Diastereomeric Resolution Yields Highly Potent Inhibitor of SARS-CoV-2 Main Protease

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic. The main protease (Mpro, 3CLpro) of SARS-CoV-2 is a key enzyme that processes polyproteins translated from the viral RNA. Mpro is therefore an attractive target for the design of inhibitors that block viral replication. We report the diastereomeric resolution of the previously designed SARS-CoV-2 Mpro α-ketoamide inhibitor 13b. The pure (S,S,S)-diastereomer, 13b-K, displays an IC50 of 120 nM against the Mpro and EC50 values of 0.8–3.4 μM for antiviral activity in different cell types. Crystal structures have been elucidated for the Mpro complexes with each of the major diastereomers, the active (S,S,S)-13b (13b-K), and the nearly inactive (R,S,S)-13b (13b-H); results for the latter reveal a novel binding mode. Pharmacokinetic studies show good levels of 13b-K after inhalative as well as after peroral administration. The active inhibitor (13b-K) is a promising candidate for further development as an antiviral treatment for COVID-19.


List of Contents:
1. Figure S1: 1 H NMR spectrum of compound 11 (13b) -S3 2. Figure S2: Single-crystal X-ray diffraction data for 12 (13b-H) -S4 3. Figure S3: Dose-response curves from the FRET-based assay -S5 4. Figure S4: X-ray co-crystal structures of 13b-K and 13b-H in the substrate-binding cleft of the M pro -S6 5. Figure S5: Levels of 13b-K in urine and BALF -S7 6. Scheme S1: Alternative synthetic sequence towards 6 -S8 7. Associated synthetic procedures -S9 8. Scheme S2: A stereo-controlled approach to analogue 16 -S12 9. Figure S6: SFC purity trace for 12 (13b-H) -S13 10. Figure S7: SFC purity trace for 13 (13b-K) -S14 11. Table S1: Diffraction data and model refinement statistics -S15 12. References -S16 S3 Figure S1. 1 H NMR spectrum of compound 11 (13b). Inset, chemical structure of compound 11 (13b). Highlighted regions indicate where two sets of peaks are observed that correspond to a single proton. Assignment of these peaks is indicated by the numbering. Figure S2. Single-crystal X-ray diffraction data for compound 12 (13b-H). Figure S3. Dose-response curves from the FRET-based cleavage assay, for 13b-K (A), 13b-H (B) and an equimolar mixture of 13b-K and 13b-H (C). Figure S4. The crystal structures of 13b-K (A) and 13b-H (B) show the substrate-binding cleft of the M pro . The same orientation of the view was chosen in both cases (A and B). Panel (C) shows the surface of pocket S2 in gold, and the cyclopropyl (P2) perfectly fits inside, in the case of 13b-K. Additional representation of the carbon atoms of the cyclopropyl (P2) as spheres (depicted in red) representing the Van der Waal radii of the carbon atoms of the cyclopropyl.

S6
The carbon atoms of the compounds are depicted in yellow and those of the protein in cyan, with the exception of the Ser1 of the other monomer (green). Blue symbols (S1, S2) indicate the canonical binding pockets for moieties P1-P4 (red symbols) of the inhibitors. The 2Fo-Fc map carved around the inhibitor is depicted as a blue mesh (contoured at 1.0). Polar contacts are represented as red dashed lines.

Associated synthetic procedures (R)-3-Cyclopropyl-2-hydroxypropanoic acid (1)
D-Cyclopropylalanine (20.50 g, 158.7 mmol, 1.00 eq) was dissolved in 1 M sulfuric acid (158.7 mL, 158.7 mmol, 1.00 eq) and chilled in a NaCl/ice cooling bath. A solution of sodium nitrite (87.61 g, 1.270 mol, 8.00 eq) in water (200 mL) was added in a manner that the internal temperature stays around −10°C. The reaction mixture was allowed to warm to room temperature and stirred for further 14 hours. The mixture was extracted with MTBE (3 x 300 mL) and the combined organic extracts were dried over Na2SO4. Evaporation of the solvent afforded the title compound (19.68 g, assumed quantitative), which was used without any further purification. All spectroscopic data matched those previously reported.

Methyl (R)-3-cyclopropyl-2-((triisopropylsilyl)oxy)propanoate
TIPSCl (3.018 g, 15.65 mmol, 1.12 eq) was added under an inert atmosphere to NMI (3.00 mL, 37.6 mmol, 2.69 eq), followed by addition of methyl (R)-3-cyclopropyl-2-hydroxypropanoate (2) (2.015 g, 13.98 mmol, 1.00 eq). The biphasic reaction mixture and was stirred vigorously with a SmCo stirring bar to ensure a microemulsion. After a reaction time of 14 hours and complete consumption of the starting material by TLC, methanol (1 mL) was added and the reaction was stirred vigorously for further 15 minutes. The crude mixture was partitioned between pentane (150 mL) and H2O (75 mL), and the organic phase was separated. The aqueous component was extracted pentane (75 mL), and the combined organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. Finally, MeOTIPS was removed on the rotary evaporator at 50°C and a final pressure of 2 mbar to provide the title compound as a colorless oil (4.199 g, quantitative), which was used in the next step without further purification. 1

(R)-3-Cyclopropyl-2-((triisopropylsilyl)oxy)propanoic acid (14)
Methyl (R)-3-cyclopropyl-2-((triisopropylsilyl)oxy)propanoate (4.190 g, 13.94 mmol, 1.00 eq) was dissolved in THF (20 mL), diluted with methanol/water(1:1, 40 mL), and lithium hydroxide (617.7 mg, 25.79 mmol, 1.85 eq) was added. After stirring the emulsion vigorously for 8 hours until complete consumption of the starting material by TLC, most of the THF and methanol was removed at ambient temperature under reduced pressure, and the resulting mixture was partitioned between MTBE (100 mL) and H2O (250 mL). After separation of the phases, the aqueous layer was adjusted to pH = 2 with 2 M HCl, extracted with petrol ether (2 x 250 mL) and the combined organic phases were dried over anhydrous sodium sulfate. Removal of all volatiles afforded the title compound as a colorless oil (3.657 g, 12.77 mmol, 91% over two steps), which was used directly in the next step without further purification. 1