The First N-Glycosylated Indoxyls and Their Application to the Synthesis of Indirubin-N-glycosides (Purple Sugars)

Abstract

The first indirubin-N-glycosides (purple sugars), containing a sugar moiety located at the amine-type nitrogen atom, are prepared by condensation of hitherto unknown indoxyl-N-glycosides with isatines.

Key words

carbohydrates - heterocycles - indirubin - indol

References and Notes

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13

General Procedure for the Synthesis of N-Glycosylated Indoxyl-3-acetates Silver acetate (6.0 equiv) was added to a stirred solution of 3-iodoindolylrhamnoside in glacial acetic acid. The resulting suspension was stirred at 80 ˚C for 4 h. After the reaction was completed, the solution was allowed to cool to r.t., and ice water was subsequently added. The suspension was filtered, and the filtrate was extracted with EtOAc (3×). The combined organic layers were washed with an aq solution of NaHCO₃ and H₂O and dried over Na₂SO₄. The solution was filtered and the solvent of the filtrate was removed under reduced pressure. The residue was purified by column chromatography.
3-Acetoxy-1-(2′,3′,4′-tri- O -benzyl-β- l -rhamnopyranosyl) indole (8β)
Starting with 6β (2.30 g, 3.5 mmol), AcOH (20 mL), and AgOAc (3.50 g, 21.0 mmol), 8β was isolated after column chromatography (heptanes-EtOAc = 10:1 → 6:1) as colorless crystals (1.23 g, 60%), mp 125-126 ˚C; [α]_D ^²²
-24.24 (c 1.13, CHCl₃); R _f  = 0.36 (heptanes-EtOAc = 3:1). ^¹H NMR (250 MHz, CDCl₃): δ = 7.66 (s, 1 H, H-2); 7.54 (m, ^³ J = 7.6 Hz, 1 H, Hetar), 7.39-7.10 (m, 18 H, Hetar, Ph), 5.55 (d, ^³ J _{1 ′ ,2 ′} = 0.9 Hz, 1 H, H-1′), 5.01, 4.73 (2 d, ^² J _Ha,Hb = 10.9 Hz, 2 H, CH ₂Ph), 4.68 (s, 2 H, CH ₂Ph), 4.41, 4.26 (2d, ^² J _Ha,Hb = 11.1 Hz, 2 H, CH ₂Ph), 4.00 (‘dd’, ^³ J _{1 ′ ,2 ′} = 0.9 Hz, ^³ J _{2 ′ ,3 ′} = 2.4 Hz, 1 H, H-2′), 3.80-3.75 (m, ^³ J _{2 ′ ,3 ′} = 2.3 Hz, ^³ J _{4 ′ ,5 ′} = 9.3 Hz, 2 H, H-3′, H-4′), 3.67-3.55 (m, ^³ J _{5 ′ ,6 ′} = 6.1 Hz, ^³ J _{4 ′ ,5 ′} = 9.3 Hz, 1 H, H-5′), 2.36 [s, 3 H, C(O)CH₃], 1.42 (d, ^³ J _{5 ′ ,6 ′}= 6.1 Hz, 1 H, H-6′). ^¹³C NMR (75 MHz, CDCl₃): δ = 168.4 [C(O)CH₃], 138.3, 138.0, 137.7 (C_qPh), 132.2, 130.3 (C_q-Hetar), 128.4, 128.1, 128.0, 127.7, 127.5 (CHPh), 122.4 (CH-Hetar), 120.9 (C_q-Hetar), 120.0, 117.8 (2 s, 2 × CH-Hetar), 116.0 (C-2), 109.5 (CH-Hetar), 83.1, 82.9, 79.6, 76.0, (C-1′, C-2′, C-3′, C-4′), 75.5 (CH₂Ph), 75.0 (C-5′), 74.6, 72.3 (CH₂Ph), 20.9 [C(O)CH₃], 18.1
(C-6′). MS (EI, 70 eV): m/z (%) = 591 (82) [M⁺], 549 (20) [benzylated indoxyl-N-rhamnoside⁺], 133 (16) [indoxyl⁺], 91 (100) [Bn⁺]. HRMS (EI, 70 eV): m/z calcd for C₃₇H₃₇NO₆ [M⁺]: 591.26154; found: 591.26148. In addition, 10β (0.41 g, 21%) was isolated as a byproduct, R _f  = 0.23 (heptane-EtOAc = 3:1).

16

Libnow S.; Hein, M.; Flemming, A.; Köckerling, M.; Langer, P., unpublished results.

18

General Procedure for the Synthesis of Indirubin- N -glycosides To a stirred dioxane solution of N-glycosylated indoxyl-3-acetate was added an aq solution of Na₂CO₃. The reaction mixture was stirred at 80 ˚C. After the reaction was completed, a fluorescent spot was visible on the TLC plate. Ice-water was added, and the mixture was extracted by degassed EtOAc. The combined organic layers were dried over Na₂SO₄, filtered, and the solvent of the filtrate was removed under reduced pressure. The residue was dissolved in degassed benzene and isatine (2.0 equiv), and a catalytic amount of piperidine was added. The solution was stirred at 80 ˚C for 2 h. After completion of the reaction, the mixture was diluted with toluene. The solvent was removed under reduced pressure, and the residue was purified by column and thin-layer chromatography (Figure  [³] ).
N -(2′′,3′′,4′′-Tri- O -benzyl-β- l -rhamnopyranosyl) indirubin 12β
Starting with 8β (250 mg, 0.42 mmol), dioxane (5 mL), and Na₂SO₃ (160 mg, 1.27 mmol, dissolved in 5 mL of H₂O) and isatine (124 mg, 0.84 mmol), 12β was isolated as a purple solid (95 mg, 33%). The purification was carried out by column chromatography (heptanes-EtOAc = 9:1 → 4:1) and TLC (heptanes-EtOAc = 3.5:1 → 1:1), mp 93-95 ˚C (heptanes-EtOAc); R _f  = 0.37 (heptanes-EtOAc = 3:1). ^¹H NMR (300 MHz, C₆D₆): δ = 8.78 (dd, ⁴ J _{4 ′ ,6 ′} = 1.2 Hz, ^³ J _4,5 = 7.7 Hz, 1 H, H-4′), 8.36 (d, ^³ J _6,7 = 8.4 Hz, 1 H, H-7), 7.82 (s, 1 H, NH), 7.60 (dd, ⁴ J _4,6 = 1.2 Hz, ^³ J _4,5 = 7.4 Hz,
1 H, H-4), 7.52 (m, ^³ J = 7.7 Hz, 2 H, Hetar), 7.39-6.84 (m, 15 H, Hetar), 6.63 (‘t’, ^³ J = 7.3 Hz, 1 H, Hetar), 6.28 (‘d’, ^³ J _{6 ′ ,7 ′} = 7.4 Hz, 1 H, H-7′), 5.65 (d, ^³ J _{2 ′′ ,3 ′′} = 2.2 Hz, 1 H,
H-2′′), 5.56 (s, 1 H, H-1′′), 5.07, 4.59 (2 d, ^² J _Ha,Hb = 11.4 Hz, 2 H, CH₂Ph), 4.87, 4.62 (2 d, ^² J _Ha,Hb = 10.9 Hz, 2 H, CH₂Ph), 4.86, 4.79 (2d, ^² J _Ha,Hb = 11.9 Hz, 2 H, CH₂Ph), 3.95 (dd, ^³ J _{2 ′′ ,3 ′′} = 2.4 Hz, ^³ J _{3 ′′ ,4 ′′} = 9.5 Hz, 1 H, H-3′′), 3.87 (‘t’, ^³ J _{3 ′′ ,4 ′′} = ^³ J _{4 ′′ ,5 ′′} = 9.2 Hz, 1 H, H-4′′), 3.31 (dq, ^³ J _{5 ′′ ,6 ′′} = 6.2 Hz, ^³ J _{4 ′′ ,5 ′′} = 8.8 Hz, 1 H, H-5′′), 1.23 (d, ^³ J _{5 ′′ ,6 ′′} = 6.2 Hz, 3 H, H-6′′). ^¹³C NMR (75 MHz, C₆D₆): δ = 187.4 (C-3), 169.2, 152.2, 144.0, 140.1 (4 s, C-2, C-2′, C-7a, C-7a′), 139.3, 139.1, 138.8 (3 s, 3 × C _qPh), 135.5 (C-6), 129.9 (C-6′), 129.1, 128.6, 128.6, 128.4, 128.4, 128.4, 128.3, 128.3, 128.3, 128.1, 128.1, 128.1, 127.9, 127.8, 127.7 (15 s, 15 × CHPh), 126.9 (C-4′), 123.9 (C-4), 123.6, 122.6 (C-3a, C-3a′), 122.9 (C-5), 121.9 (C-5′), 120.4 (C-7), 112.0 (C-3′), 109.3 (C-7′), 89.9 (C-1′′), 83.9 (C-3′′), 79.8 (C-4′′), 78.3
(C-2′′), 75.4 (C-5′′), 75.6, 75.3, 71.9 (3 s, 3 × CH₂Ph), 18.2 (C-6′′). MS (EI, 70eV): m/z (%) = 678 (17) [M⁺], 548 (9) [benzylated N-rhamnosyl indolone⁺], 262 (83) [indirubin⁺], 91 (100) [Bn⁺]. HRMS (EI, 70 eV): m/z calcd for C₄₃H₃₈N₂O₆ [M⁺]: 678.27244; found: 678.27242.

22

Synthesis of N -β- l -Rhamnopyranosylindirubin (14β) To a cooled (-78 ˚C) CH₂Cl₂ solution (8 mL) of 12β (90 mg, 0.133 mmol) was added BBr₃ (1 M solution in CH₂Cl₂, 2.0 mmol). After stirring for 3.5 h at -78 ˚C, an aq solution of NaHCO₃ was added at -78 ˚C. The mixture was allowed to warm to 20 ˚C and was extracted with EtOAc. The combined organic layers were dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (CHCl₃-EtOH = 20:1 → 5:1) to give 14β (36 mg, 67%) as a purple solid, mp 148-151 ˚C; R _f  = 0.39 (CHCl₃-EtOH = 5:1). ^¹H NMR (300 MHz, DMSO-d ₆): δ = 10.70 (s, 1 H, NH), 8.33 (d, ^³ J _{4 ′ ,5 ′} = 8.1 Hz, 1 H, H-4′), 8.20 (d, ^³ J _6,7 = 8.3 Hz, 1 H, H-7), 7.58 (d, ^³ J _4,5 = 7.6 Hz, 1 H, H-4), 7.53 (‘dt’, ⁴ J _4,6 = 1.4 Hz, ^³ J _5,6 = ^³ J _6,7 = 7.9 Hz, 1 H, H-6), 7.22 (‘dt’, ⁴ J _{4 ′ ,6 ′} = 1.2 Hz, ^³ J _{5 ′ ,6 ′} = ^³ J _{6 ′ ,7 ′} = 7.6 Hz, 1 H, H-6′), 7.08 (t, ^³ J _4,5 = ^³ J _5,6 = 7.5 Hz, 1 H, H-5), 6.90 (‘t’, ^³ J _{4 ′ ,5 ′} = ^³ J _{5 ′ ,6 ′}= 7.6 Hz, 1 H, H-5′), 6.84 (d, ^³ J _{6 ′ ,7 ′} = 7.7 Hz, 1 H, H-7′), 5.49 (s, H-1′′), 5.23 (d, ^³ J _x,OH = 5.0 Hz, 1 H, OH), 4.83 (s, 1 H, OH), 4.75 [s(br), 2 H, H-2′′, OH], 3.50-3.25 (m, 3 H, H-3′′, H-4′′, H-5′′), 1.17 (d, ^³ J _{5 ′′ ,6 ′′} = 5.1 Hz, 3 H, H-6′′). ^¹³C NMR (75 MHz, DMSO-d ₆): δ = 187.6 (C-3), 168.1 (C-2′), 153.0 (C-7a), 142.4 (C-2), 141.6 (C-7a′), 135.7 (C-6), 130.3 (C-6′), 125.6 (C-4′), 123.3 (C-4), 122.7, 121.5 (C-3a, C-3a′), 122.5 (C-5), 120.7 (C-5′), 120.5 (C-7), 112.8 (C-3′), 109.5 (C-7′), 89.6 (C-1′′), 75.5, 73.7, 71.8 (C-3′′, C-4′′, C-5′′), 71.4 (C-2′′), 18.3 (C-6′′). MS (EI, 70eV): m/z (%) = 408 (3) [M⁺], 262 (100) [indirubin⁺]. HRMS (EI, 70 eV): m/z calcd for C₂₂H₂₀N₂O₆ [M⁺]: 408.13159; found: 408.13156.