A cyclin-dependent kinase inhibitor inducing cancer cell differentiation: Biochemical identification using Xenopus egg extracts -- Rosania et al. 96 (9): 4797 Data Supplement - Supplemental Materials and Methods -- Proceedings of the National Academy of Sciences

Supplementary material for Rosania et al. (1999) Proc. Natl. Acad. Sci. USA 96(9), 4797-4802.

Supplemental Materials and Methods
2-Fluoro-6-chloro-9-isopropylpurine (1).
Anhydrous tetrahydrofuran (60.0 ml) is added to a flame-dried flask under N₂ containing 2-fluoro-6-chloropurine (0.900 g, 5.2 mmol) and triphenylphosphine (3.0 g, 10.4 mmol). To this 2-propanol (800 ml, 10.4 mmol) is added, and the mixture is then cooled to -10°C by using an ethylene glycol/dry-ice bath. After dropwise addition of diethyl azodicarboxylate (850 ml, 10.4 mmol), the mixture is warmed gradually to room temperature. After 12 h, the reaction is quenched with 500 ml of water and the solvent is removed in vacuo. The resulting yellow oil is purified by column chromatography (600 ml of SiO₂, eluted with 100% CH₂Cl₂). The resulting solid is triturated with methanol (to remove the diethyl hydrazine-N,N¢-dicarboxylate ester by-products) to yield 630 mg (57%) of 1 as a white crystalline solid. ¹H NMR (500 MHz, CDCl₃): d 1.65 (d, 6H, J = 6.8), 4.85 (m, 1H), 8.15 (s, 1H); ¹³C NMR (500 MHz, CDCl₃): d 22.3, 48.4, 130.5, 143.7, 152.3, 153.1, 156.9 (J_C-F = 870 Hz); mass spectrum (EI-) m/e 214 (M⁺); Anal. Calcd. for C₈H₈N₄FCl: C, 44.77; H, 3.76; N, 26.10; Cl, 16.52. Found: C, 44.97; H, 3.76; N, 26.09; Cl, 16.37. 2-Fluoro-6-(3-chloro-4-carboxyanilno)-9-isopropylpurine (2).
4-Amino-2-chlorobenzoic acid (76 mg, 0.44 mmol), compound 1 (95 mg, 0.44 mmol), and diisopropylethylamine (200 ml, 1.12 mmol) are combined in n-butanol in a sealed tube and heated for 12 h at 80°C. After removal of the solvent in vacuo, the crude product is resuspended in CH₂Cl₂, collected by filtration, and washed with cold 50 mM HCl, CH₂Cl₂, and ether to yield 107 mg (70%) of 2 as a white solid. ¹H NMR (500 MHz, DMSO-d₆): d 1.58 (d, 6H, J = 6.8), 4.76 (m, 1H), 7.86 (d, 1H, J = 8.4), 7.95-7.98 (m, 1H), 8.18-8.19 (m, 1H), 8.48 (s, 1H), 10.76 (s, 1H), 13.07 (s, 1H); mass spectrum (FAB⁺) m/e 350 (MH⁺); HRMS Calcd. for (C₁₅H₁₃N₅O₂)H⁺: 350.0820, Found: 350.0815. 2-(1R-Isopropyl-2-hydroxyethylamino)-6-(3-chloro-4-carboxyanilino)-9-isopropylpurine (3).
Compound 2 (1.09 g, 2.27 mmol), R-(-)-2-amino-3-methyl-1-butanol (1.17.g, 11.4 mmol), and diisopropylethylamine (2.0 ml, 11.4 mmol) are dissolved in n-butanol in a sealed tube and heated at 110°C for 18 h. After removal of the solvent in vacuo, the crude product is resuspended in CH₂Cl₂ and filtered to yield a solid which is then dissolved in 100 ml of 80:20 ddH₂O/CH₃CN. This solution is acidified to pH 2 with dilute aqueous H₃PO₄. The mixture is then cooled at -20°C for 20 min, after which the resulting precipitate is collected by filtration and washed with ethyl acetate to yield 795 mg (85%) of 3 as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): d 0.93 (d, 3H, J = 7.0), 0.95 (d, 3H, J = 7.0), 1.52 (d, 6H, J = 6.3), 1.97 (m, 1H), 3.55 (m, 2H), 3.86 (m, 1H), 4.66 (m, 1H), 6.89 (bs, 1H), 7.83 (d, 1H, J = 8.6), 7.90-8.5 (br, 2H), 8.56 (bs, 1H), 10.50 (bs, 1H); mass spectrum (FAB⁺) m/e 433 (M-); HRMS Calcd. for (C₂₀H₂₅N₆O₃Cl)H⁺: 433.1755, Found: 433.1748. 1-tert-Butyloxycarbonyl-1,8-diamino-3,6-dioxaoctane (4).
A stirred solution of 1,8-diamino-3,6-dioxaoctane (20.1 ml, 13.7 mmol) and diisopropylethylamine (8 ml, 27 mmol) in CH₂Cl₂ is cooled to 0°C. A solution of di-tert-butyldicarbonate (3.0 g, 14.0 mmol) in 100 ml of CH₂Cl₂ is added dropwise over 4 h, after which the mixture is allowed to warm up to room temperature. Upon removal of the solvent, the resulting white precipitate is purified by column chromatography (300 ml of SiO₂, eluted with 90:9:1 CH₂Cl₂/MeOH/NH₄OH) to yield 1.29 g (40%) of 4 as a colorless oil. ¹H NMR (400 MHz, CDCl₃): d 1.38 (s, 9H), 1.73 (s, 2H), 2.81-2.84 (m, 2H), 3.25-3.26 (m, 2H), 3.45-3.50 (m, 4H), 3.56 (s, 4H), 5.21 (bs, 1H); mass spectrum (FAB⁺) m/e 249 (MH)⁺; HRMS Calcd. for (C₁₁H₂₄N₂O₄)H⁺: 249.1814, Found: 249.1811. Compound 5.
To a solution of 3 (156 mg, 0.36 mmol) in 45 ml of a 1:1:1 mixture of dimethylformamide/CH₂Cl₂/1,4-dioxane at 0°C is added hydroxybenzotriazole (50.4 mg, 0.37 mmol), diisopropylcarbodiimide (59.0 ml, 0.37 mmol), diisopropylethylamine (124 ml, 0.70 mmol), 4 (165 mg, 2.01 mmol), and catalytic 4-dimethylaminopyridine. The mixture is stirred for 16 h at room temperature, after which the solvent is removed in vacuo and the crude product is purified by column chromatography (150 ml of SiO₂, eluted with 93:7 CH₂Cl₂/MeOH) to yield 280 mg (88%) of the tert-butoxycarbonyl-protected product (BOC-5) contaminated with 25% diisopropylurea by-product (as judged by ¹H NMR integration). Without further purification, BOC-5 (280 mg) is stirred at room temperature in 4 ml of CH₂Cl₂/trifluoroacetic acid/H₂O/MeS (49:49:1:1, vol/vol/vol/vol) for 12 h. The reaction mixture is concentrated in vacuo and purified by column chromatography (150 ml of SiO₂, eluted with 92:6:2 CH₂Cl₂/MeOH/NH₄OH, vol/vol/vol) to yield 206 mg (100%) of 5 as a colorless oil. ¹H NMR (500 MHz, CD₃OD): d 1.02 (m, 6H), 1.54 (m, 6H), 2.03 (m, 1H), 2.73 (t, 2H, J = 5.5), 3.49 (t, 2H, J = 5.5), 3.55 (t, 2H, J = 5.5), 3.58-3.67 (m, 6H), 3.69-3.79 (m, 2H), 4.00 (m, 1H), 4.63 (m, 1H), 7.41 (d, 2H, J = 8.5), 7.68 (bs, 1H), 7.86 (s, 1H), 8.20 (bs, 1H); mass spectrum (FAB⁺) m/e 563 (MH⁺). HRMS Calcd. for (C₂₀H₄₀N₈O₄Cl)H⁺: 563.286, Found: 563.285. Compound 6.
The procedure is identical to that used for the preparation of compound 5 except that compound 2 is used as the starting material. ¹H NMR (400 MHz, CD₃OD): d 1.61 (d, 6H, J = 6.7), 2.78-2.81 (m, 2H), 3.52-3.58 (m, 4H), 3.64-3.69 (m, 6H), 4.74-4.81 (m, 1H), 7.47-7.50 (m, 1H), 7.56-7.79 (m, 1H), 8.11 (s, 1H), 8.24 (s, 1H); mass spectrum (FAB⁺) m/e 480 (MH⁺). HRMS Calcd. for (C₂₁H₂₇N₇O₃FCl)H⁺: 480.1926, Found: 480.1904. 2-Fluoro-6-(3-chloro-5-aminoanilino)-9-isopropylpurine (7).
5-Chloro-1,3-phenylenediamine (45 mg, 0.31 mmol), compound 1 (68 mg, 0.31 mmol), and diisopropylethylamine (163 ml, 0.93 mmol) are combined in n-butanol in a sealed tube and heated for 24 h at 85°C. The solvent is removed in vacuo, dried further under high vacuum, and resuspended with the aid of Vortex and sonication in CH₂Cl₂/MeOH (95:5, vol/vol). The solid product is isolated by filtration and washed with CH₂Cl₂ (three times, 5 ml), aqueous HCl (five times, 5 ml), and CH₂Cl₂ (three times, 5 ml) followed by drying under high vacuum to yield 65 mg (65%) of 7 as a white/gray solid. ¹H NMR (500 MHz, DMSO-d₆): d 1.52 (d, 6H, J = 6.8). 3.20-3.60 (br, 2H), 4.69 (m, 1H), 6.37 (s, 1H), 7.07-7.10 (m, 2H), 8.39 (s, 1H), 10.16 (s, 1H); mass spectrum (FAB⁺) m/e 322 (MH⁺). 2-Fluoro-6-(N-methyl-3-chloro-5-dimethylaminoanilino)-9-isopropylpurine (8).
To a solution of 7 (50 mg, 0.15 mmol) in dry dimethylformamide (5 ml), NaH (103 mg, 2.6 mmol, 60% dispersion in mineral oil) is added, followed by dropwise addition of MeI (100 ml, 1.6 mmol). The mixture is stirred for 12 h at room temperature, diluted with CH₂Cl₂ (20 ml), filtered through a plug of SiO₂, and concentrated in vacuo with heating (70°C). The resulting yellow oil is purified by preparative thin-layer chromatography (20 ´ 20 mm, 0.5 mm, developed twice with 95:5 CH₂Cl₂/MeOH) to yield 20 mg (37%) of 8 as a colorless oil. ¹H NMR (500 MHz, CD₃Cl): d 1.55 (d, 6H, J = 7.0), 2.95 (s, 6H), 3.78 (s, 3H), 4.74 (m, 1H), 6.52 (t, 1H, J = 2.0), 6.62 (t, 1H, J = 2.0), 6.63 (t, 1H, J = 2.0), 7.71 (s, 1H); mass spectrum (FAB⁺) m/e 363 (MH⁺). 2-(1R-Isopropyl-2-hydroxyethylamino)-6-(N-methyl-3-chloro-5-dimethylaminoanilino)-9-isopropylpurine (9).
Compound 8 (20 mg, 0.055 mmol) and R-(-)-2-amino-3-methyl-1-butanol (200 ml, 1.80 mmol) are dissolved in n-butanol (2 ml) in a sealed tube and heated at 135-140°C for 12 h. After removal of the solvent in vacuo, the crude product is purified by preparative thin-layer chromatography (two 20 ´ 20 mm plates, 0.5 mm, eluted with 95:5 CH₂Cl₂/MeOH) to yield 15 mg (61%) of 9 as a colorless oil. ¹H NMR (500 MHz, CD₃Cl): d 0.98 (d, 6H, J = 7.0), 1.52 (dd, 6H, J = 2.0, J = 7.0), 1.95 (m, 1H), 2.94 (s, 6H), 3.64 (dd, 1H, J = 8.0, J = 10.5), 3.71 (m, 1H), 3.78 (s, 3H), 4.62 (m, 1H), 4.90 (brs, 1H), 6.52 (t, 1H, J = 2.0), 6.59 (t, 2H, J = 1.0), 6.64 (t, 1H, J = 2.0), 7.50 (s, 1H); mass spectrum (FAB⁺) m/e 446 (MH^-). HRMS Calcd. for (C₂₂H₃₂N₇OCl)H⁺: 446.2435, Found: 446.2414. Loading Purine to Solid Support.
Pierce ReactiGel 6´ (1.5 ml, no. 20259, 1,1¢-carbonyldiimadazole-activated 6% cross-linked beaded agarose, 50 mmol/ml) is transferred to a fritted vial and washed with ice-cold water (three times, 5 ml). The resin is then resuspended in 1.5 ml of aqueous K₂CO₃ (0.1 M) to which purine 5 (or 6) (38.0 mg, 67 mmol), dissolved in 100 ml of dimethylformamide, is added. The resulting mixture is rocked for 12 h at room temperature, after which time the resin is washed with PBS (five times, 5 ml), capped with ethanolamine (0.1 M) in aqueous K₂CO₃ (0.1 M) for 12 h, and washed again with PBS (five times, 5 ml). The resulting derivatized resin is stored in PBS with 0.02% NaN₃ at 4°C.