Chem. Pharm. Bull. 63, 134–142 (2015)

134

Vol. 63, No. 2

Note

Synthesis of New Nebularine Analogues and Their Inhibitory Activity against Adenosine Deaminase Nikolaos Lougiakis,a Panagiotis Marakos,a Nicole Pouli,*,a Elisabeth Fragopoulou,b and Roxane Tentab a

 School of Health Sciences, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Athens; Panepistimiopolis, Zografou 15771, Greece: and b Department of Nutrition & Dietetics, School of Health Science & Education, Harokopio University; 70 El. Venizelou Ave., Athens 17671, Greece. Received October 21, 2014; accepted November 27, 2014 A number of new 2,6-disubstituted-1-deazanebularine analogues as well as two structurally related pyrazole-fused tricyclic nucleosides were prepared. Their synthesis was carried out by the conversion of 6-amino-2-picoline to a suitable 1-deazapurine, followed by a Vorbrüggen type glycosylation and subsequent elaboration of the condensed pyrazole ring. The synthesized nebularine analogues proved to be weak adenosine deaminase inhibitors. Key words

1-deazanebularine; tricyclic nucleoside; imidazo[4,5-b]pyridine; adenosine deaminase

Nebularine (9-β-D -ribofuranosylpurine, Fig. 1), the simplest member of the purine nucleosides, was first isolated from the fungus Lepista nebularis1) and has been found to exhibit interesting antibiotic,2) antiviral,3) antiamebal,4) antiparasitic5) and cytotoxic properties.6,7) Since it can form a hydrogen bond with each of the four DNA heterocyclic bases, nebularine has the potential to serve as a universal base.8) On the other hand, 2′-deoxynebularine has been used in oligonucleotide synthesis9) and the analysis of DNA structural determinants, which are important for the recognition of damaged DNA by repair enzymes and for the formation of triple-helices at GC sequences.10) Nebularine is also known as a competitive inhibitor of adenosine deaminase11,12) and many purine metabolizing enzymes, including S-adenosylhomocysteine hydrolase,13) herpes simplex DNA polymerase,14) xanthine oxidase15) and adenylyl cyclase.16) As adenosine deaminase (ADA) regulates both intra- and extra-cellular adenosine concentrations, a decrease or increase in its levels triggers a number of pathological conditions and inhibitors of this enzyme demonstrate therapeutic potential for the treatment of several health disorders.17) Highly potent inhibitors have already been identified, including 2′-deoxycorfomycin (pentostatin, Fig. 1), the only clinically useful ADA inhibitor.18,19) However, most of these compounds suffer from poor pharmacokinetics and they bind to the enzyme so tightly, that their activity is nearly irreversible, giving rise to toxic effects. In recent years research efforts have mainly focused on the investigation of ADA–ligand interactions aiming at the design of less toxic inhibitors of the enzyme.20,21) Based on the interesting ADA inhibitory activity of several 2-substituted-adenosine and nebularine derivatives21) and as a part of our ongoing research project directed towards the preparation of novel purine ribosides,22–26) we were prompted to synthesize a number of new nebularine analogues and evaluate their potential ADA inhibitory activity. Thus, we present here the synthesis and biological evaluation of new 1-deazanebularine analogues, having in mind that the isosteric replacement of a heterocyclic nitrogen by a carbon unit is a

well-established procedure for the study of the interactions of purines with their biological targets.27–29)

Results and Discussion

Chemistry The new compounds were prepared from 6-amino-2-picoline (1, Chart 1), which was nitrated using excess nitric acid to provide the dinitropyridine 2.30) The 3-nitro group of compound 2 was then selectively reduced using ammonium sulfide in methanol31) and the resulting diamine 3 was condensed with triethyl orthoformate to give rise to the imidazopyridine 4. This compound was then treated with tetracetylribose in the presence of trimethylsilyl trifluoromethanesulfonate and N,O-bis-(trimethylsilyl)acetamide32,33) to provide both regioisomers 5 and 6 in 4 : 1 ratio, together with a small amount of the corresponding α-anomers 5a and 6a (Fig. 2). The structures of the above mentioned protected nucleosides were unambiguously determined using 1D and 2D-NMR experiments. Thus, for each pair of anomers (5/5a, 6/6a), a clear crosscoupling signal of 1′-H with 4′-H was observed in the nuclear Overhauser effect (NOE) spectra concerning the β-anomers, whereas for the corresponding α-anomers the characteristic correlation peak of 1′-H with the 5′-methylene appeared. The identification of the regio-isomers was effected using the NOE spectral data of 5 and 6. In the case of 5 a coupling of 5-methyl group with the 5′-methylene was observed, on the contrary,

Fig. 1.

Structures of Nebularine and Pentostatin

 To whom correspondence should be addressed.  e-mail: [email protected] *  © 2015 The Pharmaceutical Society of Japan

Chem. Pharm. Bull. Vol. 63, No. 2 (2015)135

Reagents and conditions. a) HNO3, H 2SO4, 60°C; b) (NH4)2S, CH3OH, 40 min r.t, then 1 h reflux; c) triethyl orthoformate, HCl, r.t.; d) (1) N,O-bis(trimethylsilyl)acetamide, ACN, reflux, (2) tetra-O-acetyl-β-D -ribofuranose, TMSOTf, reflux.

Chart 1

Fig. 2.

Structures of the α-Anomers 5a and 6a

Reagents and conditions. a) NH3, CH3OH, r.t.; b) H 2, Pd–C, EtOH; c) Ac2O, CH 2Cl 2, r.t; d) AcOK, Ac2O, isoamyl nitrite, toluene, 95°C.

Chart 2

in the case of 6 the 5′-methylene showed a clear correlation with the 7-H. The nitroderivative 5 was deprotected with the use of methanolic ammonia to give the nucleoside analogue 7 (Chart 2). On the other hand, 5 was reduced to the aminoderivative 8, which was either deacetylated to give the nucleoside 10, or converted to the acetamide 9. This acetamide was heated in toluene in the presence of isoamyl nitrite and acetic anhydride34) and through the rearrangement of the intermedi-

ate N-nitroso compound35) produced the fully protected tricyclic nucleoside 11, together with a non-negligible amount of a byproduct that was identified as 12. This derivative was possibly a result of the thermal deamination of an intermediate diazonium salt prior to cyclisation. Finally, 11 was treated with methanolic ammonia to result in the target compound 13. Using an analogous synthetic procedure, starting from compound 6, the isomeric tricyclic nucleoside 18 was prepared (Chart 3). In this case, the byproduct 17 was also isolated, yet

136

Chem. Pharm. Bull.

Vol. 63, No. 2 (2015)

Reagents and conditions. a) H 2, Pd–C, EtOH; b) Ac2O, CH 2Cl 2, r.t; c) AcOK, Ac2O, isoamyl nitrite, toluene, 95°C; d) NH3, CH3OH, r.t.

Chart 3

Reagents and conditions. a) (1) H 2, Pd–C, EtOH, (2) (CF3CO)2O, THF, r.t; b) m-CPBA, CH 2Cl 2, r.t; c) fuming HNO3, 55°C; d) PCl3, ACN, reflux; e) (1) N,Obis(trimethylsilyl)acetamide, ACN, reflux, (2) tetra-O-acetyl-β- D -ribofuranose, TMSOTf, reflux; f) K 2CO3, CH3OH–H 2O (10 : 1, v/v), reflux; g) K 2CO3, CH3OH–H 2O (1 : 1, v/v), reflux.

Chart 4

in a comparable yield to the desired cyclization product 16. In an effort to insert an additional substituent to position 7 of the bicyclic core, the nitro imidazopyridine 4 was reduced and the resulting aminoderivative was converted without any further purification to the trifluoracetamide 19 (Chart 4). The N-oxide of 19 was then formed upon treatment with m-chloroperbenzoic acid and the resulting derivative 20 was nitrated with fuming nitric acid to provide the 7-nitro-derivative 21. Treatment of compound 21 with phosphorous trichloride resulted in the simultaneous reduction of the N-oxide together with the substitution of the nitro group by a chlorine atom. The chloro-derivative 22 was glycosylated using the same conditions as described for the synthesis of 5. However, in this case, only the 3-regio-isomer 23 was formed, apparently

due to the steric effect of the chlorine atom. Furthermore, the β-anomer was the main product of the reaction and was isolated almost quantitatively. Finally, the protecting groups were successively cleaved to provide the nucleosides 24 and 25. Biological Evaluation A number of the reported new nucleosides were selected in order to investigate their potential inhibition of calf spleen ADA. The experiments were performed at a concentration of 400 µM, but even at this rather high concentration the compounds proved to be weak inhibitors of ADA, as shown in Table 1 and therefore K i determinations were not performed. It seems that the presence of an electron withdrawing group at position 6- of the imidazopyridines is in favour of ADA inhibitory activity. Thus, compounds 5, 7, 23 and 24 were among the most active deriv-

Chem. Pharm. Bull. Vol. 63, No. 2 (2015)137 Table 1.

Compounds Inhibitory Activities against Calf Spleen ADA

Compound 5 7 8 10 13 18 23 24 25

ADA Inibition (%) at 400 μM 31 25 19 6 25 7 20 23 15

atives within this class of nucleosides, providing a 31%, 25%, 20% and 23% inhibition of the enzyme’s activity respectively. On the other hand, the comparison of the tricyclic nucleosides 13 and 18 reveals that only 13 exhibits a 25% ADA inhibition, suggesting that the arrangement of heteroatoms among these two regio-isomers is important for the activity. In conclusion, a number of new nucleosides have been prepared, showing a structural analogy to the bioactive compound nebularine. The synthetic procedure involved the use of a suitable picoline, which upon conversion to 6-methyl-5-nitropyridine-2,3-diamine and treatment with triethyl orthoformate provided the corresponding imidazo[4,5-b]pyridine. The later was glycosylated under Vorbrüggen type conditions and the resulting glycosides were ring-closed to give the isomeric imidazo[4,5-b]pyrazolo[3,4-e]pyridine nucleosides. In parallel, the structurally related 6-chloro-1-deazapurine was prepared, which was converted to the corresponding nucleoside. Selected derivatives were evaluated as ADA inhibitors and proved to possess weak inhibitory activity.

Experimental

Melting points were determined on a Büchi apparatus and are uncorrected. 1H-NMR spectra and 2-D spectra were recorded on a Bruker Avanche 400 instrument, whereas 13 C-NMR specra were recorded on a Bruker AC 200 spectrometer in deuterated solvents and were referenced to tetramethylsilane (TMS) (δ scale). The signals of 1H and 13C spectra were unambiguously assigned by using 2D-NMR techniques: 1 H–1H correlation spectroscopy (COSY), NOESY, 1H-detected heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond connectivity (HMBC). Mass spectra were recorded with a LTQ Orbitrap Discovery instrument, possessing an Ionmax ionization source. Flash chromatography was performed on Merck silica gel 60 (0.040–0.063 mm). Analytical thin layer chromatography (TLC) was carried out on precoated (0.25 mm) Merck silica gel F-254 plates. Elemental analyses were within ±0.4% of the theoretical values. 6-Methyl-5-nitropyridine-2,3-diamine (3) A solution of (NH4)2S (20% in water, 47.7 mL) was added dropwise to a suspension of 230) (5.5 g, 27.78 mmol) in methanol (230 mL) and the mixture was stirred at room temperature for 40 min. The resulting red colored mixture was refluxed for 1 h and upon cooling the solvent was removed under reduced pressure. Water (200 mL) was added and the solid was filtered and washed with water and a small amount (20 mL) of ethanol. The crude solid was then dry-packed and purified by column chromatography, using a mixture of cyclohexane–ethyl acetate (4 : 6, v/v) as the eluent, providing the diamine 3 (4.3 g,

92%). mp: 231–232°C (dec.) (EtOAc). 1H-NMR (DMSO-d6) δ: 2.52 (3H, s, CH3), 5.10 (2H, br s, D2O exchang., 3-NH2), 6.89 (2H, brs, D2O exchang., 2-NH2), 7.39 (1H, s, H-4). 13C-NMR (50 MHz, DMSO-d6) δ: 24.19 (CH3), 112.43 (C-4), 128.07 (C-3), 134.71 (C-5), 143.62 (C-6), 151.64 (C-2). Anal. Calcd for C6H8N4O2: C, 42.86; H, 4.80; N, 33.32. Found: C, 42.99; H, 4.66; N, 33.15. 5-Methyl-6-nitro-3H-imidazo[4,5-b]pyridine (4) Hydrochloric acid (36%, 0.5 mL) was added dropwise to a suspension of compound 3 (4 g, 23.81 mmol) in triethyl orthoformate (25 mL) and the reaction mixture was stirred at room temperature for 48 h. Upon completion of the reaction, the solvents were evaporated, the residue was dissolved in a mixture of dichloromethane and methanol, dry-packed and purified by column chromatography, using a mixture of cyclohexane–ethyl acetate (2 : 8, v/v) as the eluent, providing compound 4 (3.6 g, 85%) as a white solid. mp: 230–231°C (EtOAc). 1H-NMR (DMSO-d6) δ: 2.80 (3H, s, CH3), 8.66 (1H, s, H-2), 8.68 (1H, s, H-7), 13.50 (1H, br s, D2O exchang., NH). 13C-NMR (50 MHz, DMSO-d6) δ: 24.06 (CH3), 122.93 (C-7), 129.66 (C-7α), 141.08 (C-6), 148.12 (C-2, C-5), 151.90 (C-3α). Anal. Calcd for C7H6N4O2: C, 47.19; H, 3.39; N, 31.45. Found: C, 47.26; H, 3.44; N, 31.36. Synthesis of Nucleosides 5, 5a, 6 and 6a N,O-Bis(trimethylsilyl)acetamide (2.78 mL, 11.33 mmol) was added under argon to a suspension of compound 4 (2 g, 11.24 mmol) in anhydrous acetonitrile (75 mL) and the reaction mixture was heated at 80°C for 15 min resulting in a clear solution. Upon cooling at room temperature, tetra-O-acetyl-β-Dribofuranose (3.93 g, 12.35 mmol) was added, followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate (2.04 mL, 11.24 mmol) at 0°C and the mixture was refluxed for 3 h. The solvents were then evaporated, the crude material was diluted with ethyl acetate (300 mL) and extracted with a saturated NaHCO3 solution (300 mL). The aqueous layer was extracted twice with ethyl acetate (250 mL) and the combined organic extracts were dried over sodium sulfate and evaporated under reduced pressure. The crude mixture was purified by column chromatography, using a mixture of cyclohexane–ethyl acetate (from 7 : 3 up to 2 : 8, v/v) as the eluent, providing 3.8 g of a mixture of anomers 5 and 5a (ratio 10 : 1, as indicated by NMR), as well as 1 g of a mixture of 6 and 6a (ratio 7 : 1, as indicated by NMR). The mixture of anomers 5 and 5a was triturated with diethyl ether (3×100 mL) to provide 3.1 g of pure 5, whereas the α-anomer could not be isolated in pure form. The mixture of 6 and 6a was repurified by column chromatography using a mixture of dichloromethane–methanol (100 : 1, v/v) as the eluent, providing 850 mg of pure 6 and 120 mg of pure 6a. 5-Methyl-6-nitro-3-(2,3,5-tri-O-acetyl-β- D -ribofuranosyl)-3H-imidazo[4,5-b]pyridine (5) White solid. Yield 63%. mp: 129–130°C (Et2O). [α]D −18.13° (c=0.557, CHCl3). 1 H-NMR (CDCl3) δ: 2.01, 2.05, 2.10 (3×3H, 3×s, 3×CH3CO), 2.89 (3H, s, CH3), 4.30 (1H, dd, J=4.8 Hz, J=12.1 Hz, H-5′), 4.38–4.46 (2H, m, H-4′+H-5′), 5.75 (1H, m, H-3′), 5.95 (1H, m, H-2′), 6.19 (1H, d, J=4.6 Hz, H-1′), 8.29 (1H, s, H-2), 8.64 (1H, s, H-7). 13C-NMR (50 MHz, CDCl3) δ: 20.50 (CH3CO), 20.61 (CH3CO), 20.78 (CH3CO), 24.69 (CH3), 63.08 (C-5′), 70.52 (C-3′), 73.24 (C-2′), 80.19 (C-4′), 87.14 (C-1′), 125.76 (C-7), 133.82 (C-7α), 143.03 (C-6), 145.76 (C-2), 147.37 (C-3α), 150.64 (C-5), 169.48 (COCH3), 169.65 (COCH3), 170.38

138

Chem. Pharm. Bull.

(COCH3). Anal. Calcd for C18H20N4O9: C, 49.54; H, 4.62; N, 12.84. Found: C, 49.71; H, 4.69; N, 12.77. 5-Methyl-6-nitro-1-(2,3,5-tri-O-acetyl-β- D -ribofuranosyl)-1H-imidazo[4,5-b]pyridine (6) White solid. Yield 17%. mp: 108–109°C (CHCl3/Et2O). [α]D −55.12° (c=0.477, DMSO). 1H-NMR (CDCl3) δ: 1.98 (3H, s, CH3CO), 2.05 (6H, s, 2×CH3CO), 2.78 (3H, s, CH3), 4.26 (1H, dd, J=2.4 Hz, J=12.5 Hz, H-5′), 4.42 (1H, dd, J=3.3 Hz, J=12.5 Hz,H-5′), 4.46 (1H, m, H-4′), 5.31 (1H, m, H-3′), 5.44 (1H, m, H-2′), 6.08 (1H, d, J=5.8 Hz, H-1′), 8.51 (1H, s, H-2), 8.60 (1H, s, H-7). 13C-NMR (50 MHz, CDCl3) δ: 20.17 (CH3CO), 20.35 (CH3CO), 20.53 (CH3CO), 24.37 (CH3), 62.51 (C-5′), 69.84 (C-3′), 73.14 (C-2′), 80.80 (C-4′), 87.78 (C-1′), 117.52 (C-7), 122.36 (C-7α), 141.62 (C-6), 147.60 (C-2), 150.30 (C-5), 157.69 (C-3α), 169.34 (COCH3), 169.47 (COCH3), 170.17 (COCH3). Anal. Calcd for C18H20N4O9: C, 49.54; H, 4.62; N, 12.84. Found: C, 49.39; H, 4.51; N, 12.92. 5-Methyl-6-nitro-1-(2,3,5-tri-O-acetyl-α- D -ribofuranosyl)-1H-imidazo[4,5-b]pyridine (6a) Pale yellow gum. Yield 2%. [α]D +45.67° (c=0.366, CHCl3). 1H-NMR (CDCl3) δ: 1.82, 2.09, 2.15 (3×3H, 3×s, 3×CH3CO), 2.95 (3H, s, CH3), 4.30 (1H, dd, J=4.0 Hz, J=12.3 Hz, H-5′), 4.37 (1H, dd, J=3.2 Hz, J=12.3 Hz, H-5′), 4.72 (1H, m, H-4′), 5.54 (1H, m, H-3′), 5.77 (1H, m, H-2′), 6.56 (1H, d, J=5.6 Hz, H-1′), 8.62 (1H, s, H-7), 8.68 (1H, s, H-2). 13C-NMR (50 MHz, CDCl3) δ: 20.16 (CH3CO), 20.62 (CH3CO), 20.92 (CH3CO), 24.62 (CH3), 63.44 (C-5′), 71.00 (C-3′), 71.48 (C-2′), 81.33 (C-4′), 85.51 (C-1′), 117.83 (C-7), 123.40 (C-7α), 141.99 (C-6), 148.94 (C-2), 150.64 (C-5), 156.80 (C-3α), 169.06 (COCH3), 169.58 (COCH3), 170.32 (COCH3). Anal. Calcd for C18H20N4O9: C, 49.54; H, 4.62; N, 12.84. Found: C, 49.70; H, 4.72; N, 12.71. 5-Methyl-6-nitro-3-(β- D -ribofuranosyl)-3H-imidazo[4,5b]pyridine (7) Compound 5 (0.35 g, 0.8 mmol) was dissolved in a saturated solution of ammonia in methanol (15 mL) and the resulting solution was stirred at room temperature for 16 h. The solvent was removed in vacuo and the residue was purified by column chromatography using a mixture of ethyl acetate–methanol (10 : 1, v/v) as the eluent, providing 7 (0.24 g, yield 97%) as a pale yellow solid. mp: 162–163°C (EtOAc). [α]D −30.81° (c=0.318, DMSO). 1H-NMR (DMSO-d6) δ: 2.84 (3H, s, CH3), 3.55–3.60 (1H, m, H-5′), 3.66–3.73 (1H, m, H-5′), 3.97 (1H, m, H-4′), 4.18 (1H, m, H-3′), 4.63 (1H, m, H-2′), 5.10 (1H, t, D2O exchang., J=5.5 Hz, OH-5′), 5.27 (1H, d, D2O exchang., J=4.9 Hz, OH-3′), 5.53 (1H, d, D2O exchang., J=6.0 Hz, OH-2′), 6.08 (1H, d, J=5.9 Hz, H-1′), 8.82 (1H, s, H-7), 8.93 (1H, s, H-2). 13C-NMR (50 MHz, DMSO-d6) δ: 21.15 (CH3), 61.02 (C-5′), 70.67 (C-3′), 73.93 (C-2′), 86.08 (C-4′), 87.65 (C-1′), 124.76 (C-7), 133.35 (C-7α), 142.00 (C-6), 147.44 (C-2), 147.99 (C-3α), 148.47 (C-5). HR-MS (ESI) m/z: Calcd for C12H15N4O6: [M1+H]+=311.0986, found 311.0987. Anal. Calcd for C12H14N4O6: C, 46.45; H, 4.55; N, 18.06. Found: C, 46.57; H, 4.71; N, 17.87. 5-Methyl-3-(2,3,5-tri-O-acetyl-β- D -ribofuranosyl)-3Himidazo[4,5-b]pyridin-6-amine (8) A solution of 5 (1.1 g, 2.52 mmol) in dry ethanol (50 mL) was hydrogenated in the presence of 10% Pd/C (110 mg) under a pressure of 40 psi at room temperature for 5 h. The solution was filtered through a celite pad to remove the catalyst and the filtrate was evaporated to dryness to give pure 7 (950 mg, 93%) as a pale yellow oil. A small amount of the amine 8 was purified by column chromatography (eluent dichloromethane–methanol 100 : 2,

Vol. 63, No. 2 (2015)

v/v) in order to provide an analytical sample, while the bulk of the amine was used for the next step with no further purification. [α]D −33.60° (c=0.539, CHCl3). 1H-NMR (CDCl3) δ: 1.99, 2.04, 2.08 (3×3H, 3×s, 3×CH3CO), 2.45 (3H, s, CH3), 3.90 (2H, br s, D2O exchang., NH2), 4.29 (1H, dd, J=5.3 Hz, J=12.0 Hz, H-5′), 4.38 (1H, m, H-4′), 4.44 (1H, dd, J=3.7 Hz, J=12.0 Hz, H-5′), 5.91 (1H, m, H-3′), 6.05 (1H, m, H-2′), 6.08 (1H, d, J=4.4 Hz, H-1′), 7.29 (1H, s, H-7), 8.00 (1H, s, H-2). 13 C-NMR (50 MHz, CDCl3) δ: 20.45 (CH3CO), 20.54 (CH3CO), 20.67 (CH3CO), 20.81 (CH3), 63.23 (C-5′), 70.61 (C-3′), 73.03 (C-2′), 79.67 (C-4′), 87.10 (C-1′), 112.33 (C-7), 134.90 (C-7α), 138.03 (C-6), 138.90 (C-3α), 141.58 (C-2), 141.83 (C-5), 169.43 (COCH3), 169.58 (COCH3), 170.48 (COCH3). Anal. Calcd for C18H22N4O7: C, 53.20; H, 5.46; N, 13.79. Found: C, 53.36; H, 5.54; N, 13.62. N-[5 -Methyl-3 -(2, 3, 5 -tri- O-acetyl-β- D -ribofurano­ syl)-3H-imidazo[4,5-b]pyridin-6-yl]acetamide(9) Acetic anhydride (0.267 mL, 2.83 mmol) was added to a solution of the amine 8 (0.82 g, 2.02 mmol) in dry dichloromethane (50 mL) and the reaction mixture was stirred at room temperature for 40 h. The solvent was vacuum-evaporated and the residue was purified by column chromatography using a mixture of ethyl acetate–methanol (100 : 1, v/v) as the eluent to give the acetamide 9 (0.8 g, 88%) as a colorless oil. [α]D −30.05° (c=0.625, CHCl3). 1H-NMR (CDCl3) δ: 1.99, 2.03, 2.08 (3×3H, 3×s, 3×CH3CO), 2.14 (3H, s, CH3CONH), 2.51 (3H, s, CH3), 4.28 (1H, dd, J=4.8 Hz, J=11.6 Hz, H-5′), 4.37–4.43 (2H, m, H-4,′ H-5′), 5.83 (1H, m, H-3′), 6.01 (1H, m, H-2′), 6.12 (1H, d, J=4.5 Hz, H-1′), 8.08 (1H, s, H-2), 8.14 (1H, s, H-7), 8.16 (1H, s, D2O exchang., NH). 13C-NMR (50 MHz, CDCl3) δ: 20.43 (CH3CO), 20.53 (CH3CO), 20.67 (CH3CO), 21.30 (CH3), 23.67 (CH3CONH), 63.17 (C-5′), 70.56 (C-3′), 73.00 (C-2′), 79.84 (C-4′), 87.00 (C-1′), 124.57 (C-7), 128.63 (C-6), 133.56 (C-7α), 142.79 (C-2), 142.92 (C-3α), 149.49 (C-5), 169.47 (NHCOCH3), 169.53 (COCH3), 169.63 (COCH3), 170.51 (COCH3). Anal. Calcd for C20H24N4O8: C, 53.57; H, 5.39; N, 12.49. Found: C, 53.68; H, 5.49; N, 12.63. 5-Methyl-3-(β- D -ribofuranosyl)-3H-imidazo[4,5-b]pyridin-6-amine (10) This compound was prepared by an analogous procedure as described for the preparation of 7, starting from 8. Yield 84% (white solid). mp: 201–202°C (MeOH). [α]D −76.41° (c=0.363, DMSO). 1H-NMR (DMSO-d6) δ: 2.36 (3H, s, CH3), 3.53–3.58 (1H, m, H-5′), 3.65–3.70 (1H, m, H-5′), 3.97 (1H, m, H-4′), 4.14 (1H, m, H-3′), 4.69 (1H, m, H-2′), 4.82 (2H, br s, D2O exchang., NH2), 5.15 (1H, d, D2O exchang., J=4.3 Hz, OH-3′), 5.35 (1H, d, D2O exchang., J=6.5 Hz, OH-2′), 5.57 (1H, m, D2O exchang., OH-5′), 5.86 (1H, d, J=6.7 Hz, H-1′), 7.27 (1H, s, H-7), 8.29 (1H, s, H-2). 13C-NMR (50 MHz, DMSO-d6) δ: 20.41 (CH3), 62.05 (C-5′), 71.03 (C-3′), 72.72 (C-2′), 85.88 (C-4′), 88.05 (C-1′), 111.07 (C-7), 135.43 (C-7α), 138.10 (C-3α), 139.32 (C-6), 139.66 (C-5), 142.71 (C-2). HR-MS (ESI) m/z: Calcd for C12H17 N4O4: [M1+H]+=281.1244, found 281.1244. Anal. Calcd for C12H16N4O4: C, 51.42; H, 5.75; N, 19.99. Found: C, 51.51; H, 5.90; N, 19.84. N-[5-(2,3,5-Tri- O-acetyl-β- D -ribofuranosyl)-1H,5Himidazo[4,5-b]pyrazolo[3,4-e]pyridin-1-yl]acetamide (11) Potassium acetate (213 mg, 2.17 mmol) and acetic anhydride (0.205 mL, 2.17 mmol) were added to a solution of 9 (750 mg, 1.67 mmol) in dry toluene (40 mL) under argon. The reaction mixture was first heated at 80°C, isoamyl nitrite (0.4 mL, 3 mmol) was added and the resulting mixture was heated at

Chem. Pharm. Bull. Vol. 63, No. 2 (2015)139

95°C for 12 h. The insoluble material was then filtered off, the solvent was vacuum evaporated and the residue was purified by column chromatography (silica gel), using a mixture of cyclohexane–ethyl acetate (3 : 7, v/v) as the eluent to result in the tricyclic nucleoside 11 and the byproduct 12 as colorless oils. Data for 11: Yield 25% (190 mg). [α]D −19.11° (c=0.403, CHCl3). 1H-NMR (CDCl3) δ: 2.04 (6H, s, 2×CH3CO), 2.12 (3H, s, CH3CO), 2.75 (3H, s, CH3CO-pyrazole), 4.37 (1H, dd, J=4.8 Hz, J=12.5 Hz, H-5′), 4.41–4.46 (2H, m, H-4,′ H-5′), 5.70 (1H, m, H-3′), 6.05 (1H, m, H-2′), 6.29 (1H, d, J=5.0 Hz, H-1′), 8.31 (1H, s, H-3), 8.40 (1H, s, H-6), 9.00 (1H, s, H-8). 13C-NMR (50 MHz, CDCl3) δ: 20.42 (CH3CO), 20.55 (CH3CO), 20.74 (CH3CO), 22.43 (CH3CO-pyrazole), 63.11 (C-5′), 70.63 (C-3′), 72.77 (C-2′), 80.09 (C-4′), 86.61 (C-1′), 114.79 (C-8), 131.02 (C-8α), 137.17 (C-7α), 139.51 (C-3), 140.80 (C-3α), 146.23 (C-4α), 146.36 (C-6), 169.40 (COCH3), 169.60 (COCH3), 170.32 (COCH3), 170.97 (pyrazole-COCH3). Anal. Calcd for C20H21N5O8: C, 52.29; H, 4.61; N, 15.24. Found: C, 52.06; H, 4.48; N, 15.40. 5-Methyl-3-(2,3,5-tri-O-acetyl-β- D -ribofuranosyl)-3Himidazo[4,5-b]pyridine (12) Yield 10% (65 mg). [α]D −24.27° (c=0.940, CHCl3). 1H-NMR (CDCl3) δ: 2.06, 2.09, 2.14 (3×3H, 3×s, 3×CH3CO), 2.65 (3H, s, CH3), 4.36 (1H, dd, J=4.9 Hz, J=11.9 Hz, H-5′), 4.44 (1H, m, H-4′), 4.48 (1H, dd, J=3.6 Hz, J=11.9 Hz, H-5′), 5.89 (1H, m, H-3′), 6.07 (1H, m, H-2′), 6.21 (1H, d, J=4.7 Hz, H-1′), 7.11 (1H, d, J=8.2 Hz, H-6), 7.92 (1H, d, J=8.2 Hz, H-7), 8.10 (1H, s, H-2). 13C-NMR (50 MHz, CDCl3) δ: 20.57 (CH3CO), 20.68 (CH3CO), 20.83 (CH3CO), 24.48 (CH3), 63.37 (C-5′), 70.80 (C-3′), 73.21 (C-2′), 80.02 (C-4′), 86.87 (C-1′), 119.35 (C-6), 128.46 (C-7), 133.94 (C-7α), 141.94 (C-2), 145.84 (C-3α), 154.55 (C-5), 169.52 (COCH3), 169.69 (COCH3), 170.55 (COCH3). Anal. Calcd for C18H21N3O7: C, 55.24; H, 5.41; N, 10.74. Found: C, 55.33; H, 5.55; N, 10.60. 5 -( β- D -R ibofuranosyl)-1H, 5H-imidazo[4, 5 -b]pyrazolo[3,4-e]pyridine (13) This compound was prepared by an analogous procedure as described for the preparation of 7, starting from 11. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane– methanol (10 : 2, v/v) as the eluent. Yield: 79% (white solid). mp: 252–253°C (MeOH). [α]D −33.38° (c=0.246, DMSO). 1 H-NMR (DMSO-d6) δ: 3.59 (1H, m, H-5′), 3.72 (1H, m, H-5′), 3.98 (1H, m, H-4′), 4.20 (1H, m, H-3′), 4.70 (1H, m, H-2′), 5.20–5.25 (2H, m, D2O exchang., OH-3′, OH-5′), 5.50 (1H, d, D2O exchang., J=6.1 Hz, OH-2′), 6.11 (1H, d, J=5.9 Hz, H-1′), 8.26 (1H, s, H-8), 8.28 (1H, s, H-3), 8.84 (1H, s, H-6), 13.30 (1H, br s, D2O exchang., NH). 13C-NMR (50 MHz, DMSO-d6) δ: 61.45 (C-5′), 70.41 (C-3′), 73.11 (C-2′), 85.34 (C-4′), 87.47 (C-1′), 107.97 (C-8), 131.45 (C-8α), 132.18 (C-3), 135.60 (C-7α), 137.46 (C-3α), 145.52 (C-4α), 147.29 (C-6). HR-MS (ESI) m/z: Calcd for C12H14N5O4: [M1+H]+=292.1040, found 292.1040. Anal. Calcd for C12H13N5O4: C, 49.48; H, 4.50; N, 24.04. Found: C, 49.54; H, 4.41; N, 24.17. 5-Methyl-1-(2,3,5-tri-O-acetyl-β- D -ribofuranosyl)-1Himidazo[4,5-b]pyridin-6-amine (14) This compound was prepared by an analogous procedure as described for the preparation of 8, starting from 6. Yield: 89% (pale yellow oil). A small amount of the amine was purified by column chromatography (eluent dichloromethane–methanol, 100 : 3, v/v) in order to provide an analytical sample, while the bulk of the amine was used for the next step with no further puri-

fication. [α]D −38.01° (c=0.524, CHCl3). 1H-NMR (methanold4) δ: 2.05, 2.08, 2.13 (3×3H, 3×s, 3×CH3CO), 2.47 (3H, s, CH3), 4.38 (1H, dd, J=3.2 Hz, J=12.3 Hz, H-5′), 4.43 (1H, dd, J=3.9 Hz, J=12.3 Hz, H-5′), 4.47 (1H, m, H-4′), 5.44 (1H, m, H-3′), 5.61 (1H, m, H-2′), 6.13 (1H, d, J=5.7 Hz, H-1′), 7.37 (1H, s, H-7), 8.27 (1H, s, H-2). 13C-NMR (50 MHz, methanold4) δ: 20.29 (CH3CO), 20.34 (CH3CO), 20.49 (CH3CO), 20.74 (CH3), 64.12 (C-5′), 71.51 (C-3′), 74.12 (C-2′), 81.53 (C-4′), 88.77 (C-1′), 105.41 (C-7), 126.27 (C-7α), 140.97 (C-6), 141.85 (C-2), 143.13 (C-5), 148.42 (C-3α), 171.15 (COCH3), 171.43 (COCH3), 172.13 (COCH3). Anal. Calcd for C18H22N4O7: C, 53.20; H, 5.46; N, 13.79. Found: C, 53.09; H, 5.29; N, 13.93. N-[5 -Methyl-1-(2, 3, 5 -tri- O -acetyl-β- D -ribofuranosyl)-1H-imidazo[4,5-b]pyridin-6-yl]acetamide (15) This compound was prepared by an analogous procedure as described for the preparation of 9, starting from 14. Yield: 68% (colorless oil). [α]D −9.33° (c=0.493, CHCl3). 1H-NMR (CDCl3) δ: 2.03, 2.06, 2.11 (3×3H, 3×s, 3×CH3CO), 2.23 (3H, s, CH3CONH), 2.53 (3H, s, CH3), 4.31 (1H, dd, J=2.9 Hz, J=12.5 Hz, H-5′), 4.36 (1H, dd, J=3.4 Hz, J=12.5 Hz, H-5′), 4.43 (1H, m, H-4′), 5.35 (1H, m, H-3′), 5.46 (1H, m, H-2′), 6.02 (1H, d, J=6.0 Hz, H-1′), 8.28 (1H, s, H-2), 8.38 (1H, s, H-7), 8.66 (1H, br s, D2O exchang., NH). 13C-NMR (50 MHz, CDCl3) δ: 20.36 (CH3CO), 20.57 (CH3CO), 20.74 (CH3CO), 20.98 (CH3), 24.13 (CH3CONH), 63.00 (C-5′), 70.44 (C-3′), 73.46 (C-2′), 80.66 (C-4′), 87.09 (C-1′), 115.20 (C-7), 123.77 (C-7α), 128.92 (C-6), 142.49 (C-2), 147.52 (C-5), 151.60 (C-3α), 169.50 (COCH3), 169.69 (COCH3), 169.72 (NHCOCH3), 170.36 (COCH3). Anal. Calcd for C20H24N4O8: C, 53.57; H, 5.39; N, 12.49. Found: C, 53.44; H, 5.31; N, 12.40. N-[7-(2, 3,5 -Tri- O-acetyl-β- D -ribofuranosyl)-1H,7Himidazo[4,5-b]pyrazolo[3,4-e]pyridin-1-yl]acetamide (16) This compound was prepared by an analogous procedure as described for the preparation of 11, starting from 15 and the tricyclic nucleoside 16 together with the byproduct 17 were obtained as colorless oils. Data for 16: Yield 18%. [α]D −17.24° (c=0.620, CHCl3). 1 H-NMR (CDCl3) δ: 2.10, 2.16, 2.19 (3×3H, 3×s, 3×CH3CO), 2.82 (3H, s, CH3CO-pyrazole), 4.40 (1H, dd, J=2.6 Hz, J=12.6 Hz, H-5′), 4.50 (1H, dd, J=3.2 Hz, J=12.6 Hz, H-5′), 4.56 (1H, m, H-4′), 5.43 (1H, m, H-3′), 5.59 (1H, m, H-2′), 6.21 (1H, d, J=6.0 Hz, H-1′), 8.45 (1H, s, H-3), 8.67 (1H, s, H-6), 8.99 (1H, s, H-8). 13C-NMR (50 MHz, CDCl3) δ: 20.49 (CH3CO), 20.69 (CH3CO), 20.96 (CH3CO), 22.55 (CH3COpyrazole), 62.95 (C-5′), 70.52 (C-3′), 73.53 (C-2′), 81.08 (C-4′), 87.40 (C-1′), 105.57 (C-8), 126.35 (C-7α), 129.94 (C-8α), 140.86 (C-3), 142.25 (C-3α), 146.14 (C-6), 155.93 (C-4α), 169.50 (COCH3), 169.72 (COCH3), 170.45 (COCH3), 171.40 (pyrazoleCOCH3). Anal. Calcd for C20H21N5O8: C, 52.29; H, 4.61; N, 15.24. Found: C, 52.42; H, 4.71; N, 15.06. 5-Methyl-1-(2,3,5-tri-O-acetyl-β- D -ribofuranosyl)-1Himidazo[4,5-b]pyridine (17) Yield 21%. [α]D −33.39° (c=0.647, CHCl3). 1H-NMR (CDCl3) δ: 2.07, 2.14, 2.15 (3×3H, 3×s, 3×CH3CO), 2.68 (3H, s, CH3), 4.38 (1H, dd, J=2.6 Hz, J=12.5 Hz, H-5′), 4.44 (1H, dd, J=3.1 Hz, J=12.5 Hz, H-5′), 4.47 (1H, m, H-4′), 5.39 (1H, m, H-3′), 5.53 (1H, m, H-2′), 6.04 (1H, d, J=5.8 Hz, H-1′), 7.12 (1H, d, J=8.3 Hz, H-6), 7.85 (1H, d, J=8.3 Hz, H-7), 8.29 (1H, s, H-2). 13C-NMR (50 MHz, CDCl3) δ: 20.46 (CH3CO), 20.64 (CH3CO), 20.89 (CH3CO), 24.50 (CH3), 62.92 (C-5′), 70.20 (C-3′), 73.08 (C-2′), 80.48 (C-4′), 87.62 (C-1′), 118.86 (C-6), 119.59 (C-7), 122.94 (C-7α),

140

Chem. Pharm. Bull.

142.77 (C-2), 154.91 (C-5), 156.45 (C-3α), 169.43 (COCH3), 169.67 (COCH3), 170.30 (COCH3). Anal. Calcd for C18H21N3O7: C, 55.24; H, 5.41; N, 10.74. Found: C, 55.44; H, 5.57; N, 10.51. 7- ( β- D -R ibofuranosyl) -1H,7H-imidazo[4, 5 -b]pyrazolo[3,4-e]pyridine (18) This compound was prepared by an analogous procedure as described for the preparation of 7, starting from 16. Purification was performed by column chromatography (silica gel) using a mixture of dichloromethane– methanol (10 : 2, v/v) as the eluent. Yield: 75% (beige solid). mp: 224–225°C (MeOH). [α]D −37.21° (c=0.108, DMSO). 1 H-NMR (DMSO-d6) δ: 3.64–3.73 (2H, m, 2×H-5′), 4.02 (1H, m, H-4′), 4.15 (1H, m, H-3′), 4.42 (1H, m, H-2′), 5.19 (1H, m, D2O exchang., OH-5′), 5.23 (1H, d, D2O exchang., J=4.7 Hz, OH-3′), 5.51 (1H, d, D2O exchang., J=6.4 Hz, OH-2′), 5.95 (1H, d, J=6.3 Hz, H-1′), 8.29 (2H, s, H-3, H-8), 8.82 (1H, s, H-6), 13.22 (1H, br s, D2O exchang., NH). 13C-NMR (50 MHz, DMSO-d6) δ: 61.16 (C-5′), 70.08 (C-3′), 73.25 (C-2′), 85.67 (C-4′), 89.22 (C-1′), 99.71 (C-8), 125.25 (C-7α), 130.24 (C-8α), 133.19 (C-3), 138.68 (C-3α), 147.70 (C-6), 154.99 (C-4α). HR-MS (ESI) m/z: Calcd for C12H14N5O4: [M1+H]+=292.1040, found 292.1042. Anal. Calcd for C12H13N5O4: C, 49.48; H, 4.50; N, 24.04. Found: C, 49.60; H, 4.59; N, 23.89. 2,2,2-Trifluoro-N-(5-methyl-3H-imidazo[4,5-b]pyridin6-yl)acetamide (19) A solution of 4 (3.7 g, 20.79 mmol) in dry ethanol (80 mL) was hydrogenated in the presence of 10% Pd/C (440 mg) under a pressure of 50 psi at room temperature for 45 h. The solution was filtered through a celite pad and the filtrate was evaporated to dryness. The resulting amine (3g, 20.27 mmol) was suspended in dry THF (130 mL), trifluoroacetic anhydride (5.67 mL, 40.54 mmol) was added dropwise at 0°C and the mixture was stirred at room temperature for 16 h. Upon completion of reaction, the solvents were evaporated, water was added to the residue and it was made alkaline (pH=8) with the addition of a saturated sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate (4×200 mL), the combined organic layers were dried over sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography (silica gel) using a mixture of ethyl acetate–methanol (10 : 1, v/v) as the eluent to give pure 19 (4.1 g, 81% for two steps) as a white solid. mp: 249–252°C (EtOAc). 1H-NMR (DMSO-d6) δ: 2.45 (3H, s, CH3), 7.95 (1H, s, H-7), 8.44 (1H, s, H-2), 11.18 (1H, br s, D2O exchang., NHCOCF3). 13C-NMR (50 MHz, DMSO-d6) δ: 20.74 (CH3), 107.50, 113.29, 119.01, 124.76 (CF3), 123.08 (C-7), 124.27 (C-6), 129.41 (C-7α), 144.66 (C-2), 148.95 (C-5), 149.24 (C-3α), 154.67, 155.40, 156.13, 156.86 (COCF3). Anal. Calcd for C9H7F3N4O: C, 44.27; H, 2.89; N, 22.95. Found: C, 44.02; H, 3.06; N, 22.69. N 4 -Oxide of 2,2,2-Trifluoro-N-(5-methyl-3H-imidazo[4,5b]pyridin-6-yl)acetamide (20) m-Chloroperbenzoic acid (77%, 3.14 g, 14 mmol) was added to a cooled (0°C) suspension of the trifluoroacetamide 19 (2.9 g, 11.89 mmol) in dichloromethane (300 mL) and the mixture was stirred at room temperature in the absence of light, for 7 d. The precipitate was filtered and washed with dichloromethane (2×100 mL). The crude compound was dry-packed and purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (100 : 15, v/v) as the eluent to give pure 20 (2.8 g, 91%) as a white solid. mp: 300–301°C (EtOH). 1 H-NMR (DMSO-d6) δ: 2.38 (3H, s, CH3), 7.68 (1H, s, H-7), 8.42 (1H, s, H-2), 11.40 (1H, br s, D2O exchang., NHCOCF3),

Vol. 63, No. 2 (2015)

13.20 (1H, br s, D2O exchang., NH). 13C-NMR (50 MHz, DMSO-d6) δ: 12.52 (CH3), 109.60 (C-7), 113.08, 114.99, 116.90, 118.81 (CF3), 125.73 (C-6, C-7α), 140.38 (C-5), 143.77 (C-2), 145.96 (C-3α), 155.49, 155.73, 155.97, 156.21 (COCF3). Anal. Calcd for C9H7F3N4O2: C, 41.55; H, 2.71; N, 21.53. Found: C, 41.71; H, 2.59; N, 21.34. N 4 -Oxide of 2,2,2-Trifluoro-N-(5-methyl-7-nitro-3Himidazo[4,5-b]pyridin-6-yl)acetamide (21) The N-oxide 20 (2.6 g, 10 mmol) was added in portions into fuming nitric acid (13 mL) at 0°C and the mixture was heated at 55°C for 5 h. Upon cooling, the solution was poured into ice (40 mL) and the pH was adjusted to 3 by dropwise addition of ammonium hydroxide 25%. The precipitate was filtered, washed with cold water and vacuum-dried to provide the nitro-compound 21 (1.6 g, 52%) as a yellow solid. A small amount of 21 was purified by column chromatography (eluent dichloromethane–methanol, 100 : 12, v/v) in order to obtain an analytical sample, while the bulk of the compound was used for the next step with no further purification. mp: >320°C (dec) (EtOAc). 1 H-NMR (DMSO-d6) δ: 2.43 (3H, s, CH3), 8.61 (1H, s, H-2), 11.96 (1H, br s, D2O exchang., NHCOCF3), 13.92 (1H, br s, D2O exchang., NH). 13C-NMR (50 MHz, DMSO-d6) δ: 12.65 (CH3), 112.82, 114.73, 116.64, 118.55 (CF3), 120.44 (C-6), 121.51 (C-7α), 127.24 (C-7), 142.52 (C-5), 146.09 (C-2), 148.68 (C-3α), 155.49, 155.74, 155.99, 156.24 (COCF3). Anal. Calcd for C9H6F3N5O4: C, 35.42; H, 1.98; N, 22.95. Found: C, 35.26; H, 1.87; N, 23.19. 2,2,2-Trifluoro-N-(7-chloro-5-methyl-3H-imidazo[4,5b]pyridin-6-yl)acetamide (22) Phosphorus trichloride (4.12 mL, 47.25 mmol) was added dropwise to a suspension of trifluoroacetamide 21 (1.6 g, 5.25 mmol) in dry acetonitrile (20 mL) at 0°C and the mixture was refluxed for 5 h. Upon completion of the reaction, the solvents were vacuumevaporated, a saturated solution of sodium bicarbonate was added to the residue, followed by extraction with ethyl acetate (4×200 mL). The combined organic layers were dried over sodium sulfate, evaporated to dryness and the residue was purified by column chromatography, using a mixture of ethyl acetate–methanol (100 : 5, v/v) as the eluent, providing the chloroderivative 22 as a white solid (1.2 g, 82%). mp: 243–244°C (EtOAc). 1H-NMR (DMSO-d6) δ: 2.48 (3H, s, CH3), 8.52 (1H, s, H-2), 11.41 (1H, br s, D2O exchang., NHCOCF3). 13C-NMR (50 MHz, DMSO-d6) δ: 21.15 (CH3), 113.16, 115.07, 116.98, 118.89 (CF3), 121.88 (C-7), 129.61 (C-7α), 130.38 (C-6), 145.14 (C-2), 148.01 (C-3α), 151.41 (C-5), 155.31, 155.56, 155.81, 156.05 (COCF3). Anal. Calcd for C9H6ClF3N4O: C, 38.80; H, 2.17; N, 20.11. Found: C, 38.98; H, 2.40; N, 19.99. 2,2,2-Trif luoro-N-[7-chloro-5-methyl-3-(2,3,5-tri-Oacetyl-β- D -ribofuranosyl)-3H-imidazo[4,5-b]pyridin-6-yl]acetamide (23) This compound was prepared by an analogous procedure as described for the preparation of 5, starting from 22. Purification was performed by column chromatography, using a mixture of cyclohexane–ethyl acetate (6 : 4 to 4/6, v/v) as the eluent. Yield 95% (pale yellow oil). [α]D −20.48° (c=0.415, CHCl3). 1H-NMR (CDCl3) δ: 2.08, 2.10, 2.15 (3×3H, 3×s, 3×CH3CO), 2.61 (3H, s, CH3), 4.35 (1H, dd, J=5.7 Hz, J=13.0 Hz, H-5′), 4.44–4.48 (2H, m, H-4,’ H-5′), 5.80 (1H, m, H-3′), 6.01 (1H, m, H-2′), 6.18 (1H, d, J=4.6 Hz, H-1′), 8.10 (1H, br s, D2O exchang., NH), 8.21 (1H, s, H-2). 13C-NMR (50 MHz, CDCl3) δ: 20.36 (CH3CO), 20.47 (CH3CO), 20.61

Chem. Pharm. Bull. Vol. 63, No. 2 (2015)141

(CH3CO), 21.64 (CH3), 63.03 (C-5′), 70.48 (C-3′), 73.11 (C-2′), 80.07 (C-4′), 87.18 (C-1′), 113.12, 115.03, 116.94, 118.85 (CF3), 123.19 (C-6), 132.30 (C-7α), 133.36 (C-7), 143.70 (C-2), 145.01 (C-3α), 153.70 (C-5), 155.97, 156.21, 156.46, 156.71 (COCF3), 169.50 (COCH3), 169.66 (COCH3), 170.53 (COCH3). Anal. Calcd for C20H20ClF3N4O8: C, 44.75; H, 3.76; N, 10.44. Found: C, 44.54; H, 3.92; N, 10.61. 2,2,2-Trifluoro-N-[7-chloro-5-methyl-3-(β- D -ribofuranosyl)-3H-imidazo[4,5-b]pyridin-6-yl]acetamide (24) Potassium carbonate (0.46 g, 3.36 mmol) was added to a solution of 23 (0.45 g, 0.84 mmol) in a mixture of methanol (10 mL) and water (1 mL) and the resulting mixture was refluxed for 2 h. The solvent was then vacuum evaporated and the residue was neutralized with diluted hydrochloric acid (2 N), followed by extraction with ethyl acetate (3×150 mL). The combined organic extracts were dried over sodium sulfate and the solvent was vacuum-evaporated. The residue was purified by column chromatography, using a mixture of ethyl acetate–methanol (9 : 1, v/v) as the eluent, providing trifluoroacetamide 24 as a white solid (0.28 g, 81%). mp: 239°C (EtOAc). [α]D −28.38° (c=0.359, DMSO). 1H-NMR (DMSO-d6) δ: 2.51 (3H, s, CH3), 3.56–3.61 (1H, m, H-5′), 3.67–3.72 (1H, m, H-5′), 3.99 (1H, m, H-4′), 4.18 (1H, m, H-3′), 4.62 (1H, m, H-2′), 5.12 (1H, m, D2O exchang., OH-5′), 5.23 (1H, d, D2O exchang., J=4.8 Hz, OH-3′), 5.50 (1H, d, D2O exchang., J=6.0 Hz, OH-2′), 6.04 (1H, d, J=5.9 Hz, H-1′), 8.79 (1H, s, H-2), 11.49 (1H, br s, D2O exchang., NH). 13C-NMR (50 MHz, DMSO-d6) δ: 21.13 (CH3), 61.43 (C-5′), 70.52 (C-3′), 73.55 (C-2′), 85.85 (C-4′), 87.68 (C-1′), 113.12, 115.03, 116.94, 118.85 (CF3), 122.77 (C-6), 131.94 (C-7), 132.26 (C-7α), 145.24 (C-3α), 145.40 (C-2), 151.84 (C-5), 155.30, 155.54, 155.78, 156.02 (COCF3). HR-MS (ESI) m/z: Calcd for C14H15ClF3N4O5: [M1+H]+=411.0678, found 411.0676. Anal. Calcd for C14H14ClF3N4O5: C, 40.94; H, 3.44; N, 13.64. Found: C, 41.09; H, 3.60; N, 13.56. 7-Chloro-5-methyl-3-( β- D -ribofuranosyl)-3H-imidazo[4,5-b]pyridin-6-amine (25) This compound was prepared analogously to 24, using longer reaction time (48 h reflux) and equal percentage of methanol–water. The residue was purified by column chromatography, using a mixture of ethyl acetate–methanol (100 : 15, v/v) as the eluent. Yield: 76% (pale yellow solid). mp: 224°C (EtOAc). [α]D −67.49° (c=0.356, DMSO). 1H-NMR (DMSO-d6) δ: 2.46 (3H, s, CH3), 3.53–3.58 (1H, m, H-5′), 3.65–3.70 (1H, m, H-5′), 3.97 (1H, m, H-4′), 4.14 (1H, m, H-3′), 4.64 (1H, m, H-2′), 5.04 (2H, br s, D2O exchang., NH2), 5.17 (1H, d, D2O exchang., J=4.4 Hz, OH-3′), 5.32 (1H, m, D2O exchang., OH-5′), 5.39 (1H, d, D2O exchang., J=6.3 Hz, OH-2′), 5.90 (1H, d, J=6.4 Hz, H-1′), 8.42 (1H, s, H-2). 13C-NMR (50 MHz, DMSO-d6) δ: 21.35 (CH3), 61.77 (C-5′), 70.75 (C-3′), 73.05 (C-2′), 85.77 (C-4′), 87.86 (C-1′), 115.40 (C-7), 132.28 (C-7α), 135.79 (C-6), 137.65 (C-3α), 140.82 (C-5), 142.77 (C-2). HR-MS (ESI) m/z: Calcd for C12H16ClN4O4: [M1+H]+=315.0855, found 315.0856. Anal. Calcd for C12H15ClN4O4: C, 45.80; H, 4.80; N, 17.80. Found: C, 45.69; H, 4.92; N, 17.87. Biological Assays Adenosine deaminase (ADA, EC 3.5.4.4), adenosine and bovine serum albumin (BSA) were purchased from Sigma. The target compounds were screened against calf spleen ADA in vitro. The total volume of the reaction in 50 m M phosphate buffer (pH 7.0) was 0.25 mL containing substrate concentration 60 µM, 0.003% BSA and 0.02 unit of ADA. The compounds were dissolved in DMSO

final concentration 2%. The reaction was performed at 25°C. The conversion of adenosine to inosine was monitored by following the absorbance decrease at 265 nm using a microplate reader (BioTek Power Wave XS2) and compared to the appropriate solution, which did not contain the extracts. The inhibitors, at a concentration of 0.4 m M, were used with 5 min pre-incubation with enzyme and the reactions were initiated by addition of adenosine. Assays were performed in two different experiments in triplicate and the standard deviation in absorbance determinations was less than ±10%. Acknowledgments This project is funded by NSRF 2007–2013 National action: Cooperation sub-action II: Large Scale Cooperative Project 09SYN-21-1078. Conflict of Interest interest.

References

The authors declare no conflict of

1) Löfgren N., Lüning B., Jensen K. A., Schønfeldt E., Steensgaard I., Rosenberg T., Acta Chem. Scand., 7, 225 (1953). 2) Löfgren N., Lüning B., Hedström H., Burris R. H., Acta Chem. Scand., 8, 670–680 (1954). 3) Tamm I., Folkers K., Shunk C., J. Bacteriol., 72, 59–64 (1956). 4) Das S. R., Baer H. P., Trop. Med. Parasitol., 42, 161–163 (1991). 5) el Kouni M. H., Cha S., Biochem. Pharmacol., 36, 1099–1106 (1987). 6) Paterson A. R., Paran J. H., Yang S., Lynch T. P., Cancer Res., 39, 3607–3611 (1979). 7) Biesele J. J., Slautterback M. S., Margolis M., Cancer, 8, 87–96 (1955). 8) Rahman M. S., Humayun M. Z., Mutat. Res., 377, 263–268 (1997). 9) Eritja R., Horowitz D. M., Walker P. A., Ziehler-Martin J. P., Boosalis M. S., Goodman M. F., Itakura K., Kaplan B. E., Nucleic Acids Res., 14, 8135–8153 (1986). 10) Stilz H. U., Dervan P. B., Biochemistry, 32, 2177–2185 (1993). 11) Wang Z., Quiocho F. A., Biochemistry, 37, 8314–8324 (1998). 12) Wilson D. K., Rudolph F. N., Quiocho F. A., Science, 252, 1278– 1284 (1991). 13) Guranowski A., Montgomery J. A., Cantoni G. L., Chiang P. K., Biochemistry, 20, 110–115 (1981). 14) Frank K. B., Cheng Y. C., J. Biol. Chem., 261, 1510–1513 (1986). 15) Brown E. G., Konuk M., Phytochemistry, 37, 1589–1592 (1994). 16) Backer W. S., Khan J. A., Pak. J. Med. Sci., 20, 41–45 (2004). 17) Cristalli G., Costanzi S., Lambertucci C., Lupidi G., Vittori S., Volpini R., Camaioni E., Med. Res. Rev., 21, 105–128 (2001). 18) Agarwal R. P., Spector T., Parks R. E. Jr., Biochem. Pharmacol., 26, 359–367 (1977). 19) Pragnacharyulu P. V., Varkhedkar V., Curtis M. A., Chang I. F., Abushanab E., J. Med. Chem., 43, 4694–4700 (2000). 20) La Motta C., Sartini S., Mugnaini L., Salerno S., Simorini F., Taliani S., Marini A. M., Da Settimo F., Lavecchia A., Novellino E., Antonioli L., Fornai M., Blandizzi C., Del Tacca M., J. Med. Chem., 52, 1681–1692 (2009). 21) Gillerman I., Fischer B., J. Med. Chem., 54, 107–121 (2011). 22) Kourafalos V. N., Marakos P., Pouli N., Townsend L. B., J. Org. Chem., 68, 6466–6469 (2003). 23) Korouli S., Lougiakis N., Marakos P., Pouli N., Synlett, 181–184 (2008). 24) Kourafalos V. N., Tite T., Mikros E., Marakos P., Pouli N., Balzarini J., Synlett, 3129–3132 (2008). 25) Lougiakis N., Marakos P., Pouli N., Balzarini J., Chem. Pharm. Bull., 56, 775–780 (2008). 26) Tite T., Lougiakis N., Myrianthopoulos V., Marakos P., Mikros E., Pouli N., Tenta R., Fragopoulou E., Nomikos T., Tetrahedron, 66,

142

Chem. Pharm. Bull.

9620–9628 (2010). 27) Seela F., Peng X., Curr. Top. Med. Chem., 6, 867–892 (2006). 28) Borrmann T., Abdelrahman A., Volpini R., Lambertucci C., Alksnis E., Gorzalka S., Knospe M., Schiedel A. C., Cristalli G., Müller C. E., J. Med. Chem., 52, 5974–5989 (2009). 29) Rayala R., Theard P., Ortiz H., Yao S., Young J. D., Balzarini J., Robins M. J., Wnuk S., ChemMedChem, 9, 2186–2192 (2014). 30) Ritter H., Licht H. H., J. Heter. Chem., 32, 585–590 (1995). 31) Zhou Z. L., Kher S. M., Cai S. X., Whittemore E. R., Espitia S. A.,

32) 33) 34) 35)

Vol. 63, No. 2 (2015)

Hawkinson J. E., Tran M., Woodward R. M., Weber E., Keana J. F. W., Bioorg. Med. Chem., 11, 1769–1780 (2003). Vorbrüggen H., Krolikiewicz K., Bennua B., Chem. Ber., 114, 1234–1255 (1981). Vorbrüggen H., Bennua B., Tetrahedron Lett., 19, 1339–1342 (1978). Marakos P., Pouli N., Wise D., Townsend L. B., Synlett, 561–562 (1997). Chapman D., Hurst J., J. Chem. Soc., Perkin Trans., 1, 2398–2404 (1980).