Novel Oxidation of Cyclosporin A: Preparation of Cyclosporin Methyl Vinyl Ketone (Cs-MVK)

Abstract

Cyclosporin A (CsA) was converted into cyclosporin methyl vinyl ketone (Cs-MVK) by either a biocatalytic method utilizing 1-hydroxybenzotriazole-mediated laccase oxidation or by a chemical oxidation using t-butyl hydroperoxide and potassium ­periodate as co-oxidants. Cs-MVK is a novel, versatile synthetic intermediate that can be used for the preparation of many novel cyclosporin analogues possessing therapeutic potential as immunosuppressive agents.

cyclosporin A - cyclosporin methyl vinyl ketone - lac­case oxidation - allylic oxidation - immunosuppression

References and Notes:

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Biocatalytic Method: Cyclosporin A (1.0 g) and 1-hydroxybenzotriazole (500 mg) were dissolved in tert-butanol (70 mL) in a 500 mL reaction vessel equipped with a stir bar. Sodium citrate/sodium phosphate buffer (80 mM, 250 mL, pH 5.6) was added while stirring, resulting in a thick white suspension. Laccase C (1.8 g, ASA Spezialenzyme) was added as a solution in 35.5 mL of the same buffer, turning the reaction mixture slightly yellow in appearance. The reaction was mechanically stirred enough to create a vortex, open to ambient atmosphere at room temperature for a period of 20 h, after which time the reaction mixture became orange in appearance. After removing a portion of the tert-butanol via rotavapor, the orange reaction mixture was loaded onto a pre-conditioned VARIAN Bond-Elut^® C8 solid-phase extraction cartridge (60 cc, 10 g of sorbent). After a wash with water, the cyclosporin-related products were eluted using acetonitrile. The acetonitrile eluate was concentrated in vacuo, and the residue was transferred to a tared scintillation vial and dried in vacuo inside a Savant dryer to provide 913 mg of crude product as tan solids. The solids were re-dissolved in a minimal volume of acetonitrile and purified by reversed-phase semi-prep chromatography to provide 551 mg of CsA-MVK.
Chemical Method: Cyclosporin A (5 g, 4.2 mmol) was dissolved in acetone (25 mL), benzene (25 mL) and H₂O (25 mL). tert-Butyl hydroperoxide (31.25 mL of 70% aqueous solution, 258 mmol), potassium periodate (6.5 g, 28.3 mmol), and 18-crown-6 (4.38 g, 16.5 mmol) were added to the reaction mixture at room temperature. The resulting mixture was stirred vigorously at room temperature under N₂ atmosphere for 3 d. Organic solvents were removed from the reaction mixture in vacuo. The remaining mixture was poured into ice-water (1 L) and extracted twice with a mixture of EtOAc-hexanes (200 mL, 1:1). The combined extracts were stirred in a 10% sodium sulfite solution for 2 h. The organic layer was separated, dried over Na₂SO₄ and concentrated. The crude product was purified by either preparative or semi-preparative HPLC, using acetonitrile (containing 0.05% TFA)/water (containing 0.05% TFA) solvent system, to provide CsA-MVK (2.5-3.5 g, 50-70%) as light-yellow solid.
Analytical Data of 3: ^¹H NMR (300 MHz, CDCl₃): δ = 8.03 (d, J = 9.9 Hz, 1 H), 7.79 (d, J = 7.8 Hz, 1 H), 7.44 (d, J = 8.0 Hz, 1 H), 7.13 (d, J = 8.0 Hz, 1 H), 6.89 (dd, J = 16.1, 7.6 Hz, 1 H), 6.06 (d, J = 16.1 Hz, 1 H), 5.71 (dd, J = 11.0, 3.8 Hz, 1 H), 5.65 (br s, 1 H), 5.22 (dd, J = 11.5, 3.8 Hz, 1 H), 5.10 (d, J = 11.0 Hz, 2 H), 5.05 (dd, J = 15.7, 9.1 Hz, 1 H), 4.96 (dd, J = 10.1, 5.7 Hz, 1 H), 4.85 (q, J = 7.2 Hz, 1 H), 4.73 (d, J = 14.1 Hz, 1 H), 4.65 (q, J = 8.7 Hz, 1 H), 4.55 (q, J = 7.4 Hz, 1 H), 4.04 (br s, 2 H), 3.52 (s, 3 H), 3.39 (s, 3 H), 3.31 (s, 3 H), 3.20 (d, J = 13.9 Hz, 1 H), 3.12 (s, 3 H), 3.11 (s, 3 H), 2.72 (s, 3 H), 2.68 (s, 3 H), 2.54-2.34 (m, 3 H), 2.26 (s, 3 H), 2.20-1.76 (m, 11 H), 1.75-1.35 (m, 6 H), 1.32 (d, J = 7.3 Hz, 3 H), 1.26 (d, J = 7.3 Hz, 3 H), 1.10-0.81 (m, 39 H); ^¹³C NMR (90 MHz, CDCl₃): δ = 198.6, 174.7, 174.1 (2 C), 173.8, 171.7, 171.6, 171.3, 170.6, 170.5, 170.3, 170.2, 148.9, 131.8, 74.9, 59.6, 58.2, 57.8, 55.7 (2 C), 55.2, 50.6, 48.9, 48.6, 48.3, 45.3, 40.7, 39.7, 39.5, 39.2, 38.0, 36.2, 35.0, 31.7, 31.6, 31.3, 30.1 (2 C), 29.8, 29.6, 27.5, 25.3, 25.1, 24.9 (2 C), 24.6, 24.0 (4 C), 23.8, 23.6, 22.0 (2 C), 21.3, 20.8, 20.0, 18.8 (2 C), 18.6, 18.4, 16.1; MS (ESI): m/z = 1216.

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13

The murine system uses the H2 disparate inbred mouse strains: Balb/c (H2^d) and C57B1/6 (H2^b). Splenocytes of the C57B1/6 mice are γ-irradiated so as to act as stimulators of an immune response from the splenocytes from the Balb/c mice. IC₅₀ values are the concentration of test compound that inhibit ^³H-thymidine uptake by 50% relative to control cells and are determined from 7 point concentration-response curves using GraphPad software.