New Silybin Scaffold for Chemical Diversification: Synthesis of Novel 23-Phosphodiester Silybin Conjugates

Abstract

Silybin is the major component (ca. 30%) of the silymarin complex extracted from the seeds of Silybum marianum, with multiple biological activities operating at various cell levels. As an ongoing effort toward the exploitation of natural products as scaffolds for chemical diversification at readily accessible positions, we present here an efficient synthetic procedure to obtain new 23-phosphodiester silybin conjugates with different labels. A key point in our approach is the new 3,5,7,20-tetra-O-acetylsilybin-23-phosphoramidite, useful for a variety of derivatizations following a reliable and well-known chemistry. The feasibility of the procedure has been demonstrated by preparing new 23-silybin conjugates, exploiting standard phosphoramidite chemistry.

Key words

natural products - flavonolignans - silybin - phosphorylation - drugs

• References and Notes

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• 27 General Procedure for the Synthesis of Conjugates 5 (A–E) Derivative 3 (150 mg, 0.22 mmol) and the requisite compound A–E (0.21 mmol, previously dried and kept under reduced pressure, were reacted with a 0.45 M tetrazole solution in anhydrous MeCN (1.0 mL, 0.45 mmol). The reaction was left under stirring at r.t. and monitored by TLC with an eluent system n-hexane–EtOAc = 1:1 (v/v). After 1 h, a 5.5 M t-BuOOH solution in decane (100 μL) was added to the mixture and left stirring at r.t. After 30 min the reaction mixture was diluted with CHCl₃, transferred into a separatory funnel, washed three times with H₂O, concentrated under reduced pressure, and purified by flash chromatography, eluting with n-hexane–EtOAc = 7:3 (v/v), to afford pure 4a–e as a yellow-brown amorphous powder. Treatment with concd aq NH₃ in MeOH (1:1, v/v) for 1 h at r.t., led to full removal of the acetyl and 2-cyanoethyl groups. The concentrated mixture was then purified on a silica gel column, eluting with CHCl₃–MeOH containing increasing proportions of MeOH. Compounds 5a–e thus obtained were converted into the corresponding sodium salts by cation exchange on a DOWEX (Na⁺ form) resin to obtain homogeneous samples in good yield (55–72%). See the Supporting Information. Compound 5a: NMR spectra of this compound showed dramatic line broadening, diagnostic of a slow equilibrium on the NMR time scale, which could suggest a strong propensity toward aggregation in CDCl₃. ¹H NMR (500 MHz, CDCl₃, r.t., mixture of diastereomers): δ = 7.02–6.60 (6 H, complex signals), 5.85 (2 H, br s), 5.59 (1 H, dd, J = 11.9, 11.7 Hz), 5.11 (2 H, complex signals), 4.78 (1 H, br s), 4.04 (1 H, br signal), 3.95–3.63 (6 H, complex signals), 2.05 (2 H, br signal), 1.92–0.99 (26 H, complex signals), 0.85 (3 H, br s), 0.79 (3 H, d, J = 4.8 Hz), 0.74 (6 H, br s), 0.55 (3 H, s) ppm. ³¹P NMR (161.98 MHz, CDCl₃): δ = –0.83 ppm. HRMS (MALDI-TOF, negative ions): m/z calcd for C₅₂H₆₆O₁₃P: 929.4246; found: 929.4246 [M – H]^–. Compound 5b: ¹H NMR (500 MHz, CD₃OD, r.t., mixture of diastereomers): δ = 7.11–6.72 (6 H, complex signals), 5.91 (1 H, d, J = 1.5 Hz), 5.85 (1 H, m), 4.98–4.75 (2 H, complex signal), 4.41 (1 H, d, J = 11.5 Hz), 4.21–4.05 (5 H, m), 3.88–3.68 (24 H, complex signals) ppm. ³¹P NMR (161.98 MHz, CDCl₃): δ = 3.1 ppm. HRMS (MALDI-TOF, negative ions): m/z calcd for C₃₆H₄₄O₁₈P: 795.2271; found: 795.2271 [M – H]^–. Compound 5c: ¹H NMR (500 MHz, CD₃OD, r.t., mixture of diastereomers): δ = 7.79 (1 H, s), 7.11–6.72 (6 H, complex signals), 6.41 (1 H, dd, J = 6.8, 6.8 Hz), 5.91 (1 H, d, J = 1.5 Hz), 5.85 (1 H, m), 4.97–4.74 (2 H, complex signal), 4.65 (1 H, m), 4.41 (1 H, d, J = 11.5 Hz), 4.22–4.10 (4 H, m), 4.00–3.85 (5 H, complex signals), 2.44 (2 H, m), 1.99 (3 H, s) ppm. ³¹P NMR (161.98 MHz, CDCl₃): δ = 3.3 ppm. HRMS (MALDI-TOF, negative ions): m/z calcd for C₃₅H₃₄N₂O₁₇P: 785.1600; found: 785.1602 [M – H]^–. Compound 5d: ¹H NMR (500 MHz, CD₃OD, r.t., mixture of diastereomers): δ = 8.56 (1 H, s), 8.29 (1 H, s), 7.11–6.72 (6 H, complex signals), 6.05 (1 H,d, J = 6.0 Hz), 5.91 (1 H, d, J = 1.5 Hz), 5.85 (1 H, m), 4.90 (2 H, complex signals), 4.63 (1 H, m), 4.54 (1 H, m), 4.41 (1 H, d, J = 11.5 Hz), 4.35 (1 H, m), 4.20 (2 H, m), 4.21–4.05 (3 H, m), 3.88–3.68 (3 H, s) ppm. ³¹P NMR (161.98 MHz, CDCl₃): δ = 3.1 ppm. HRMS (MALDI-TOF, negative ions): m/z calcd for C₃₅H₃₃N₅O₁₆P: 810.1665; found: 810.1666 [M – H]^–. Compound 5e: ¹H NMR (500 MHz, CD₃OD, r.t., mixture of diastereomers): δ = 7.08–6.81 (6 H, complex signals), 5.95–5.89 (2 H, m), 5.07 (1 H, m), 4.97 (1 H, m), 4.52 (2 H, m), 4.24 (1 H, m), 4.06 (1 H, m), 3.95–3.68 (7 H, complex signals), 3.30 (1 H, m), 2.01–0.70 (33 H, complex signals) ppm. ³¹P NMR (161.98 MHz, CDCl₃): δ = 4.3 ppm. ESI-MS (positive ions): m/z calcd for C₄₉H₆₃O₁₆P: 938.39; found: 939.48 [MH]⁺; [MNa]⁺ = 961.37. HRMS (MALDI-TOF, negative ions): m/z calcd for C₄₉H₆₂O₁₆P: 937.3781; found: 937.3782 [M – H]^–.
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