Synthesis and Antiviral Evaluation of 2′,3′-β-C-Disubstituted Nucleoside Analogue ProTides to Test a Conformational Model of Potency Against Hepatitis C

Abstract

An analysis of nucleoside active metabolite potencies against Hepatitis C virus (HCV) versus their parent ground-state energetic conformational bias as calculated by density functional theory suggested that nucleotides with a small difference between their antipodal energies are more likely to have potent antiviral activity compared to those with larger energetic differences. This energetic conformational bias was thought to be manipulated with substitutions along the ­ribofuranose ring. From 2′-C-methyluridine, a representative nucleoside with fair anti-HCV activity, two C3′ modifications in particular (ethyne and methyl) showed contrasting antipodal biases relative to each other while originating from a common synthetic intermediate, allowing a test of reasonable extremes of the computational model with a divergent nine-step synthesis. Antiviral activity of the compounds contradicted that suggested by the model, indicating a need for further refinement with additional biostructural considerations.

Key words

nucleosides - hepatitis C - prodrugs - antiviral agents - protides - density functional theory

Publication History

Received: 02 June 2022

Accepted after revision: 19 July 2022

Accepted Manuscript online:
19 July 2022

Article published online:
27 October 2022

• References and Notes

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• 43 Procedures for the key stereoselective 3′-ketone additions: TMS-Ethynylated Intermediate 7a: A flame-dried 100-mL two-neck round-bottom flask with stir bar was charged with argon and anhydrous cerium(III) chloride (770 mg, 3.13 mmol), which was dried briefly by heating in vacuo. The powder was then suspended in anhydrous THF (5 mL) and left to stir under argon overnight. A separate oven-dried 50-mL two-neck round-bottom flask with stir bar was charged with anhydrous THF (5 mL) and trimethylsilylacetylene (453 μL, 3.23 mmol), and the colorless solution was chilled to –78 °C before being dropwise treated with n-butyllithium (1.37 mL, 3.28 mmol) as a ca. 2.4 M solution in hexanes. The reaction took on a light color; the deprotonation mixture was allowed to warm to –20 °C and stirred for 1 hour before being again cryogenically cooled and cannulated to the cerium suspension cooled to the same temperature. The heterogenous transmetalation mixture took on a dark-yellow color and was stirred for 1 hour at reduced temperature before ketone 6 (193 mg, 0.52 mmol) was cannulated as a solution in anhydrous THF (5 mL). The still heterogenous reaction was stirred at reduced temperature overnight. The next day, TLC showed partial conversion of the starting ketone to a less polar spot. The reaction was quenched with acetic acid (0.3 mL) and allowed to warm to ambient temperature before being poured into a pH 7 aqueous potassium phosphate solution. The product was extracted with ethyl acetate and washed with brine solution. The organic layer was collected, dried over sodium sulfate, filtered, and concentrated to a glass-like solid, which was brought up in DCM and purified via silica gel flash column chromatography (0–80% ethyl acetate in hexanes). The fractions of interest were pooled and concentrated to afford the desired product as a glass-like semisolid (128 mg, 80.5% yield BRSM; 67 mg, 35% starting material recovered). ¹H NMR (400 MHz, CDCl₃): δ = 8.86 (s, 1 H), 7.83 (d, J = 8.3 Hz, 1 H), 6.06 (s, 1 H), 5.68 (dd, J = 8.2, 2.3 Hz, 1 H), 4.09–4.03 (m, 2 H), 3.98 (dd, J = 6.1, 4.4 Hz, 1 H), 3.25 (s, 1 H), 2.07 (br s, 1 H), 1.40 (s, 3 H), 0.93 (s, 9 H), 0.36 (s, 3 H), 0.25 (s, 3 H), 0.19 (s, 9 H) ppm; ¹³C NMR (151 MHz, acetone-d ₆): δ = 163.5, 151.7, 141.4, 103.8, 101.3, 96.1, 93.7, 86.3, 85.3, 77.3, 63.0, 26.3, 19.1, 18.5, 0.1, –0.4, –2.1, –2.3 ppm; HRMS (ACPI): m/z [M + H]⁺ calcd for C₂₁H₃₇N₂O₆Si₂: 469.2185; found: 469.2177. Methylated Intermediate 7b: In a flame-dried 100-mL Schlenk flask with stir bar, a ca. 0.45 M solution of trichlorolanthanum bis(lithium chloride) complex (1.1 mL, 0.66 mmol) in THF was treated with ketone 6 (200 mg, 0.54 mmol) and diluted with an equivolume amount of anhydrous THF (1.1 mL). The colorless solution was stirred at ambient temperature for approximately 1 hour before being chilled to 0 °C with an ice bath and treated with MeMgCl (0.6 mL, 1.8 mmol) as a ca. 3 M solution in THF; bubbles were observed, and the reaction turned yellow. The reaction mixture was then left to stir under argon overnight. The next day, TLC of the now orange reaction mixture showed what appeared to be ca. 50% conversion of the starting material to a single more polar product. Hence, the reaction was chilled and another half-portion of MeMgCl (0.3 mL, 0.9 mmol) was added dropwise. The reaction proceeded for an additional hour before being quenched by addition of saturated aqueous ammonium chloride solution. The product mixture was diluted and extracted with ethyl acetate and washed with dilute HCl solution followed by brine solution. The organic layer was dried over magnesium sulfate, filtered, and concentrated to a yellow residue. Crude ¹H NMR analysis in d₆-acetone confirmed an almost 1:4 mixture of starting material (doublet at 7.94 ppm) to the more polar product (doublet at 7.99 ppm). The crude mixture was adsorbed onto silica gel and purified via silica gel flash column chromatography (0–10% methanol in DCM). The relevant fractions of interest were combined and concentrated for either recovered starting material (19 mg, 9.5% recovered) or desired methylated product (58 mg, 27.8% yield). ¹H NMR (500 MHz, acetone-d ₆): δ = 10.04 (s, 1 H), 7.99 (d, J = 8.3 Hz, 1 H), 5.96 (s, 1 H), 5.51 (dd, J = 8.3, 2.0 Hz, 1 H), 4.73 (s, 1 H), 4.28 (dd, J = 6.0, 4.9 Hz, 1 H), 4.12 (dd, J = 5.1, 3.9 Hz, 1 H), 4.09–3.96 (m, 2 H), 1.32 (s, 3 H), 1.25 (s, 3 H), 0.91 (s, 9 H), 0.32 (s, 3 H), 0.20 (s, 3 H) ppm; ¹³C NMR (151 MHz, acetone-d ₆): δ = 163.7, 152.1, 142.8, 100.6, 93.7, 88.0, 86.7, 80.9, 61.4, 26.2, 18.8, 18.5, 15.6, –2.0, –2.5 ppm; HRMS (ESI): m/z [M + Na]⁺ calcd for C₁₇H₃₀N₂O₆SiNa: 409.1765; found: 409.1767.