The Synthesis of a New Pyrazolo[3,4-c]pyridine C-Nucleoside, Structurally
Related to Formycin B

Abstract

The first preparation of the 4-deaza analogue of formycin B is described, via the reaction of 3-acetamido-2-methoxy-4-methylpyridine with a suitably protected ribonolactone and subsequent ring closure to result in the 3-substituted pyrazolo[3,4-c]pyridine riboside 12.

Key words

lithiation - ring closure - heterocycles - C-nucleosides - pyrazolopyridine

References

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12

For hemiacetals 3 the prefix α refers to the position of the glycosidic OH group relative to the configuration at the reference C-atom (C-4′ in 3; i.e. the methylpyridinyl moiety is in the β-position). For C-glycosides (no glycosidic OH present), the prefix α-D refers to the alkyl (or aryl) position relative to the reference C-atom.

13

Optimized procedure for the preparation of the hemiacetals 3: To a solution of 1 (0.5 g, 2.72 mmol) in dry THF (30 ml) at -78 °C was added under argon n-BuLi (4.3 ml, 6.95 mmol, 1.6 M solution in hexanes). The resulting light yellow solution was stirred at -78 °C for 15 min and the temperature then raised to -40 °C for 1 h. The orange-colored solution was then cooled to -78 °C, a solution of the d-ribonolactone 2 (1.4 g, 3.34 mmol) in dry THF (10 ml) was added dropwise and the resulting mixture was stirred at -78 °C for 1 h and at -40 °C for an additional 5 h. A saturated ammonium chloride solution was then added to the reaction mixture to quench the excess n-BuLi. The solvent was vacuum-evaporated, water was added to the residue and this was extracted with dichloromethane. The organic extracts were dried (Na₂SO₄) and concentrated to dryness to give an oil, which was purified by flash chromatography (silica gel 20 × 2 cm) using a mixture of cyclohexane-ethyl acetate, 7:3 v/v as the eluent to give the isomeric hemiacetals 3 (63%), together with 4 (6%).

14

The configuration at C-1′ was assigned on the basis of nOe spectral data. In the case of the β-D anomer clear correlation peaks between the OH-1′ and the H-2′, H-3′ and H-5′ were observed.

15

In the HMBC spectrum of 4, a strong correlation peak between the 5-aromatic H and the carbon of the methyl group (³ J coupling) is evident. The methyl group protons correlate also with the aromatic carbons 3,5 (³ J coupling) and 4 (² J coupling). The methylene group protons possess a strong correlation with both the anomeric carbon and the carbonyl. NOESY data provided evidence for the β-conformation, since we observed correlation peaks between the methylene protons attached at the anomeric position with H-4′.

16

Data of 3-[(1-hydroxy-2,3,5-tri- -benzyl)-β-d-ribofurano-syl)acetylamino]-2-methoxy-4-methylpyridine(4): Colorless oil. ¹ -NMR (400 MHz, CDCl₃) δ 1.95 (s, 3 H, 4-CH₃), 2.78 (m, 2 H, COCH₂), 3.38 (m, 2 H, H-5′), 3.73 (d, 1 H, J_{2 ′ ,3 ′} = 4.56 Hz, H-2′), 3.85 (m, 1 H, H-3′), 3.99 (s, 3 H, OCH₃), 4.29 (m, 1 H, H-4′), 4.32-4.68 (m, 7 H, 3 × CH₂-Ph, OH), 6.70 (d, 1 H, J _5,6 = 4.98 Hz, H-5), 7.2-7.4 (m, 15 H, 3 × C₆H₅), 7.84 (d, 1 H, J_6,5 = 4.98 Hz, H-6), 7.94 (br s, 1 H, D₂O exchangeable, NH). ¹³C NMR (50 MHz, CDCl₃) δ 18.0 (4-CH₃), 41.2 (COCH₂), 53.3 (CH₃O), 69.3 (C-5′), 72.3 (CH₂-Ph), 72.6 (CH₂-Ph), 73.3 (CH₂-Ph), 76.9 (C-3′), 78.9 (C-2′), 80.4 (C-4′), 105.0 (C-1′), 118.9 (C-5), 119.1 (C-3), 127.7 [CH(Ph)], 128.0 [CH(Ph)], 128.2 [CH(Ph)], 128.3 [CH(Ph)], 128.6 [CH(Ph)], 136.7 [C(Ph)], 136.9 [C(Ph)], 137.6 [C(Ph)], 145.6 (C-4), 146.0 (C-6), 158.4 (C-2), 169.2 (C=O). Anal. Calcd. For C₃₅H₃₈N₂O₇: C: 70.21, H: 6.40, N: 4.68. Found: C: 70.10, H: 6.62, N: 4.63.

17

The excess n-butyllithium (two equivalents) required for the lithiation of 1, induces the formation of an anion on the acetamide’s methyl group, which probably attacks the ribonolactone to provide 4.

18

The use of acid labile protecting groups for the 5-OH of the lactone component (e.g. TBDMS) should be avoided, since the corresponding 1′,5′-anhydro derivative is obtained from this reaction as the major product, irrespective of the reaction conditions or of the Lewis acid used for catalysis.

20

Preparation of 6: To a solution of the anomers 3 (0.5 g, 0.84 mmol) in dry CH₂Cl₂ (20 ml) at 0 °C was added under argon BF₃Et₂O (0.22 ml, 1.68 mmol). The solution was stirred at 5-10 °C for 5 h and then, was neutralized with a saturated NaHCO₃ solution. The mixture was extracted with CH₂Cl₂ and the combined organic extracts were dried (Na₂SO₄) and concentrated to dryness. The residue was purified by flash chromatography (silica gel, 18 × 1 cm) using EtOAc as the eluent to give 0.46 g (95%) of an inseparable E/Z mixture of 5. This mixture was dissolved in absolute EtOH (20 mL) and hydrogenated (10% Pd/C, 90 mg) at 50 psi for 5 h. The catalyst was filtered off, washed with EtOH, the solvent was vacuum-evaporated and the residue was purified by column chromatography (CH₂Cl₂-MeOH, 97:3, silica gel, 18 × 1 cm), to give 6, together with the corresponding α-anomer(7). 3-Acetamido-2-methoxy-4-[(β-d-ribofuranosyl)methyl]py-ridine(6): White foam. 250 mg (29%). ¹ NMR (400 MHz, CDCl₃) δ 1.98 (s, 3 H, COCH₃), 2.53 (dd, 1 H, J = 9.15 Hz, 14.64 Hz, pyCH₂), 2.78 (dd, 1 H, J = 4.03 Hz, 14.64 Hz, pyCH₂), 3.36 (d, 1 H, J_{4 ′ ,5 ′} = 5.12 Hz, J_{5 ′ ,5 ′} = 11.71 Hz, H-5′), 3.41 (d, 1 H, J_{4 ′ ,5 ′} = 4.03 Hz, J_{5 ′ ,5 ′} = 11.71 Hz, H-5′), 3.59 (m, 2 H, H-2′, H-4′), 3.81 (m, 5 H, H-1′, H-3′, OCH₃), 4.88 (m, 1 H, OH, D₂O exchangeable), 5.07 (m, 2 H, 2 × OH, D₂O exchangeable), 6.96 (d, 1 H, J_5,6 = 5.12 Hz, H-5), 7.93 (d, 1 H, J_6,5 = 5.12 Hz, H-6), 9.21 (br s, 1 H, NHAc, D₂O exchangeable). ¹³C NMR (50 MHz, CDCl₃) δ 22.6 (CH₃-CO), 36.0 (pyCH₂), 53.6 (OCH₃), 61.6 (C-5′), 70.8 (C-3′), 74.62 (C-2′), 81.45 (C-4′), 84.32 (C-1′), 118.7 (C-5), 120.1 (C-3), 143.9 (C-6), 147.6 (C-4), 159.7 (C-2), 169.0 (C=O). Anal. Calcd. for C₁₄H₂₀N₂O₆: C: 53.84, H: 6.45, N: 8.97. Found: C: 53.69, H: 6.17, N: 8.76. 3-Acetamido-2-methoxy-4-[(α-d-ribofuranosyl)methyl]pyridine(7): Mp: 187 °C (MeOH). 490 mg (57%). ¹ NMR (400 MHz, CDCl₃) δ 2.02 (s, 3 H, COCH₃), 2.72 (dd, 1 H, J = 8.05 Hz, 14.27 Hz, pyCH₂), 2.74 (dd, 1 H, J = 5.12 Hz, 14.27 Hz, pyCH₂), 3.32 (d, 1 H, J_{4 ′ ,5 ′} = 5.13 Hz, J_{5 ′ ,5 ′} = 12.08 Hz, H-5′), 3.48 (d, 1 H, J_{4 ′ ,5 ′} = 2.20 Hz, J_{5 ′ ,5 ′} = 12.08 Hz, H-5′), 3.67 (m, 1 H, H-4′), 3.78 (m, 1 H, H-2′), 3.81 (s, 3 H, OCH₃), 3.88 (m, 1 H, H-3′), 4.04 (m, 1 H, H-1′), 4.59 (m, 1 H, OH, D₂O exchangeable), 4.84 (m, 2 H, 2 × OH, D₂O exchangeable), 6.98 (d, 1 H, J_5,6 = 5.12 Hz, H-5), 7.92 (d, 1 H, J_6,5 = 5.12 Hz, H-6), 9.28 (br s, 1 H, NHAc, D₂O exchangeable). ¹³C NMR (50 MHz, CDCl₃) δ 22.7 (CH₃CO), 31.3 (pyCH₂), 53.3 (OCH₃), 61.7 (C-5′), 71.9 (C-3′), 72.1 (C-2′), 79.1 (C-4′), 81.9 (C-1′), 118.7 (C-5), 120.0 (C-3), 143.5 (C-6), 148.0 (C-4), 159.5 (C-2), 168.5 (C=O). Anal. Calcd. for C₁₄H₂₀N₂O₆: C: 53.84, H: 6.45, N: 8.97. Found: C: 53.55, H: 6.39, N: 9.11.

21

Data of 12: Yield: 90%. Mp: 253 °C (dec.) (EtOH). UV (CH₃OH) λ_max(nm) (ɛ × 10^-3): 302 (8.17), 251 (6.62). ¹ NMR (400 MHz, DMSO-d ₆) δ 3.48 (m, 2 H, H-5′), 3.81 (m, 1 H, H-4′), 3.95 (m, 1 H, H-3′), 4.12 (m, 1 H, H-2′), 4.81 (d, 1 H, J_{1 ′,2 ′} = 6.95 Hz, H-1′), 4.82 (br s, 1 H, D₂O exchangeable, OH-5′), 4.89 (br. s., 1 H, D₂O exchangeable, OH-3′), 4.98 (br. s., 1 H, D₂O exchangeable, OH-2′), 6.68 (d, 1 H, J_4,5 = 6.22 Hz, H-4), 6.88 (d, 1 H, J_5,4 = 6.22 Hz, H-5), 11.2 (br s, 1 H, D₂O exchangeable, NH-6), 13.8 (br. s., 1 H, D₂O exchangeable, NH-1). ¹³C NMR (50 MHz, DMSO-d ₆) δ 62.2 (C-5′), 71.5 (C-3′), 75.1 (C-2′), 79.5 (C-1′), 85.5 (C-4′), 99.3 (C-4), 123.4 (C-3α), 132.8 (C-7α), 125.4 (C-5), 145.8 (C-3), 154.3 (C-7). Anal. Calcd. for C₁₁H₁₃N₃O₅: C: 49.44, H: 4.90, N: 15.72. Found: C: 49.60, H: 5.12, N: 15.51.