A Nucleoside Derivative For Use As A Drug, Particularly For The Treatment Of Chronic Lymphocytic Leukemia

Abstract

We disclose nucleoside derivatives useful as drugs, particularly for the treatment of chronic lymphocytic leukemia.

Claims

1. A nucleoside derivative containing a boron cluster connected with the nucleoside structure at position 2 or 8 of purine nucleobase through a connecting group and its 5′ phosphate, thiophosphate or phosphonate derivative for use as a drug.

2. A nucleoside derivative for use according to claim 1 characterised in that the connecting group is defined by the formula: (CH.sub.2).sub.mW.sub.k(CC).sub.nT.sub.1(CH.sub.2).sub.jQ.sub.i(CC).sub.p(CH.sub.2).sub.r— where: m, j and r are whole numbers from 0 to 3; k, n, l, i and p each have a value of 0 or 1; W, T and Q independently of one another denote O, S, C(O), S(O), S(O).sub.2, Se, NR, where R denotes: H, alkyl, haloalkyl, alkoxyalkyl or aryl, the boron cluster is 1,2-dicarba-closo-dodecaborane (ortho-carboranyl), 1,7-dicarba-closo-dodecaborane (meta-carboranyl), 1,12-dicarba-closo-dodecaborane (para-carboranyl), 7, 8-dicarba-nido-undecaborane (nido-carboranyl), closo-dodecaborane or their derivatives substituted on a carbon or boron atom, that the nucleoside part of the agent is selected from a group containing a combination of guanine, adenine or cytosine and ribose, deoxyribose or arabinose residue.

3. A nucleoside derivative for use according to claim 1, characterised in that it is the compound defined by the formula 1: ##STR00001## where: R1 and R2 denote: H or a group with the formula: (CH.sub.2).sub.mW.sub.k(CC).sub.nT.sub.1(CH.sub.2).sub.jQ.sub.i(CC).sub.p(CH.sub.2).sub.r— boron cluster where: m, j and r are whole numbers from 0 to 3; k, n, l, i and p each have a value of 0 or 1; W, T and Q independently of one another denote O, S, C(O), S(O), S(O).sub.2, Se, NR, where R═H, alkyl, haloalkyl, alkoxyalkyl or aryl, and the boron cluster constitutes a compound selected from a group encompassing: 1,2-dicarba-closo-dodecaborane (orto-carboranyl), 1,7-dicarba-closo-dodecaborane (meta-carboranyl), 1, 12-dicarba-closo-dodecaborane (para-carboranyl), 7,8-dicarba-nido-undecaborane (nido-carboranyl), closo-dodecaborane or their derivatives substituted at the carbon or boron atom. R3 denotes XP(Z)(Y)X.sup.1 where X and X.sup.1 denote: O, S, Se, alkyl, haloalkyl, alkoxyalkyl, aryl, CH═CH, CC, N═N, CHOH or CHN.sub.3; Z denotes: O, S, Se; Y denotes: OH, SH, SeH, H, alkyl, haloalkyl, alkoxyalkyl, aryl, a fluoride atom, particularly fluorine, CH═CH.sub.2, CCH, N═NH, CHOH or CHN.sub.3, R4 and R5 denote: H, OH, F, N.sub.3, SH, Cl, Br, J, NHR or .sup.15NHR where R denotes: H, alkyl, haloalkyl, alkoxyalkyl or aryl.

4. A nucleoside derivative for use according to claim 3, characterised in that R3 denotes: OH or O (PO.sub.3)m where m is a whole number from 0 to 3, and R4 and R5 respectively denote: H and OH or OH and H.

5. A nucleoside derivative for use according to claim 1, characterised in that it is a compound selected from the group encompassing: 2-ethynyl-(1,12-dicarba-c/oso-dodecaboran-2-yDadenosine, 8-ethynyl-(1,12-dicarba-doso-dodecaboran-2-yl)-2′ -deoxyadenosine, 2-ethynyl-(1,12-dicarba-doso-dodecaboran-2-yparabinoadenosine, 2-ethyl -(1,12-dicarba-c/oso-dodecaboran-2-yparabinoadenosine or phosphates thereof.

6. A method for treatment or prophylaxis of neoplasms a comprising administering the nucleoside derivative of claim 1.

7. A compound selected from a group encompassing: 2-ethynyl-(1,12-dicarba-c/oso-dodecaboran-2-yparabinoadenosine, 2-ethyl-(1,12-dicarba-doso-dodecaboran-2-yparabinoadeno sine or phosphates thereof.

8. The method of claim 6, where said neoplasm is chronic lymphocytic leukemia.

Description

EXAMPLE 1

Cytotoxicity and Apoptosis Assay

[0010] PBMC cells were isolated from peripheral blood using centrifugation via a Histopaque gradient according to the manufacturer's instructions. Next, PBMC samples were cultured in RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum, L-glutamine, and antibiotics (streptomycin 100 μg/mL, penicillin 100 U/mL) at 37° C., 5% CO.sub.2, fully humidified atmosphere. For cytotoxicity evaluation the leukemic cells were incubated in culture medium without tested compounds (control) or were exposed to DMSO (vehicle control) at concentrations of 0.08%-0.4%, as well as to cladribine, fludarabine, and adenosine modified with boron cluster at C-8 (compound 5) and C-2 (compound 3) positions of purine ring; all at concentrations of 0.1-60 μM. The cytotoxicity was assessed cytometrically after 24 and 48 h incubation.

[0011] Firstly, it was analyzed by propidium iodide staining. Then, Membrane Permeability/Dead Cell Apoptosis Kit (Molecular Probes, Invitrogen™) was used to examine the contribution of apoptosis to cell death induction after exposure to the used agents. The analyses were performed on BD LSR II flow cytometry using FACSDiva Version 6.1.2 software. Ten thousand events were examined for each analysis. The distinction between living (YO-PRO-negative/propidium iodide-negative), early apoptotic (YO-PRO-positive/propidium iodide-negative) and dead (YO-PRO-positive/propiodium iodide-positive) cells was based on the differences in their permeability for fluorescent dyes. The results are shown as the percent of viable and early apoptotic cells in gated PBM cell population, respectively.

[0012] The cytotoxicity of normal PBMCs (isolated from volunteers without leukemia) was evaluated using the same test after 24 and 48 h exposure to the examined agents at the concentrations of 20 μM, 40 μM, and 60 μM.

[0013] For further analyses the concentration of 15 μM of all tested agents was chosen. In addition, 0.15 μM concentration of cladribine was used, which corresponds to cladribine concentration in serum of leukemic patients during the anticancer therapy.

[0014] Cladribine (Biodrybina) and fludarabine were purchased from the Institute of Biotechnology and Antibiotics Bioton (Warsaw, Poland) and Schering AG (Berlin, Germany), respectively. Adenosine derivatives modified with boron clusters were synthesized by Prof. Zbigniew J. Leśnikowski (Institute for Medical Biology of the Polish Academy of Sciences, Lodz, Poland) (see results in FIG. 1) and Institute of Biochemistry and Biophysics of the Polish Academy of Sciences (Dr. Adam Mieczkowski, compounds 7 and 8). FIG. 1 shows the viability and apoptosis induction in normal (a), CLL (b), and PLL (c) PBMCs after their exposure to conventional antileukemic agents: cladribine and fludarabine, as well as the novel compounds at chosen concentrations (15 μM, in the case of cladribine, also 0.15 μM) assessed by Membrane Permeability/Dead Cell Apoptosis Kit

EXAMPLE 2

Caspase-3 Activation Assay

[0015] The percent of leukemic PBMCs with active form of caspase-3 was measured cytometrically by PE Active Caspase-3 Apoptosis Kit, BD Pharmingen according to manufacturer's protocol. The analyses were conducted after 48 h incubation with/without (control) the agents or DMSO (vehicle control). BD LSR II flow cytometer was used and the analyses were performed using FACSDiva Version 6.1.2 software (see results in FIG. 2). FIG. 2 shows Percent of apoptotic cells in PLL cells after their exposure to conventional antileukemic agents: cladribine and fludarabine, as well as the novel compounds at chosen (15 μM) concentration assessed by PE Active Caspase-3 Apoptosis Kit.

EXAMPLE 3

Western Blot Analysis

[0016] Control PBMCs (without the agents), as well as the cells samples exposed to DMSO (vehicle control) and the tested agents were lysed (4° C., 1 h) in a buffer containing 10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1% Triton X-100, 2 mM MgCl.sub.2, 0.1 M DTT, and protease inhibitors as described previously (A., Kobylinska, J., Bednarek, J. Z., Blonski, M., Hanausek, Z., Walaszek, H., Piekarski, T., Robak, Z. M., Kilianska. 2006. In vitro sensitivity of B-cell chronic lymphocytic leukemia to cladribine and its combinations with mafosfamide and/or mitoxantrone, Oncol. Rep., 16, 1389-1395). Protein content was estimated according to Lowry method (O. H., Lowry, N. J., Rosebrough, A. L., Farr, R. J., Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275) and the cell lysates were prepared for subsequent Western blotting analysis. Protein samples (60 μg/lane) were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on 8.0 or 12.5% slab gels and electrotransfered onto Immobilon P (H., Towbin, T., Staechlin, J., Gordon, 1979, Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76, 4350-4354). Membrane staining with 0.5% Ponceau S solution was done to confirm equal protein loading and completeness of the transfer. The membranes were saturated in 5.0% skim milk in TBS (10 mM Tris-HC1, pH 7.5, 150 mM NaC1) for 1 h at ambient temperature, and then incubated overnight with antibodies specific to Mcl -1 (1:1000), Bcl-2 (1:1000), Bax (1:1000), PARP-1 (1:1000), all from Santa Cruz Biotechnology Inc. Antigen recognition was performed with appropriate secondary antibodies conjugated with horseradish peroxidase. The antigen-antibodies complexes were detected with Novex HRP Chromogenic substrate (TMB) from Invitrogen or, alternatively, by chemiluminescence method (see results in FIG. 3). FIG. 3 shows the expression of apoptosis-related proteins in CLL (a) and PLL (b) PBMCs after 48-hour exposure to conventional antileukemic agents: cladribine and fludarabine, as well as the novel compounds at chosen (15 μM) concentration assessed by Western blot.

EXAMPLE 4

Synthesis of 2-iodoadenosine, Compound 1

[0017] Synthesis of compound 1 was performed according to literature procedure (Robins, M. J.; Uznański, B. Nucleic Acid Related Compounds. 33. Conversions of Adenosine And Guanosine to 2,6-Dichloro, 2-Amino-6-Chloro, and Derived Purine Nucleosides. Can. J. Chem. 1981, 59, 2601-2606; Matsuda A., Shinozaki M., Yamaguchi T., Homma H., Nomoto R., Miyasaka T., Watanabe Y., Asiru T., Nucleosides and Nucleotides. 103. 2-Alkynyladenosines: A Novel Class of Selective Adenosine A2 Receptor Agonists with Potent Antihypertensive Effects, J. Med. Chem., 1992, 35, 241-252).

EXAMPLE 5

Synthesis of 8-bromo-2′-deoxyadenosine, Compound 4

[0018] Synthesis of compound 4 was performed according to literature procedure (Ikehara, M.; Kaneko, M. Studies of Nucleosides and Nucleotides. XLIV. Purine Cyclonucleosides. Synthesis of Cyclonucleosides having 8,3′-O- and -S-Anhydro Linkage derived from 2′-Deoxyadenosine. Chem. Pharm. Bull. 1970, 18, 2441-2446).

EXAMPLE 6

Synthesis of 2-iodo-arabinoadenosine, Compound 6

[0019] Synthesis of compound 6 was performed according to literature procedure ((Robins, M. J.; Uznański, B. Nucleic Acid Related Compounds. 33. Conversions of Adenosine And Guanosine to 2,6-Dichloro, 2-Amino-6-Chloro, and Derived Purine Nucleosides. Can. J. Chem. 1981, 59, 2601-2606; Matsuda A., Shinozaki M., Yamaguchi T., Homma H., Nomoto R., Miyasaka T., Watanabe Y., Asiru T., Nucleosides and Nucleotides. 103. 2-Alkynyladenosines: A Novel Class of Selective Adenosine A2 Receptor Agonists with Potent Antihypertensive Effects, J. Med. Chem., 1992, 35, 241-252).

EXAMPLE 7

Synthesis of 2-ethynyl-1,12-dicarba-closo-dodecaborane, Compound 2

[0020] Synthesis of compound 2 was performed according to the literature procedure (Jiang, W.; Knobler, C. B.; Curtis, C. E.; Mortimer, M. D.; Hawthorne, M. F. Iodination Recation of Icosahedral para-carborane and the Synthesis of Carborane Derivatives with Boron-Carbon Bonds. Inorg. Chem. 1995, 34, 3491-3498).

EXAMPLE 8

Synthesis of 2-ethynyl-(1,12-dicarba-closo-dodecaboran-2-yl)adenosine, Compound 3

[0021] Synthesis of compound 3 was performed according to the literature procedure (K. Bednarska, A. B. Olejniczak, A. Piskala, M. Klink, Z. Sulowska, Z. J. Leśnikowski, Effect of adenosine modified with a boron cluster pharmacophore on reactive oxygen species production by human neutrophils, Bioorg. Med. Chem., 2012, 20, 6621-6629).

[0022] This synthesis was performed under anhydrous conditions in an argon atmosphere. 2-lodoadenosine (1, 50 mg, 0.13 mmol), 2-ethynyl-1,12-dicarba-closo-dodecaborane (2, 23.56 mg, 0.14 mmol), CuI (3.06 mg, 0.016 mmol) and Pd(PPh.sub.3).sub.4 (6.36 mg, 0.0055 mmol) were placed in a flame-dried flask and dissolved in anhydrous DMF (0.9 mL) and Et.sub.3N (0.3 mL). The resultant solution was stirred for 75 min at room temperature and then for 70 min at 80° C. After the reaction was complete, the volatiles were evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (12 mL). The resultant solution was extracted with deionised water (2×6 mL). The organic fractions were collected, washed with 0.5% aqueous EDTA (1×2 mL), dried over anhydrous magnesium sulphate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (2 g, 230-400 mesh) using a linear gradient of CH.sub.3OH in CH.sub.2Cl.sub.2 (0-10%) as the eluent. Yield: 37.68 mg (68%). TLC (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1 v/v): R.sub.f=0.11; UV-Vis (95% C.sub.2H.sub.5OH, nm): λ.sub.min=280, 251, λ.sub.max=300, 270, 239; .sup.1H NMR ((CD.sub.3).sub.2CO.sub.3 ppm): δ=8.31, (s, 1H, H-8), 6.81 (bs, 2H, NH.sub.2), 5.97 (d, 1H, .sup.1J.sub.1′:2′=7.50, H-1′), 5.10-5.05 (m, 2H-5′, 1H—OH), 4.82-4.73 (m, 2H, 1H-2′, 1H—OH), 4.39-4.37 (m, 2H, 1H-3′, 1H—OH), 4.16-4.14 (m, 1H, H-4′), 4.16-4.14 (m, 1H, H-4′), 3.84-3.71 (m, 3H, 2H-5′, 5″, C(.sub.1)H.sub.carborane) , 3.51 (bs, 1H, c(12)H.sup.carborane), 3.50-1.00 (m, 9H, BH.sup.carborane); .sup.13C NMR ((CD.sub.3).sub.2CO, ppm): δ=157.02 (C-6), 150.00 (C-2), 146.21 (C-4), 142.15 (C-8), 121.00 (C-5), 90.38 (C-1′), 87.81 (C-4′), 75.17 (C-2′), 72.38 (C-3′), 67.53 (CH.sup.carborane); 64.90 (CH.sup.carborane), 63.20 (C-5′); .sup.11B NMR ((CD.sub.3).sub.2CO, ppm): coupled δ=−12.18 (s, 2B), −13.90 (s, 4B), −15.90 (s, 4B); MS (FAB): m/z =435.40 (M+2).sup.+, 497.4 (M+Na+K+2).sup.+, C.sub.14H.sub.23B.sub.10N.sub.5O.sub.4 [433.28].

EXAMPLE 9

Synthesis of 8-ethynyl-(1,12-dicarba-closo-dodecaboran-2-yl-2′-deoxyadenosine, Compound 5

[0023] Synthesis of compound 5 was performed according to the literature procedure (Bednarska K., Olejniczak A. B., Wojtczak B. A., Sulowska Z., Leśnikowski Z. J., Adenosine and 2′-deoxyadenosine modified with boron cluster pharmacophores as new classes of human blood platelet function modulators, ChemMedChem., 2010, 5, 749-756).

[0024] Nucleoside substrate, 8-bromo-2′-deoxyadenosine (4, 46.2 mg, 0.4 mmol) lyophilized from water (0.5 mL), 2-ethynyl-para-carborane (2, 25.2 mg, 0.15 mmol), cuprous iodide (3.2 mg, 0.017 mmol) and tetrakis(triphenylphosphine)palladium(0) (6.8 mg, 0.006 mmol) were placed in 25 mL burner-dried round bottom flask, then anhydrous dimethyloformamide (0.9 cm.sup.3) and triethylamine (0.3 mL) were added. The reaction was carried out under argon atmosphere, with stirring, at 80° C. After 30 min the reaction was quenched by evaporation of the solvent. Dichloromethane (10 cm.sup.3) was added to the residue then resultant solution was extracted with distilled water (2×5 mL). Organic layer was collected and extracted with 0.5% aqueous EDTA (2×5 mL) then dried over MgSO.sub.4. Drying agent was filtered off and washed with dichloromethane. The organic fractions and washing were combined then solvent was evaporated under reduced pressure. Crude product 5 was purified by column chromatography on silica gel (4 g, 230-400 mesh); as an eluting solvent a linear gradient of CH.sub.3OH in CH.sub.2Cl.sub.2 was used (0-14%). Yield: 41.5 mg (71%) as an opalescent, colorless oil: R.sub.f=0.46 (CH.sub.2Cl.sub.2/CH.sub.3OH, 1:9); RP-HPLC: R.sub.t=20.08 min; .sup.1H-NMR (250.131 MHz, CD.sub.3OD, Me.sub.4Si): δ=0-4 (11H, BH-carborane), 2.2-2.4 (m, 1H, 2′-H), 3.03-3.09 (m, 1H, 2″-H), 3.62-3.69 (m, 2H, 5′-H, 5″-H), 3.85-3.88 (m, 1H, 4′-H), 4.43-4.45 (m, 1H, 3′-H), 6.39 (t, 1H, 1′-H, J.sup.3.sub.1′2′=7.78, J.sup.3.sub.1′2′=6.62), 8.15 (s, 1H, 2-H); .sup.13C-NMR (62.90 MHz, CD.sub.3OD, Me.sub.4Si): δ=40.31 (C-2′), 64.16 (C-5′), 65.45, 67.90 (CH-carborane), 73.60 (C-3′), 88.12 (C-1′), 90.34 (C-4′), 120.77 (C-5), 134.90 (C-8), 149.45 (C-4), 154.31 (C-2), 157.45 (C-6); .sup.11B-NMR (80.20 MHz, CD.sub.3OD, BF.sub.3/Et.sub.2O): δ=−12.99 (9H, BH); UV/Vis (EtOH): λ.sub.min=250 nm, λ.sub.max=236, 301 nm; MS (CI, isobutane) m/z [M+1].sup.+ calcd for C.sub.14H.sub.24B.sub.10N.sub.5O.sub.3: 418.482, found: 419. 3.

EXAMPLE 10

Synthesis of 2-ethynyl-(1,12-dicarba-closo-dodecaboran-2-yl)arabinoadenosine, Compound 7

[0025] This synthesis was performed under anhydrous conditions in an argon atmosphere. 2-iodo-9-(β-D-arabinofuranosyl)adenine (6, 144.5 mg, 0.37 mmol), 2-ethynyl-1,12-dicarba-closo-dodecaborane (2, 75.3 mg, 0.45 mmol), CuI (8.8 mg, 0.046 mmol), Pd(PPh.sub.3).sub.4 (18.4 mg, 0.016 mmol) were placed in the oven dried flask (130° C.) and dissolved in anhydrous DMF (2.6 mL) and Et.sub.3N (0.9 mL). The resultant solution was stirred for 75 min at room temperature and then for 70 min at 80° C. After the reaction was complete, the solvents were evaporated under reduced pressure in 40° C. water bath and residue was dissolved in ethyl acetate 50 mL. The resultant solution was extracted with deionised water (2×20 mL). The organic fractions were collected, washed with 0.5% aqueous EDTA (1×10 mL), dried over anhydrous magnesium sulphate and concentrated under the reduced pressure. The crude product was purified by silica gel column chromatography (8 g, 230-400 mesh) using 10% methanol in chloroform as an eluent. Yield: 125.5 mg (79%). TLC (CH.sub.2Cl.sub.2/CH.sub.3OH, 4:1): R.sub.f=0.49, (CH.sub.2Cl.sub.2/CH.sub.3OH, 9:1): R.sub.f=0.18; ATR-FTIR (cm.sup.−1): v=3500-3000 (OH, NH), 2953 (CH.sub.2), 2919 (CH.sub.2), 2850 (CH.sub.2), 2614 (BH), 1655 (NH.sub.2), 1590, 1504, 1454 (C═C.sup.arom), 1042 (C—O); .sup.1H NMR (CD.sub.3OD, ppm): δ=1.700-2.880 (m, 10H, B—H), 3.399, 3.646 (2s, 2H, C—H.sup.p-carborane) 3.863-3.933 (qd, 2H, 5′-H), 3.998-4.019 (q, 1H, 4′-H), 4.298-4.336 (2t, 2H, 2′-H, 3′-H), 6.412-6.420 (d, 1H, l′-H), 8.437 (s, 1H, 8-H); .sup.11B{H} NMR (CD.sub.3OD, ppm): δ=−14.89 (s), −14.16 (s), −13.25 (s); .sup.13C{H} NMR (CD.sub.3OD, ppm): δ=63.40 (C-5′), 66.13 (C.sup.p-carborane) 68.91 (C.sup.p-carborane), 77.95, 78.55 (C-2′, C-3′), 86.92, 87.38 (C-1′, C-4′), 120.42 (C-5), 144.24 (C-8), 147.86 (C-4), 151.57 (C-2), 157.94 (C-6); MS (FAB, Gly): m/z=433.3 [M], calc. for C.sub.14H.sub.23B.sub.10N.sub.5O.sub.4=433.7.

EXAMPLE 11

Synthesis of 2-ethyl-(1,12-dicarba-closo-dodecaboran-2-aarabinoadenosine, Compound 8

[0026] 2-Ethynyl-(1,12-dicarba-closo-dodecaboran-1-yl)-9-(β-D-arabinofuranosyl)adenine (7) (52.3 mg, 012 mmol) was dissolved in 10 mL of ethanol and 30 mg of palladium on activated charcoal (10% Pd basis) were added to the reaction mixture. The reaction vessel was connected to the balloon filled with hydrogen and reaction mixture was stirred in room temperature for the 20 hrs. The resultant solution was filtered through Celite, evaporated and purified by silica gel column chromatography (2 g, 230-400 mesh) using 10% methanol in chloroform as an eluent. Yield: 30.4 mg (58%). TLC (CH.sub.2Cl.sub.2/CH.sub.3OH, 4:1): R.sub.f=0.48, (CH.sub.2Cl.sub.2/CH.sub.3OH, 9:1): R.sub.f=0.18; ATR-FTIR (cm.sup.−1): v=3500-3000 (OH, NH.sub.2), 2953 (CH.sub.2), 2923 (CH.sub.2), 2849 (CH.sub.2), 2602 (BH), 1667 (NH.sub.2), 1601, 1581, 1507, 1454 (C=C.sup.arn, 1043 (C—O); .sup.1HNMR (CD.sub.3OD, ppm): δ=1.480-1.509 (t, 2H, CH.sub.2.sup.linker), 1.550-2.850 (m, 10H, B—H), 2.887-2.915 (m, 2H, CH.sub.2.sup.linker), 3.220 (s, 1H, C—H.sup.p-carborane), 3.861-3.929 (qd, 2H, 5′-H), 4.000-4.021 (q, 1H, 4′-H), 4.294-4.341 (2t, 2H, 2′-H, 3′-H), 6.444-6.452 (d, 1H, 1′-H), 8.313 (s, 1H, 8-H); .sup.11B{H} NMR (CD.sub.3OD, ppm): δ=−18.08 (s), −15.31 (s), −14.66 (s), −13.70 (s), −3.91 (s); .sup.13C{H} NMR (CD.sub.3OD, ppm): δ=40.18 (C.sup.linker) 63.62 (C-5′), 65.97 (C.sup.p-carberane), 68.74 (C.sup.p-carbonrane), 78.34, 78.62 (C-2′, C-3′), 86.96, 87.28 (C-1′, C-4′), 118.83 (C-5), 143.19 (C-8), 152.43 (C-4), 157.98 (C-2), 169.04 (C-6); MS (FAB, Gly): m/z=436.4 [M−1].sup.−, calc. for C.sub.14H.sub.27B.sub.10N.sub.5O.sub.4 =437.7.

EXAMPLE 12

Synthesis 5′-monophosphates 9, 10, 11 and 12

[0027] Yoshikawa's procedure for unprotected nucleoside phosphorylation with phosphorus oxychloride was used (Yoshikawa M, Kato T, Takenishi T. A novel method for phosphorylation of nucleosides to 5′-nucleotides. Tetrahedron Lett. 1967, 50, 5065-5068). Suitable nucleoside (0.1 mmol) was dissolved in freshly distilled triethyl phosphate (1 mL). The resulting solution was cooled to 0° C. and further POCl.sub.3 (23 μL, 0.25 mmol) was added. Then the mixture was stirred at 0° C. The reaction progress was monitored by TLC (isopropyl alcohol/water/aq. ammonia, 7:1:2). After completion of the reaction (usually 1.5-2 h), 1M triethylammonium biscarbonate buffer (TEAB, pH 7.5) was added (2 mL) and the mixture was concentrated by evaporation. The crude product 2 was purified by FPLC using a HiPrep 16/10 DEAE FF column (Et.sub.3HN.sup.+ form,)Pharmacia® equilibrated with TEAB. Chromatography was performed with a linear gradient of TEAB from 0.05M to 0.1M TEAB. The fractions containing the product were combined, concentrated under vacuum and then co-evaporated with ethanol (3×3 mL) to remove traces of buffer. 9: TLC (isopropyl alcohol/water/aq. ammonia, 7:1:2): R.sub.f=0.25; UV-vis (95% C.sub.2H.sub.5OH, nm): λ.sub.min=229.4, λ.sub.max=238.5; ATR-FTIR (cm.sup.−1): v=2604 (BH); .sup.31P NMR (D.sub.2O, ppm): δ=0.51; MS (FAB, Gly, -VE): m/z (%)=513.5 (100%) [M].sup.−, calc. for C.sub.14H.sub.24B.sub.10N.sub.5O.sub.7P=513.45; 10: TLC (isopropyl alcohol/water/aq. ammonia, 7:1:2): R.sub.f=0.35; UV-vis (95% C.sub.2H.sub.5OH, nm): λ.sub.min=231.4, 245.5, 270.3, λ.sub.max=238.3, 252.5; ATR-FTIR (cm.sup.−1): v=2614 (BH); .sup.31P NMR (D.sub.2O, ppm): δ=0.31; MS (FAB, Gly, -VE): m/z (%)=497.4 (40%) [M−2].sup.−, calc. for C.sub.14H.sub.25B.sub.10N.sub.5O.sub.6P=498.46; 11: TLC (isopropyl alcohol/water/aq. ammonia, 7:1:2): R.sub.f=0.27; UV-vis (95% C.sub.2H.sub.5OH, nm): λ.sub.min230.0, λ.sub.max=238.3; ATR-FTIR (cm.sup.−1): v=2605 (BH); .sup.31P NMR (D.sub.2O, ppm): δ=0.09; MS (FAB, Gly, -VE): m/z (%)=513.3 (100%) [M].sup.−, calc. for C.sub.14H.sub.24B.sub.10N.sub.5O.sub.7P=513.45; 12: TLC (isopropyl alcohol/water/aq. ammonia, 7:1:2): R.sub.f=0.28; UV-vis (95% C.sub.2H.sub.5OH, nm): λ.sub.min=233.9, λ.sub.max=251.8; ATR-FTIR (cm.sup.−1): v=2603 (BH); .sup.31P NMR (D.sub.2O, ppm): δ=0.1; MS (FAB, Gly, -VE): m/z (100%)=516.5 (100%) [M−1].sup.−, calc. for C.sub.14H.sub.28B.sub.10N.sub.5O.sub.7P=517.48.