Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication

Abstract Zinc pyrithione (1a), together with its analogues 1b–h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC50=1.88 ± 0.49 µM) and PLPro (IC50=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b–h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.


General information
Ligand pyrithione a, starting materials and other reagents as well as solvents for the synthesis of ligands b-h, complexes 1a-h and 2a, were purchased from commercial suppliers (Fluorochem, Alfa Aesar, Riedel-de-Haën, Merck) and used as received without further purification.
The progress of the reaction was monitored by thin layer chromatography using pre-coated TLC sheets ALUGRAM® SIL G/UV254 (Macherey-Nagel) visualised with UV lamp (254 nm). The column chromatography of the ligands was carried out on Merck silica gel 60 (35-70 mm). 1 H NMR spectra for compound characterization were acquired on Bruker Avance III 500 spectrometer at room temperature at 500 MHz. 1 H NMR stability spectra were obtained on NMR Bruker AvanceNeo 600 MHz spectrometer at room temperature at 600 MHz. All chemical shifts (δ) in the spectra of the complexes are referenced to the residual peaks of deuterated solvent (CD3)2SO, CDCl3 and D2O at 2.50 (referenced to the central line of a quintet), 7.26 and 4.79 ppm, respectively and are reported in ppm. Coupling constants (J) are reported in Hz. Spectra were processed in MestReNova 11.0.4. The multiplicity of the signals is abbreviated as ssinglet, ddoublet, ttriplet and mmultiplet. IR spectra were obtained on a Bruker FTIR Alpha Platinum ATR spectrometer. High resolution mass spectrometry was performed on an Agilent 6224 Accurate Mass TOF LC/MS system. Elemental analysis (C, H, N) was carried out on a PerkinElmer 2400 II instrument. UV-vis spectroscopy was performed on a PerkinElmer LAMBDA 750 UV/Vis/near-IR spectrophotometer. For X-ray structural analysis, single crystals were surrounded with silicon grease, mounted onto the tip of glass fibres and transferred to the goniometer head in the liquid nitrogen cryostream (150(2) K). Data were collected on a SuperNova diffractometer equipped with Atlas detector using CrysAlis software with monochromated Mo Kα (0.71073 Å). 1 The initial structural models were obtained via direct methods using the Olex2 graphical user interface 2 implemented in SHELXT. A full-matrix least-squares refinement on F 2 magnitudes with anisotropic displacement parameters for all non-hydrogen atoms using Olex2 or SHELXL-2018/3 was employed. 2,3 All non-hydrogen atoms were refined anisotropically, while the hydrogen atoms were placed at calculated positions and further treated as riding on their parent atoms. Figures depicting the structures were prepared with Mercury. 4 The crystal structures have been submitted to the CCDC and have been allocated the deposition numbers 2143703-2143706. These data are provided free of charge by The Cambridge Crystallographic Data Centre. 5 Synthetic fluorogenic substrates benzyloxycarbonyl-Leu-Arg-7-amino-4-methylcoumarin (Z-LR-AMC; Cat. No. 4034611) and benzyloxycarbonyl-Arg-Leu-Arg-Gly-Gly-7-amino-4-methylcoumarin (Z-RLRGG-AMC; Cat. No. 4027158) were purchased from Bachem. Ni-affinity column HisTrap was purchased from GE Healthcare.
OD600 measurements were obtained with Varian Cary 50 Bio spectrophotometer (Agilent). Protein elution was performed on ÄKTA FPLC system (GE Healthcare). Enzymatic kinetic measurements were performed on a PerkinElmer LS55 fluorescence spectrometer with PTP-1 Fluorescence Peltier System and Peltier Controlled Fluid Circulator (PCB1500).

Synthesis and Characterization
Ligands b-g were synthesized as previously reported. [6][7][8] Newly prepared ligand h was synthesized according to the same procedure as ligands b-e.
2-Bromo-3-methoxypiridine N-oxide.The starting material 2-bromo-3-methoxypyridine (2.659 mmol, 1 mol. equiv.) was dissolved in dichloromethane (30 mL) and m-chloroperoxybenzoic acid (2 mol. equiv., 70% purity) was added to the solution and stirred over the night at room temperature. Next day, reaction mixture was first washed with Na2S2O3 aqueous solution (0.5 M, 1 x 50 mL; water phase WP1) and then with saturated NaHCO3 aqueous solution (1 x 25 mL; WP2), obtaining organic phase OP1. WP1 was further extracted with dichloromethane (1 x 50 mL), which was consequently washed with saturated NaHCO3 aqueous solution (1 x 50 mL). The latter water phase was then extracted with dichloromethane (2 x 50 mL), obtaining OP2. WP2 was extracted with dichloromethane (2 x 25 mL), obtaining organic phase OP3. OP1-3 were joined together, dried over Na2SO4, filtered, dichloromethane was evaporated and the oily residue was purified by column chromatography (stationary phase: silica gel, mobile phase: 2% MeOH/DCOM). After the evaporation of the selected fractions white solid was obtained.  (100 mg) was dissolved in saturated NaSH(aq) solution (10 mL) together, followed by the addition of distilled water (10 mL). Orange solution was stirred overnight at the room temperature. Next day the solution was acidified with HCl(aq) (4 M) and extracted with CHCl3 (2 x 30 mL). The combined organic layers were dried over Na2SO4 and filtered followed by solvent evaporation. Yellow solid was triturated with acetone (5 mL), by-product S8 was filtered off and the mother liquor was evaporated. To ensure required purity of the ligand for the synthesis of the zinc complex, ligand h was purified by column chromatography (stationary phase: SiO2; mobile phase: hexane/ethyl acetate=7/3). To the selected fractions mobile phase was evaporated and yellow solid was dried over night at 45 ºC. Zinc complexes 1a-h have been prepared according to the following modified procedure. 9 Appropriate ligand a-h (0.786 mmol, 2 mol. equiv.) was dissolved in methanol (10 mL) to which 1M NaOH aqueous solution was added dropwise to obtain pH ~ 8. Then, aqueous solution (10 mL) of Zn(CH3COO)2·2H2O (0.393 mmol, 1 mol. equiv.) was added to the solution of the ligand upon which white precipitate appeared immediately. Suspension was further stirred for 1 h at room temperature. White solid was filtered off under reduced pressure and first washed with methanol (10 mL) to eliminate a by-product CH3COONa and additionally with diethyl ether (10 mL). Obtained zinc complexes 1a-h were left to dry overnight at 45 ºC.

Crystal structures
1a* 1c** 1d 1f Supplementary Figure 1: Crystal structure of complexes 1a, 1c, 1d and 1f. Ellipsoids are drawn at the 35% probability level. *As the structure report for 1a dates back to 1977, 10 we decided to re-measure data on a crystal sample of this compound to obtain high-quality data. **The reported structure is an ethanol solvate containing a heavily disordered solvent molecule. Crystals of 1c were obtained by liquid diffusion of hexane into a chloroform solution. Single crystal X-ray diffraction shows the same structure even in the absence of ethanol. The coordinates of solvent molecules could not be reliably determined. Utilization of the solvent mask function in Olex2 (equivalent to Squeeze function from ShelX) shows the presence of a 41Å 3 void containing 8 ecorresponding most likely to approx. 1 water molecule per void. Figure 2: Crystal structure of complexes 1a and 1d (together with zoom of trigonal bipyramidal and tetrahedral coordination, respectively). Ellipsoids are drawn at the 35% probability level.  (6) 16.8962 (9) 13.5512 (6) 16.2539 (9)  The checkcif report contains B/C-type alerts due to residual peaks of electron density (Q1 2.58, Q2 2.56). These peaks cannot be refined by insertion of for example solvate water molecules. The absence of solvate water molecules is confirmed by CHN elemental analysis. The anomalies are attributed to lower quality of the single crystal. Despite that, all bond lengths and angles are within the value ranges typical for zinc complexes. At present this is the best data we were able to obtain.

UV-vis and NMR stability
Supplementary Figure 11: UV-vis stability spectra of ligand pyrithione a in 1% DMSO/acetate buffer.