Synthesis of Isofagomine and a New C₆ Pyrrolidine Azasugar with Potential Biological Activity

Abstract

An efficient asymmetric synthesis of isofagomine, based on a precursor containing three differentiated hydroxyl functions, is described. The side product in the key alkylation step is converted into (2S,3R,4R)-2,4-bis(hydroxymethyl)-3-hydroxypyrrolidine, a new C₆ pyrrolidine azasugar, which inhibits α-glucosidase from yeast.

asymmetric synthesis - azasugars - glycosidase inhibitors - hydroboration - ring-closing metathesis

References and Notes

1 Stütz AE. Iminosugars as Glycosidase Inhibitors   Wiley-VCH; Weinheim: 1999. 

2 Compain P. Martin OR. Iminosugars   Wiley; Chichester: 2007. 

3 Dong W. Jespersen T. Bols M. Skrydstrup T. Sierks MR. Biochemistry  1996,  35:  2788 

4 Ouchi H. Mihara Y. Takahata H. J. Org. Chem.  2005,  70:  5207 

5

[α]_D +20 (c = 1, pentane).

6 Schaus S. Brandes B. Larrow J. Tokunaga M. Hansen K. Gould A. Furrow M. Jacobsen E. J. Am. Chem. Soc.  2002,  124:  1307 

7

Chiral HPLC analysis: Chiralcel OD-H; n-hexane-EtOH, 99:1 (isocratic); T = 35 ˚C; UV (λ = 214 nm).

8

Incomplete stereoselective epoxidation [4] and incomplete regioselective epoxide opening with vinyl cuprate [4] both yielded an inseparable mixture of product and starting material.

9 For a recent related approach towards racemic isofagomine (rac-1), see: Imahori T. Ojima H. Tateyama H. Mihara Y. Takahata H. Tetrahedron Lett.  2008,  49:  265 

10 Jautelat M, Artl D, and Wieland H. inventors; Eur Patent, EP  0110245. 

11 Friedrich M. Savchenko A. Wächtler A. de Meijere A. Eur. J. Org. Chem.  2003,  11:  2138 

12

Selected Experimental Details:
O-Benzylation and Staudinger Reduction of 14: To an ice-cooled solution of 14 (12.17 g, 107.6 mmol, colorless oil) in anhyd DMF (180 mL) was added NaH (6.45 g, 60% dispersion on mineral oil, 161.3 mmol). After stirring for 30 min at 0 ˚C, benzyl bromide (25.6 mL, 214 mmol) was added at 0 ˚C and the reaction was stirred overnight (0 ˚C → r.t.). NaOH (60 mL, 2 M aq solution) was added and the mixture was stirred at 80 ˚C during 1 h. The reaction mixture was diluted with H₂O (1 L) and extracted with Et₂O (3 × 1 L). The organic phase was washed with brine (1 L) and dried (Na₂SO₄). The drying agent was filtered and the resulting clear solution was evaporated under reduced pressure. The oil was filtered over silica gel (n-hexane-EtOAc, 9:1) and after evaporation, the crude residue was dissolved in anhyd THF (570 mL). Ph₃P (56.4 g, 215.2 mmol) was added and the mixture was stirred overnight at r.t. NaOH (190 mL, 2 M aq solution) was added and the mixture was refluxed for 1 h. The mixture was diluted with H₂O (1 L) and extracted with CH₂Cl₂ (3 × 1 L). The organic phase was washed once with brine (0.6 L) and dried (MgSO₄). The drying agent was filtered and the resulting clear solution was evaporated under reduced pressure. After flash column chromatography on silica gel and purification (EtOAc-CH₂Cl₂, 8:2 + 5% Et₃N), 2-benzyloxymethylallylamine (8; 15 g, 84.63 mmol, 78%) was isolated as a colorless oil.
8: ^¹H NMR (300 MHz, CDCl₃): δ = 7.18-7.30 (m, 5 H), 5.03 (d, 2 H), 4.43 (s, 2 H), 3.97 (s, 2 H), 3.28 (s, 2 H), 1.21 (s, 2 H). ^¹³C NMR (75 MHz, CDCl₃): δ = 147.6 (C), 138.3 (C), 128.4 (CH), 127.7 (CH), 127.7 (CH), 111.3 (CH₂), 72.1 (CH₂), 72.1 (CH₂), 44.8 (CH₂). HRMS (ES): m/z [M + H]⁺ calcd for C₁₁H₁₅NO: 178.12263; found: 178.12260.
Alkylation of 2-Benzyloxymethylallylamine (8) with ( S )-Vinyloxirane [( S )-4]: 2-Benzyloxymethylallylamine (8; 5.24 g, 29.56 mmol) was mixed with H₂O (132 µL, 7.33 mmol) in a pressure tube at 0 ˚C. (S)-Vinyloxirane [(S)-4; 0.8 mL, 9.93 mmol] was slowly added at 0 ˚C and the reaction vessel was sealed, heated and stirred for 6 h at 100 ˚C. The reaction mixture was transferred to a 100-mL round-bottomed flask and dissolved in H₂O (5.8 mL), dioxane (35 mL) and NaOH (23 mL, 1 M in H₂O). Boc₂O (9.7 g, 44.44 mmol) was added at r.t. and the reaction mixture was stirred overnight at r.t. The mixture was evaporated under reduced pressure, diluted with Et₂O (500 mL), subsequently washed with H₂O (200 mL), citric acid (200 mL, 20%) and brine (200 mL). The pooled aqueous phases were extracted with Et₂O (3 × 250 mL). The organic phases were dried (MgSO₄). The drying agent was filtered and the resulting clear solution was evaporated under reduced pressure. The residue (13 g) was purified by flash column chromatography on silica gel (gradient elution: n-hexane-EtOAc, 85:15 → 6:4) to furnish 15 (2.21 g, 6.35 mmol, 64%) and 16 (558 mg, 1.61 mmol, 16%) as colorless oils.
15: [α]_D 0 (c = 1, CHCl₃). ^¹H NMR (300 MHz, CDCl₃): δ = 7.18-7.30 (m, 5 H), 5.75 (ddd, J = 5.6, 10.5, 17.1 Hz, 1 H), 5.25 (d, J = 17.1 Hz, 1 H), 5.08 (m, 2 H), 4.94 (s, 1 H), 4.42 (s, 2 H), 4.26 (m, 1 H), 3.87 (m, 4 H), 3.58 (br s, 1 H), 3.21 (m, 2 H), 1.36 (s, 9 H). ^¹³C NMR (75 MHz, CDCl₃): δ = 157.5 (C), 141.9 (C), 138.4 (CH), 138.0 (C), 128.4 (CH), 127.7 (CH), 115.7 (CH₂), 113.4 (CH₂), 80.6 (C), 72.7 (CH), 72.2 (CH₂), 71.3 (CH₂), 53.7 (CH₂), 51.5 (CH₂), 28.3 (Me). HRMS (ES): m/z [M + Na]⁺ calcd for C₂₀H₂₉NO₄: 370.19889; found: 370.19967.
16: [α]_D +9.1 (c = 1, CHCl₃). ^¹H NMR (300 MHz, CDCl₃): δ = 7.18-7.30 (m, 5 H), 5.78 (m, 1 H), 5.04-5.15 (m, 4 H), 4.43 (s, 2 H), 4.26 (m, 1 H), 3.93 (m, 2 H), 3.80 (m, 2 H), 3.70 (m, 2 H), 1.37 (s, 9 H). ^¹³C NMR (75 MHz, CDCl₃): δ = 156.3 (C), 143.0 (C), 137.9 (C), 133.9 (CH), 128.4 (CH), 127.8 (CH), 117.5 (CH₂), 113.9 (CH₂), 80.4 (C), 72.3 (CH₂), 71.7 (CH₂), 63.5 (CH₂), 62.0 (CH), 48.7 (CH₂), 28.4 (Me). HRMS (ES): m/z [M + Na]⁺ calcd for C₂₀H₂₉NO₄: 370.19889; found: 370.19897.
Ring-Closing Metathesis of 15: To a solution of 15 (1.89 g, 5.44 mmol) in anhyd and degassed CH₂Cl₂ (170 mL) was added the Grubbs second generation catalyst (150 mg, 0.177 mmol) at r.t. under an argon atmosphere and the solution was stirred for 48 h. The solvent was evaporated and purified by flash column chromatography on silica gel (n-hexane-EtOAc, 2:1; silica gel saturated with Et₃N) to furnish 9 (1.60 g, 5 mmol, 92%) as a brown solid.
9: [α]_D +52.2 (c = 1.01, CHCl₃). ^¹H NMR (300 MHz, CDCl₃): δ = 7.19-7.30 (m, 5 H), 5.84 (m, 1 H), 4.43 (s, 2 H), 4.16 (br s, 1 H), 3.93 (d, J = 18.0 Hz, 1 H), 3.89 (s, 2 H), 3.73 (d, J = 18.1 Hz, 1 H), 3.46 (br s, 2 H), 1.40 (s, 9 H). ^¹³C NMR (75 MHz, CDCl₃): δ = 155.2 (C), 137.9 (C), 136.6 (C), 128.5 (CH), 127.8 (CH), 127.8 (CH), 125.4 (CH), 80.2 (C), 72.4 (CH₂), 71.5 (CH₂), 63.7 (CH), 47.7 (CH₂), 44.0 (CH₂), 28.4 (Me). HRMS (ES): m/z [M + Na]⁺ calcd for C₁₈H₂₅NO₄: 342.16759; found: 342.16788.
Hydroboration of 18: Compound 18 (500 mg, 1.15 mmol) was dissolved in anhyd THF (2 mL) and a freshly prepared solution of thexylborane (20; [¹6] 5.77 mmol, 11.5 mL, 0.5 M in THF) was added at 0 ˚C and the reaction was stirred for 48 h (0 ˚C → r.t.). The reaction was quenched by adding 30% H₂O₂-2 M NaOH (1:1; 8 mL) at 0 ˚C and the mixture was then stirred for 2 h at 0 ˚C. The reaction was diluted with brine (100 mL) and extracted with CH₂Cl₂ (3 × 100 mL). After drying (Na₂SO₄), filtration and evaporation of the solvent, the residue was purified by flash chromatography on silica gel (n-hexane-EtOAc, 4:1), affording 19 (488 mg, 1.08 mmol, 93%) as a clear colorless oil.
19: [α]_D -5 (c = 0.97, CHCl₃). ^¹H NMR (300 MHz, CDCl₃): δ = 7.20-7.30 (m, 5 H), 4.45 (s, 2 H), 4.06 (m, 2 H), 3.53 (br s, 2 H), 3.34 (m, 2 H), 2.54 (m, 3 H), 1.76 (m, 1 H), 1.39 (s, 9 H), 0.84 (s, 9 H), 0.06 (s, 6 H). ^¹³C NMR (75 MHz, CDCl₃): δ = 154.6 (C), 138.2 (C), 128.4 (CH), 127.6 (CH), 127.5 (CH), 80.0 (C), 75.7 (CH), 73.3 (CH₂), 73.1 (CH), 69.4 (CH₂), 48.8 (CH₂), 45.1 (CH₂), 41.6 (CH), 28.4 (Me), 25.8 (Me), 18.1 (C), -4.4 (Me). HRMS (ES): m/z [M + Na]⁺ calcd for C₂₄H₄₁NO₅Si: 452.28265; found: 452.28284

13

Reported yields are isolated yields. The ratio of 15/16 was determined by RP-HPLC: 30% → 65% MeCN in H₂O in 60 min; Phenomenex Luna C18 (2), 5 µ, 250 × 4.6 mm.

14

RCM can also be accomplished by the Grubbs-Hoveyda catalyst, however, not by the Grubbs first generation catalyst.

15

Solutions of Boc-protected 17, 21, 22 and 23 consisted of rotamers complicating interpretation of NMR data.

16a Negishi E. Brown HC. Synthesis  1974,  77 

16b

Freshly prepared (according to the Aldrich Technical Bulletin AL-109) 0.5 M solution of thexylborane in THF.

17

24: [α]_D -4.0 (c = 0.72, H₂O). ^¹H NMR (700 MHz, D₂O): δ = 4.09 (app. t, J _2,3 = J _3,4 = 8.0 Hz, 1 H, H-3), 3.98 (dd, J _2,6a = 3.6 Hz, J _6a,6b = 12.6 Hz, 1 H, H-6a), 3.86 (dd, J _2,6b = 6.4 Hz, J _6a,6b = 12.6 Hz, 1 H, H-6b), 3.82 (dd, J _4,7a = 4.6 Hz, J _7a,7b = 11.6 Hz, 1 H, H-7a), 3.73 (dd, J _4,7b = 6.3 Hz, J _7a,7b = 11.6 Hz, 1 H, H-7b), 3.66 (dd, J _4,5a = 8.7 Hz, J _5a,5b = 12.2 Hz, 1 H, H-5a), 3.58 (ddd, J _2,6a = 3.6 Hz, J _2,6b = 6.4 Hz, J _2,3 = 8.0 Hz, 1 H, H-2), 3.25 (dd, J _4,5b = 9.3 Hz, J _5a,5b = 12.2 Hz, 1 H, H-5b), 2.48 (m, 1 H, H-4). ^¹³C NMR (75 MHz, D₂O): δ = 70.9 (C-3), 65.3 (C-2), 59.5 (C-7), 57.8 (C-6), 46.2 (C-4), 45.3 (C-5). IR (HATR): 3304, 2930, 2749, 1595, 1400, 1346, 1055, 1021, 956, 820, 640 cm^-¹. HRMS (ES): m/z [M + H]⁺ calcd for C₆H₁₃NO₃: 148.09681; found: 148.09671.

18a

When 19 was submitted to the same hydrogenolysis conditions, the TBDMS protecting group remained intact.

18b Ikawa T. Sajiki H. Hirota K. Tetrahedron  2004,  60:  6189 

18c Hattori K. Sajiki H. Hirota K. Tetrahedron  2001,  57:  2109 

19 Jespersen T. Bols M. Sierks MR. Skrydstrup T. Tetrahedron  1994,  50:  13449 

20

25: ^¹H NMR (700 MHz, CDCl₃): δ = 7.18-7.30 (m, 5 H, Ph), 4.46 (s, 2 H, OBn), 4.41 (dd, J = 7.4, 9.2 Hz, 1 H, H-1b), 4.27 (dd, J = 2.5, 9.2 Hz, 1 H, H-1a), 3.75 (m, 2 H, H-2, H-3), 3.62 (dd, J = 5.2, 9.0 Hz, 1 H, H-6b), 3.40 (m, 2 H, H-5a, H-6a), 3.22 (dd, J = 8.2, 11.7 Hz, 1 H, H-5b), 2.70 (br s, 1 H, OH), 2.45 (m, 1 H, H-4). ^¹³C NMR (75 MHz, CDCl₃): δ = 161.4 (C), 137.4 (C), 128.7 (CH), 128.1 (CH), 127.8 (CH), 78.9 (CH), 73.7 (CH₂), 71.5 (CH₂), 66.3 (CH₂), 64.1 (CH), 47.2 (CH), 46.7 (CH₂).

21 Zhao H. Thurkauf A. Synlett  1999,  1280 

22

The inhibition constants (K _i) were determined using four inhibitor concentrations within a limited range around the K _i value.
Inhibition of β-Glucosidase (Almond): K _i was determined at 37 ˚C using a NaH₂PO₄-Na₂HPO₄ buffer (pH 6.5; 100 mM) and 2-chloro-4-nitrophenyl-β-d-glucoside as substrate. The release of 2-chloro-4-nitrophenol was monitored continuously by measuring absorbance at λ = 405 nm. The K _i values were determined by Dixon plots.
Inhibition of α-Glucosidase (Yeast): K _i was determined at 37 ˚C using a NaH₂PO₄-Na₂HPO₄ buffer (pH 5.6; 100 mM) and 4-nitrophenyl-α-d-glucoside as substrate. The release of 4-nitrophenol was monitored by measuring absorbance at λ = 405 nm after addition of 10% Na₂CO₃ to samples of the reaction mixture at regular time intervals. The K _i values were determined by Dixon plots.
Inhibition of α-Mannosidase (Jack Bean): Reactions were performed at 37 ˚C using a HOAc-NaOAc buffer (pH 4.5; 100 mM) containing ZnCl₂ (2 mM) and 4-nitrophenyl-α-d-mannoside as substrate, and were monitored analogously to the α-glucosidase assay above.

23

No significant inhibition of the glycolysis of 4-nitrophenyl-α-d-mannoside was observed at a concentration of 1.9 mM in the presence of 24 or rac-24 (1 mM).