EXAMPLES OF THE INVENTION EMBODIMENT

List of Abbreviations

(13) ACN Acetonitrile AcOEt Ethyl acetate BrdU 5-Bromo-2-deoxyuridine DIPEA N,N-Diisopropylethylamin EtOH Ethanol HRMS High Resolution Mass Spectroscopy MALDI Matrix-assisted laser desorption/ionization NMR Nuclear magnetic resonance NTP Nucleoside triphosphate TBME tert-Butyl methyl ether TFA Trifluoroacetic acid THF Tetrahydrofurane HPLC High performance liquid chromatography DNA Deoxyribonucleic acid LED Light-emitting diode HIV Human immunodeficiency virus Aminoallyl-dUTP—Cy3 5-(3-Aminoallyl)-2′-deoxyuridin-5′-triphosphate, bearing the Cy3 fluorescent label Cy3 Cyanine fluorophore TZM-bl Cell lines of cervical cancer DMF N,N-Dimethylformamide MeOH Methanol

(14) U2-OS Human osteosarcoma cell line

Example 1

Preparation of 2.SUP.II-VII.,3.SUP.I-VII.-trideca-O-acetyl-2.SUP.I.-mono-O-allyl-6.SUP.I-VII.-hepta-azido-6.SUP.I-VII.-hepta-deoxy-β-cyclodextrin 2

(15) Sodium hydride free of oil (54.8 mg, 2.28 mmol) was added to a solution of compound 1 (2 g, 1.52 mmol) in anhydrous dimethylformamide (40 ml) and reaction mixture was stirred for 3 hours at room temperature under an argon atmosphere. Allyl bromide (200 μl, 2.30 mmol) was then dropwise added to the reaction mixture and the mixture was allowed to react for 12 hours. Then, dimethylformamide was evaporated and the product mixture purified by column chromatography (silica gel, chloroform, methanol 4:1); and the fraction containing isomers 2.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin and 3.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin (total 740 mg) was isolated. This mixture (600 mg, 0.44 mmol) was dissolved in acetonitrile (18 ml) and acetanhydride (1 ml, 10.57 mmol), N,N-diisopropylethylamin (3 ml, 17.22 mmol) and N,N-dimethylaminopyridine (70 mg, 0.57 mmol) were gradually added to the solution with stirring. The reaction mixture was stirred for 12 hours at room temperature, then evaporated on a rotary evaporator and the residue was suspended in chloroform (100 ml) and the resulting suspension was washed with water (3×50 ml). The chloroform layer was dried with sodium sulphate, then the desiccant was removed by filtration on sintered glass and the resulting solution was concentrated to a volume of about 10 ml and loaded on a chromatographic column (silica gel, chloroform/acetone 5:1). The main fraction after evaporation contained 2.sup.I-O-allyl-heptakis(6-azido-6-deoxy)-O-cyclodextrin 2 (702 mg, 31%).

(16) Characterization: HRMS (MALDI): m/z calculated for C.sub.71H.sub.93N.sub.21O.sub.41 [M+Na].sup.+: 1918.5730; found 1918.5758; elemental analysis (%): calculated for C.sub.71H.sub.93N.sub.21O.sub.41: C, 44.96; H, 4.94; N, 15.51; found C, 44.98; H, 4.92; N, 15.41. .sup.1H and .sup.13C NMR data—see FIGS. 10 and 11.

Example 2

Preparation of 2.SUP.I.-O-allyl-heptakis(6-azido-6-deoxy)-β-cyclodextrin 3

(17) Compound 2 (700 mg, 0.369 mmol) was dissolved in sodium methanolate solution in anhydrous methanol (0.1 mol.Math.l.sup.−1, 70 ml). The reaction mixture was stirred at room temperature; formation of a white precipitate was observed during the reaction. After 3 hours, the mixture was evaporated to dryness, the residue was dissolved in a mixture of methanol and dimethylformamide 1:1 (2 ml) and re-precipitated with water (100 ml). The precipitate was filtered on a sintered glass and dried under vacuum at room temperature. Compound 3 was isolated in a yield of 375 mg (75%).

(18) Characterization: HRMS (MALDI): m/z calculated for C.sub.45H.sub.67N.sub.21O.sub.28 [M+Na].sup.+: 1372.4357; found 1372.4362; elemental analysis (%: calculated for C.sub.45H.sub.67N.sub.21O.sub.28: C, 40.03; H, 5.00; N, 21.79; found C: 41.59; H, 5.44; N, 18.42. .sup.1H and .sup.13C NMR data—see FIGS. 10 and 11.

Example 3

Preparation of 2.SUP.I.-O-allyl-heptakis(6-amino-6-deoxy)-β-cyclodextrin heptakis(trifluoroacetate) 4

(19) Triphenylphosphine (190 mg, 0.72 mmol) was added to a solution of compound 3 (70 mg, 0.05 mmol) in dimethylformamide (1.4 ml). After 2 hours a solution of ammonia in water (0.5 ml of 25% solution) was added to the reaction mixture and the mixture was stirred for 16 h. The solvent was then evaporated and acetone (20 ml) was added to the resulting thick syrup. The resulting white precipitate was isolated by filtration on sintered glass and then suspended in a mixture of methanol (1.5 ml) and ammonia (1.5 ml). The mixture was heated in a pressure tube at 60° C. for 6 hrs, and then evaporated under reduced pressure. The residue was dissolved in a 0.01% aqueous solution of trifluoroacetic acid and purified by ultrafiltration on a membrane with 1 kDa pores (Ultracell; manufacturer Merck Millipore), and lyophilized. The yield of compound 4 was 53 mg (48%).

(20) Characterization: HRMS (MALDI): m/z calculated for C.sub.45H.sub.67N.sub.7O.sub.28 [M+Na].sup.+: 1190.5022; found 1190.5036; elemental analysis (%), calculated for C.sub.45H.sub.67N.sub.7O.sub.28.7TFA.5H.sub.2O: C, 34.46; H, 4.80; N, 4.77; found C, 34.42; H, 4.80; N, 4.37. .sup.1H and .sup.13C NMR data—see FIGS. 10 and 11.

Example 4

Preparation of 2.SUP.I.-O-allyl-heptakis(6-guanidino-6-deoxy)-β-cyclodextrin heptakis(trifluoroacetate) 5

(21) Compound 4 (35 mg, free base) and 1H-pyrazolcarboxamidin hydrochloride (219 mg) were suspended in a mixture of N,N-diisopropylethylamine (0.26 ml) and water (0.26 ml). The mixture was stirred at room temperature for 24 hours, then solvents were evaporated under reduced pressure. The residue was dissolved in a 0.01% aqueous solution of trifluoroacetic acid and purified by ultrafiltration on a membrane with 1 kDa pores (Ultracell; manufacturer Merck Millipore), and lyophilized. Yield of compound 5 was 33 mg (49%).

(22) Characterization: HRMS (MALDI), m/z calculated for C.sub.52H.sub.96N.sub.21O.sub.28 [M+H].sup.+: 1462.6728; found 1462.6758; elemental analysis (%), calculated for C.sub.66H.sub.102F.sub.21N.sub.21O.sub.42.7TFA.6H.sub.2O C; 33,47; H; 4.85; F; 16.84; N; 12.42; O; 32.42 .sup.1H a .sup.13C NMR data—see FIGS. 10 and 11.

Example 5

Synthesis of Conjugate 6a (FIG. 1)

(23) 2,2-Dimethoxy-2-phenylacetophenone (0.89 mg), SH—(CH.sub.2).sub.5—CO-(Arg-Aca).sub.8-NH.sub.2 thiol (44.90 mg; prepared by standard peptide solid phase synthesis using an automated ABI 433A synthesizer, Applied Biosystems) and the compound of formula 5 (34.7 mg) were dissolved in DMF. The mixture was stirred with ultrasound followed by magnetic stirring under an inert atmosphere of argon. The mixture was periodically irradiated with UV light of wavelength 365 nm generated by the LED (1 W) under constant stirring for 1 min, then the mixture was allowed to stand for 20 min; the process was repeated 6 times. The reaction mixture was monitored by HPLC after each irradiation cycle. After completion of the reaction, the product was precipitated with 10 ml of AcOEt, filtered on sintered glass and washed with AcOEt (3×0.5 ml). The crude product was dried, then dissolved in 15% ACN in water and lyophilised. The compound was purified by HPLC on a Phenomenex Gemini column 5 μm NX-C18 250×21.2 mm (manufacturer Phenomenex®); linear gradient A and B: 7-18% B in 14 min, 100% B in 15 min; 14 ml/min; 25° C.; where A was a 0.02% solution of TFA in water and B was 100% ACN. Yield of the compound: 27.8 mg (36%).

(24) Characterization: HRMS (MALDI): for C.sub.154H.sub.293N.sub.62O.sub.45S [M+H].sup.+ calculated 3763.226; found 3763.231; analytical HPLC: column ZORBAX Poroshell 120 SB-C18; 3×50 mm (manufacturer Agilent); 2.7 μm; linear gradient A and B: 0.5% B 0-1 min, then linear gradient up to 22% B during 10 min; 1 ml/min; 25° C.; where A was 0.05% TFA in water, B was 100% ACN. Retention time of compound 6a=8.6 min.

Example 6

Synthesis of Conjugate 6b (FIG. 2)

(25) Compound 6b was prepared analogously to the above described compound 6a using 2,2-dimethoxy-2-phenylacetophenone (0.12 mg), thiol SH—(CH.sub.2).sub.5—CO-(Arg).sub.8-NH—(CH.sub.2).sub.2—NH.sub.2 (2.32 mg; prepared by standard peptide synthesis on a solid phase using an automatic ABI 433A synthesizer, Applied Biosystems) and compound 5 (5.58 mg). Yield 1.60 mg (36%).

(26) Characterization: HRMS (MALDI): for C.sub.108H.sub.210N.sub.55O.sub.37S [M+H].sup.+ calculated 2901.596; found 2901.595; analytical HPLC: column ZORBAX Poroshell 120 SB-C18; 3×50 mm (manufacturer Agilent); 2.7 μm; linear gradient A and B: from 5% B to 20% B over 5 min; 1 ml/min; 25° C.; where A was a 0.02% solution of TFA in water, B was 100% ACN. Retention time of compound 6b=2.6 min.

Example 7

Synthesis of Conjugate 6c (FIG. 3)

(27) Compound 6c was prepared analogously to the above described compound 6a using 2,2-dimethoxy-2-phenylacetophenone (0.84 mg), thiol SH—(CH.sub.2).sub.5—CO-(Arg-Aca).sub.8-NH.sub.2 (29.3 mg; prepared by standard peptide synthesis on a solid phase using an automatic ABI 433A synthesizer, Applied Biosystems) and compound 4 (44.1 mg). Yield 17.4 mg (39%). Characterization: HRMS (MALDI): for C.sub.147H.sub.279N.sub.48O.sub.45S [M+H].sup.+ calculated 3469.073; found 3469.070; analytical HPLC: column ZORBAX Poroshell 120 SB-C18; 3×50 mm (manufacturer Agilent); 2.7 μm; linear gradient A and B: from 5% B to 20% B over 5 min; 1 ml/min; where A was a 0.02% solution of TFA in water, B was 100% ACN. Retention time of compound 6c=3.7 min.

Example 8

Synthesis of Conjugate 6d (FIG. 4)

(28) Compound 6d was prepared analogously to above described compound 6a using 2,2-dimethoxy-2-phenylacetophenone (0.13 mg), thiol SH—(CH.sub.2).sub.5—CO-(Arg).sub.8-NH—(CH.sub.2).sub.2—NH.sub.2 (2.22 mg; prepared by standard peptide synthesis on a solid phase using an automatic ABI 433A synthesizer, Applied Biosystems) and compound 4 (7.62 mg). Yield 1.38 mg (35%).

(29) Characterization: HRMS (MALDI): for C.sub.101H.sub.196N.sub.41O.sub.37S [M+H].sup.+ calculated 2607.443; found 2607.448; analytical HPLC: column ZORBAX Poroshell 120 SB-C18; 3×50 mm (manufacturer Agilent); 2.7 μm; linear gradient A and B: from 5% B to 20% B over 5 min; 1 ml/min; where A was a 0.02% solution of TFA in water, B was 100% ACN. Retention time of compound 6d=2.2 min.

Example 9

Transport of Nucleoside Triphosphate Carrying a Fluorescent Marker Into U2-OS Cells

(30) Preparation of buffer A for application of the complexes: The compounds N-[tris(hydroxymethyl)methyl]glycin (448.14 mg), calcium chloride (100.6 mg), magnesium sulfate (48.8 mg), potassium chloride (200.0 mg), glucose (1.001 g) and sodium chloride (3.661 g) were successively dissolved in 497 ml of deionized sterile water. The acidity of the resulting solution was adjusted with 1 mol.Math.l.sup.−1 solution of sodium hydroxide to pH 7.40 at 31° C. The solution was made up to 500.0 ml.

(31) Compound 6a was dissolved in a buffer solution A to a concentration of 20 μmol.Math.l.sup.−1. Nucleoside triphosphate (ChromaTide® Alexa Fluor® 488-5-dUTP; ThermoFisher Scientific, Cat. No.: C11397) was also dissolved in a solution of buffer A to a concentration of 20 μmol.Math.l.sup.−1. The solutions were then mixed in equal volume ratios (1:1) to a final concentration of the complex 10 μmol.Math.l.sup.−1. U2-OS cell culture prepared in a standard manner with 50-100% confluency was shortly washed with buffer A and then the cells were overlaid with the solution of the prepared complex. The sample thus prepared was immediately placed in a confocal microscope and NTP penetration into the cell was monitored at regular time intervals. Colouring of cytosol and nucleus could be observed from the first minute after beginning of observation. Fluorescence was observed in all the cells with varying intensity (FIG. 5a). In a control experiment, where 10 μM solution of NTP alone (ChromaTide® Alexa Fluor® 488-5-dUTP), without compound 6a was applied (FIG. 5b) no penetration of NTP into cells was observed, the presence of NTP was only observed in the intercellular space.

Example 10

Monitoring of Incorporation of Labeled NTP (Aminoallyl-dUTP-Cy3, Jena Bioscience) Into the DNA

(32) Complex of compound 6b and NTP (Aminoallyl-dUTP-Cy3, Jena Bioscience, Cat. No. NU-803-Cy3-S) in buffer A at a final concentration of 25 μmol.Math.l.sup.−1 was applied to TZM-bl cell culture and incubated for 3 minutes. Then, the solution of the complex was removed from the cells and the cells were overlaid with complete medium, further incubated at 37° C. for 24 hours and periodically monitored by confocal microscopy. Fluorescence of chromosomal DNA of dividing cells (FIG. 6) shows that fluorescently labelled NTP has been incorporated into the genomic DNA of the cells.

Example 11

Testing Virostatic Activity of Adefovir Diphosphate 8

(33) The subject of testing was the compound of structure formula 8 (adefovir diphosphate) which is a known (Mulato & Cherrington, 1997) active metabolite—the reverse transcriptase inhibitor—in inhibiting the replication of viral DNA of HIV. In determining activity of triphosphate 8, compound 7 (tenofovir), which is a clinically approved drug used for the treatment of HIV, was used as a standard. TZM-bl cell culture infected with HIV was exposed to the effect of a solution of 10 μmol.Math.l.sup.−1 complex of the compound 8 and the transporter—compound 6c—in buffer A for 10 minutes. Then the solution of the complex was removed and the infected cells were overlaid with medium with serum and incubated at 37° C. Parallel control experiments were performed (without infection; infection without active substance; 10 μmol.Math.l.sup.−1 tenofovir 7 throughout incubation; compound 8 without a carrier). After three days of incubation, virostatic activity of compound 8, transported by 6c for 10 min, was observed—identical with the control experiments where a solution of compound 7 was present in the medium throughout the incubation (FIG. 7). From this observation it may be concluded that compound 8, when applied as the active metabolite (triphosphate) using the 6c carrier is—due to the much shorter time of application—more active than the clinically used drug 7, used here as a standard.

Example 12

Preparation of 2.SUP.I.-O-allyl-heptakis(6-trifluoroacetamido-6-deoxy)-β-cyclodextrin 10

(34) Triphenylphosphine (141 mg, 0.53 mmol) was added to a solution of isomers 3 and 9 (52 mg, 0.038 mmol) in dimethylformamide (1.04 ml). After 2 hours a solution of ammonia in water (0.4 ml of 25% solution) was added to the reaction mixture and the mixture was stirred for 16 h. The solvent was then evaporated and acetone (15 ml) was added to the resulting thick syrup. The resulting white precipitate was isolated by filtration on sintered glass and then suspended in a mixture of methanol (1.1 ml) and ammonia (1.1 ml). The mixture was heated in a pressure tube at 60° C. for 6 hrs, and then evaporated under reduced pressure. The residue was dissolved in a 0.01% aqueous solution of trifluoroacetic acid and purified by ultrafiltration on a membrane with 1 kDa pores (Ultracell; manufacturer Merck Millipore), and lyophilized. The yield of the mixture of isomeric compounds 3 and 9 was 41 mg (55%).

(35) Crude mixture of isomeric compounds 3 and 9 (41 mg; 0.021 mmol) was dissolved in dry methanol (1 mL) and ethyl trifluoroacetate (0.12 mL) was added. DIPEA (0.75 mL) was then added to stirring reaction mixture in small portions within 20 minutes so as to prevent formation of insoluble suspension. The reaction was allowed to stir overnight and then it was evaporated to dryness on a rotatory evaporator. Then the crude product was purified by reversed phase HPLC on a Phenomenex Gemini column 5 μm NX-C18 250×21.2 mm (manufacturer Phenomenex®); isocratic elution ACN-water 48:52 (column loading: 20 mg of material per run), flow 14 mL/min. Elution time: minor isomer (O-3) 11: 11.1 min; major isomer (O-2) 10: 13.0 min. Yield of 10: 16 mg (41%).

(36) Characterization: HRMS (MALDI): m/z calculated for C.sub.59H.sub.74F.sub.21N.sub.7O.sub.35 [M+Na].sup.+: 1862.3788; found 1862.3791; elemental analysis (%), calculated for C.sub.59H.sub.74F.sub.21N.sub.7O.sub.35: C, 38.51; H, 4.05; N, 5.33; found: C, 38.22; H, 4.15; N, 5.07. .sup.1H and .sup.13C NMR data—see FIGS. 10 and 11.

Example 13

Synthesis of conjugate 6c (FIG. 3) by alternative method using 2.SUP.I.-O-allyl-heptakis(6-trifluoroacetamido-6-deoxy)-β-cyclodextrin 10

(37) Compound 10 (7.5 mg; 4.08 μmol) and thiol SH—(CH.sub.2).sub.5—CO-(Arg-Aca).sub.8-NH.sub.2 (10 mg; 3.11 μmol) were dissolved in methanol (40 μL) containing dissolved 2,2-dimethoxy-2-phenylacetophenon (0.080 mg) under argon. The solution was irradiated with UV light of wavelength 365 nm generated by the LED (1 W) under constant stirring for 15 min. Then the reaction mixture was applied to reversed phase HPLC column (Phenomenex Gemini column 5 μm NX-C18 250×21.2 mm, manufacturer Phenomenex®) and eluted with a linear gradient A to B: from 22% B to 30% B over 15 min; 14 ml/min; where A was a 0.02% solution of TFA in water, B was 100% ACN. Retention time of intermediate product (compound 12, X=CF.sub.3CONH—; Y=(-Arg-Aca).sub.8-NH—(CH.sub.2).sub.2—NH.sub.2)=11.8 min, yield 8.4 mg.

(38) This intermediate product was subsequently treated with solution of aqueous ammonia diluted with water (1:3, v/v) for 4 hours and lyophilized. The lyophilizate was purified by dialysis against water (Float-a-Lyzer G2, 0.5-1 kDa MW cutoff) and lyophilized again to obtain pure conjugate 6c (6.8 mg). Analytical data are identical with that of compound 6c prepared from compound 4.

Example 14

Synthesis of 2.SUP.I.-O-allyl-octakis(6-azido-6-deoxy)-γ-cyclodextrin 14

(39) Octakis(6-azido-6-deoxy)-γ-cyclodextrin (165 mg, 0.11 mmol) was dissolved in dry DMF (3.4 ml) and potassium tert-butoxide solution in THF was added (1.0 mol l.sup.−1, 0.12 ml, 0.120 mmol). The mixture was heated briefly to 40° C. The suspension was cooled to −15° C. and allyl iodide was added (0.01 ml, 0.109 mmol). The reaction mixture was stirred at −15° C. for 50 hours; during the reaction the precipitate dissolved completely. Resulted solution was poured into TBME (100 ml). The precipitate was filtered on a sintered glass, washed with TBME (3×10 ml) and dried under vacuum at room temperature. Crude product (157 mg) was dissolved in THF (2 ml) and resulted solution was coated on silica gel (1.3 g) and purified by flash chromatography (30 g silica gel, AcOEt:TBME:acetone:EtOH:water:THF; 36:40:7.2:9.6:7.2:1.6) Compound 14 was isolated in a yield of 26 mg (15%).

(40) Characterization: HRMS (MALDI): m/z calculated for C.sub.51H.sub.76N.sub.24O.sub.32 [M+Na].sup.+: 1559.4955; found 1559.4961; elemental analysis (%: calculated for C.sub.51H.sub.76N.sub.24O.sub.32: C, 39.85; H, 4.98; N, 21.87; O, 33.30; found C: 41.37; H, 5.41; N, 19.12. .sup.1H and .sup.13C NMR data—see FIGS. 10 and 11.

Example 15

Synthesis of 2.SUP.I.-O-allyl-octakis(6-amino-6-deoxy)-γ-cyclodextrin octakis(trifluoroacetate) 16

(41) Compound 16 was prepared by the method described above for the preparation of compound 4.

(42) Characterization: HRMS (MALDI): m/z calculated for C.sub.51H.sub.92N.sub.8O.sub.32 [M+H].sup.+: 1329.5890; found 1329.5873; elemental analysis (%: calculated for C.sub.51H.sub.92N.sub.8O.sub.32:C, 46.08; H, 6.98; N, 8.43; O, 38.; found C: 45.42; H, 7.93; N, 7.85.

Example 16

Synthesis of Conjugate 13a (FIG. 8)

(43) Compound 13a was prepared from compound 16 by the method described above for the preparation of compound 6c. The trifluoroacetate counterions were exchanged for Cl.sup.− by passing the aqueous solution of the material through column of Dowex-1 in Cl.sup.− cycle (0.5 mL of Dowex per 3 mg of material).

(44) Characterization: HRMS (MALDI): for C.sub.153H.sub.289N.sub.49O.sub.49S [M+H].sup.+ calculated 3629.1349; found 3629.1362; analytical HPLC: column ZORBAX Poroshell 120 SB-C18; 3×50 mm (manufacturer Agilent); 2.7 μm; linear gradient A and B: from 5% B to 20% B over 5 min; 1 ml/min; where A was a 0.02% solution of TFA in water, B was 100% ACN. Retention time of compound 13a is 3.4 min.

Example 17

Detection of S-phase Cell Cycle Progression With DIRECT Labelling of DNA Using Compound 6c/aminoallyl-dUTP-Cy3 Complex (FIG. 9)

(45) U2-OS cells were washed twice with the treating buffer and then treating solution of the complex (10 μM compound 6c, 10 μM aminoallyl-dUTP-Cy3 in the buffer) was added. After 3 min the solution was removed, cells were washed once with the buffer and then they were incubated in complete medium (37° C., 5% CO.sub.2) for 15 min. Cells were trypsinized, washed with PBS, fixed with ethanol, washed with PBS and then treated with DAPI solution (10 μg/mL in 0.1% Triton X 100 in PBS) for 30 min at room temperature. Cells were analyzed by flow cytometry without washing (FIG. 9). Proportions of cells in phases of cell cycle were as follows: G0/G1: 30%, S: 55%, G2/M: 9%, which is in accord to proportions obtained by standard BrdU assay (G0/G1: 28%, S: 55%, G2/M: 11%).

INDUSTRIAL APPLICABILITY

(46) Compounds of the general formulae 6 and 13 can be used in pharmaceutical research when testing the activity of novel virostatic agents based on nucleoside triphosphates. Further, they can be used in molecular and cell biology for the incorporation of labelled NTPs into DNA or RNA.

REFERENCES

(47) 1. Jordheim, L. P., Durantel, D., Zoulim, F. & Dumontet, C. (2013). Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nature Reviews Drug Discovery 12, 447-464. 2. Hillaireau, H. & Couvreur, P. (2009). Nanoencapsulation of antiviral nucleotide analogs. Journal of Drug Delivery Science and Technology 19, 385-390. 3. Vinogradov, S. V., Kohli, E. & Zeman, A. D. (2005a). Cross-linked polymeric nanogel formulations of 5′-triphosphates of nucleoside analogues: Role of the cellular membrane in drug release. Molecular Pharmaceutics 2, 449-461. 4. Vinogradov, S. V., Zeman, A. D., Batrakova, E. V. & Kabanov, A. V. (2005b). Polyplex Nanogel formulations for drug delivery of cytotoxic nucleoside analogs. Journal of Controlled Release 107, 143-157. 5. Gollnest, T., de Oliveira, T. D., Schols, D., Balzarini, J. & Meier, C. (2015). Lipophilic prodrugs of nucleoside triphosphates as biochemical probes and potential antivirals. Nature Communications 6. 6. Mulato, A. S. & Cherrington, J. M. (1997). Anti-HIV activity of adefovir (PMEA) and PMPA in combination with antiretroviral compounds: in vitro analyses. Antiviral Research 36, 91-97.