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.

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

References

1a Revankar GR. Robins RK. In Chemistry of Nucleosides and Nucleotides   Vol. 2:  Townsend LB. Plenum Press; New York: 1994.  p.161-398  

1b Srivastava PC. Robins RK. Meyer RB. In Chemistry of Nucleosides and Nucleotides   Vol. 3:  Townsend LB. Plenum Press; New York: 1994.  p.421-535  

1c Perigaud C. Gosselin G. Imbach JL. Nucleosides Nucleotides  1992,  11:  903 

1d De Clercq E. Nucleosides Nucleotides  1994,  12:  1271 

2a Hori M. Ito E. Takida T. Koyama Y. Takeuchi T. Umezawa H. J. Antibiot.  1964,  17A:  96 

2b Long RA. Lewis AF. Robins RK. Townsend LB. J. Org. Chem.  1971,  36:  2443 

2c Lewis AF. Townsend LB. J. Am. Chem. Soc.  1982,  104:  1073 

2d Orozco M. Canela EI. Franco R. Mol. Pharmacol.  1989,  35:  257 

3a Robins RK. Townsend LB. Cassidy F. Gersrter FG. Lewis AT. Miller RL. J. Heterocycl. Chem.  1966,  3:  110 

3b Koyama G. Umezawa H. Antibiot. Ser. A  1965,  18:  175 

4a Ishizuka M. Sawa T. Hori S. Takayama T. Takeuchi T. Umezawa H. J. Antibiot.  1968,  21:  5 

4b Sheen MR. Martin FH. Parks HF. Mol. Pharmacol.  1970,  6:  255 

5 Otter BA. Patil SA. Klein RS. Abstract CARB 046, the 4^th Chemical Congress of North America and the 202^nd American Chemical Society Meeting   ACS; New York: August 1991. 

6 Ward DC. Fuller W. Reich E. Proc. Natl. Acad. Sci. U.S.A.  1969,  62:  581 

7a Gudmundsson KS. Drach JC. Townsend LB. J. Org. Chem.  1997,  62:  3453 

7b Walker JAII. Liu W. Wise DS. Drach JC. Townsend LB. J. Med. Chem.  1998,  41:  1236 

8 Marakos P. Pouli N. Wise SD. Townsend LB. Synlett  1997,  561 

9 Chapman D. Hurst J. J. Chem. Soc., Perkin Trans. 1  1980,  2398 

10 Beumel OF. Smith WN. Rubalka B. Synthesis  1974,  43 

11a Cousineau TJ. Secrist JA. J. Org. Chem.  1979,  44:  4351 

11b Timpe W. Dax K. Wolf N. Weidmann H. Carbohydr. Res.  1975,  69:  51 

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.

19 Krohn K. Heidi H. Wielkens K. J. Med. Chem.  1992,  35:  511 

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.