SARS-CoV-2 3CL protease

Ensitrelvir dose-dependently inhibits intrapulmonary replication of SARS-CoV-2 in mice[2]. The antiviral efficacy of S-217622 was evaluated in vivo in mice infected with SARS-CoV-2 Gamma strain. Five-week-old BALB/c mice were intranasally inoculated with SARS-CoV-2 Gamma strain (hCoV-19/Japan/TY7-501/2021), and S-217622 was administered orally as a 0.5% methylcellulose suspension immediately and 12 hours after infection. S-217622 treatment reduced the intrapulmonary viral titers dose-dependently. The mean viral titer was significantly lower in the S-217622 treatment groups than in the vehicle treatment group (2 mg/kg vs vehicle, p = 0.0289; 8, 16, and 32 mg/kg vs vehicle, p < 0.0001). Viral titers reached near the lower limit of quantification (1.80 – log10 50% tissue culture infectious dose [TCID50]/mL) at 16 and 32 mg/kg in the S-217622 treatment group. Although twice-daily treatment was applied in this mouse model, a once-daily treatment model could be applicable in clinical treatment because S-217622 showed a much lower clearance and longer elimination half-lives in nonrodents than in rodents. [3]

3CL Protease Inhibition Assay[3]
The 3CL protease inhibition assay was conducted in 384-well plates. The substance solution (10 mM dimethyl sulfoxide [DMSO] solution) was diluted to 250 μmol/L stepwise with a threefold dilution with DMSO. Finally, the solutions were mixed with 20 mmol/L Tris-HCl (pH 7.5) as a compound solution. Ten microliters of compound solution was added manually to each well, and then 5 μL of 16 μM substrate in inhibition buffer (2 mM EDTA, 20 mM DTT, 0.02% BSA, and 20 mM Tris-HCl, pH 7.5) was added. The reaction was initiated by adding 5 μL of 12 nM 3CL protease) in an inhibition buffer and incubated at room temperature for 3 h. The following operations were the same as those described in the Biological Screening.

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Biological Screening[3]
The compound screening assay was performed in 384-well plates. Testing compound (159 nL) at various concentrations was added to each well by an ECHO 555 dispenser. Next, 7.5 μL of 8 μM substrate (Dabcyl-KTSAVLQSGFRKME [Edans]-NH2, 3249-v.) in assay buffer (100 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 10 mM dl-dithiothreitol (DTT), 0.01% bovine serum albumin [BSA], and 20 mM Tris-HCl, pH 7.5) was dispensed using Multidrop Combi. The reaction was initiated by adding 7.5 μL of 6 or 0.6 nM 3CL protease in assay buffer and incubated at room temperature for 3 h. After incubation, the reaction was stopped by adding 45 μL of water solution containing 0.1% formic acid, 10% acetonitrile, and 0.05 μmol/L Internal Standard (IS) peptide (Dabcyl-KTSAVLeu [13C6,15N]-Q). The reactions were analyzed with MS using a RapidFire 360 high-throughput sampling robot connected to an iFunnel Agilent 6550 accurate mass quadrupole time-of-flight mass spectrometer using electrospray. Peak areas were acquired and analyzed using a RapidFire Integrator. Reaction product peak areas were acquired from m/z 499.27; IS peak areas were acquired from m/z 502.78. IC50 values were determined by plotting the compound concentration versus inhibition and fitting data with a four-parameter logistical fit.

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Human Protease Enzyme Assay[3]
Selectivity tests against a variety of host protease activity were conducted by Eurofins Panlabs Discovery Services Taiwan, Ltd., on behalf of Shionogi Co. & Ltd. as per established protocols. S-217622 was tested on a set of seven proteases (caspase-2, chymotrypsin, cathepsin B/D/G/L, and thrombin) at 100 μM.

Cellular Antiviral Activity[3]
Antiviral activity against SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-229E was assessed by monitoring the cell viability; that against HCoV-OC43 was assessed by monitoring viral RNA in a cell suspension. EC50 values were determined by plotting the compound concentration versus inhibition and fitting data with a four-parameter logistical fit. EC90 values against HCoV-OC43 were determined from the resulting dose–response curves and calculated with the two-point method.
Antiviral activities against SARS-CoV-2 were evaluated using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells (1.5 × 104/well) suspended in minimum essential medium (MEM) supplemented with heat-inactivated 2% FBS were seeded into 96-well plates with diluted compounds in each well. Cells were infected with each SARS-CoV-2 at 30–3000 TCID50/well and cultured at 37 °C with 5% CO2 for 3 days or 4 days. Cell viability was assessed using a CellTiter-Glo 2.0 assay. The CC50 was assessed in the absence of viruses after being cultured for 3 days.
Antiviral activities against SARS-CoV and MERS-CoV were evaluated at Hokkaido University using VeroE6/TMPRSS2 cells as previously reported.23 VeroE6/TMPRSS2 cells (1.5 × 104/well) suspended in 2% FBS-containing MEM were seeded into 96-well plates with diluted compounds in each well. Cells were infected with each SARS-CoV at 1000 TCID50/well or MERS-CoV 2500 TCID50/well and cultured at 37 °C with 5% CO2 for 3 days. Cell viability was assessed via (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as previously described.
Antiviral activity against HCoV-229E was evaluated using MRC-5 cells. MRC-5 cells (2.0 × 104/well) suspended in 2% FBS-containing MEM were seeded into 96-well plates and incubated at 37 °C with 5% CO2 overnight. The next day, the cells were infected with HCoV-229E at 1000 TCID50/well and incubated at 37 °C with 5% CO2 for 1 h, followed by removal of the inoculum and addition of 2% FBS-containing MEM with the diluted compounds. Cells infected with HCoV-229E were incubated at 37 °C with 5% CO2 for 3 days. Cell viability was assessed using a CellTiter-Glo 2.0 assay.
Antiviral activity against HCoV-OC43 was evaluated using MRC-5 cells. MRC-5 cells (2.0 × 104/well) suspended in 2% FBS-containing MEM were seeded into 96-well plates and incubated at 37 °C with 5% CO2 overnight. The next day, the cells were infected with HCoV-OC43 at 100 TCID50/well and incubated at 37 °C with 5% CO2 for 1 h, followed by removal of the inoculum and addition of 2% FBS-containing MEM with the diluted compounds. Cells infected with HCoV-OC43 were incubated at 37 °C with 5% CO2 for 42 h, and viral RNA was extracted from the supernatants using a Quick-RNA Viral Kit. Viral RNA was quantified via real-time PCR with specific primers and probes for HCoV-OC43 detection.

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Cellular Antiviral Activity in the Presence of Mouse Serum[3]
Antiviral activity against SARS-CoV-2 in the presence of mouse serum was assessed by monitoring cell viability. S-217622 was diluted with 3.125%, 6.25%, 12.5%, and 25% mouse serum in MEM supplemented with heat-inactivated 2% FBS. One hundred microliters of serially diluted compound solutions was added to a 96-well plate and incubated at room temperature for approximately 1 h. Each 50 μL/well of VeroE6/TMPRSS2 cells was adjusted to 3.0 × 105 cells/mL with MEM supplemented with heat-inactivated 2% FBS and dispensed on the plate. Each 50 μL/well of SARS-CoV-2 was added at 10000 TCID50/well and cultured at 37 °C with 5% CO2 for 3 days. Cell viability was assessed using a CellTiter-Glo 2.0 assay, followed by the determination of the EC50 value from the cell viability. PA-EC50 extrapolated to 100% serum was calculated by linear regression using the EC50 value of each serum concentration. PS extrapolated to 100% serum was calculated by dividing the PA-EC50 (extrapolated value of 100% mouse serum) by EC50 (in the presence of mouse serum).

In Vivo SARS-CoV-2 Infection and Treatment Studies[3]
In vivo SARS-CoV-2 infection experiments were conducted in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The animal study protocol was approved by the director of the institute based on the report of the Institutional Animal Care and Use Committee of Shionogi Research Laboratories.
Mouse in vivo SARS-CoV-2 infection studies were done at Shionogi Pharmaceutical Research Center. Five-week-old female BALB/cAJcl mice (n = 5 or 10 per group) were intranasally inoculated with SARS-CoV-2 Gamma strain (hCoV-19/Japan/TY7-501/2021) (10000 TCID50/mouse) under anesthesia. Immediately after infection, the mice were orally administered S-217622 fumaric acid (2, 8, 16, or 32 mg/kg q12h; n = 5 per group) or vehicle (0.5 w/v% methyl cellulose in aqueous solution q12h; n = 10 per group) for 1 day. Twenty-four hours postinfection, the mice were euthanized via cervical dislocation under anesthesia; their lungs were removed, and the viral titers in the lung homogenates were determined using VeroE6/TMPRSS2 cells. Viral titers are expressed as log10 TCID50/mL.

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PK Study in Infected Mice[3]

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PK experiments in infected mice were conducted in accordance with the guidelines provided by AAALAC and were approved by IACUC of Shionogi Research Laboratories.
Mouse PK studies were done at Shionogi Pharmaceutical Research Center (Osaka, Japan). BALB/cAJcl mice were intranasally inoculated with SARS-CoV-2 Gamma strain (hCoV-19/Japan/TY7-501/2021) (10000 TCID50/mouse) and orally administered with S-217622 fumaric acid (2, 8, 16, or 32 mg/kg) immediately after infection. Blood was taken at 0.5, 1, 2, 4, 6, 12, 18, and 24 h after dosing (n = 4 per group per time point), and plasma concentrations of S-217622 were determined by LC/MS/MS. LC/MS/MS analysis was performed using a Vanquish Binary Flex system equipped with TSQ Altis (Thermo Fisher Scientific). The plasma concentrations of all dosing groups in the in vivo SARS-CoV-2 infection and treatment studies were simulated by nonparametric analysis from plasma concentration data obtained in the PK study using Phoenix WinNonlin.

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Rat PK Studies[3]
The animal study protocol was approved by the director of the institute after reviewing the protocol by the Institutional Animal Care and Use Committee in terms of the 3R (Replacement/Reduction/Refinement) principles.
Rat PK studies were done at Shionogi Pharmaceutical Research Center. Eight-week-old male Sprague–Dawley rats were purchased from Charles River Laboratories. For oral administration, the dosing vehicle was dimethyl sulfoxide/0.5% methylcellulose (400 cP) = 1:4. The compound was orally administered at 1–2 μmol/5 mL/kg (n = 2) under nonfasted conditions. Blood samples (0.2 mL) were collected with 1 mL syringes containing anticoagulants (EDTA-2K and heparin) at 0.5, 1, 2, 4, 8, and 24 h after dosing. For intravenous administration, compounds were formulated as solutions in dimethyl sulfoxide/propylene glycol (1:1, v/v) and intravenously administered via the tail vein at 0.5–1.0 μmol/mL/kg (n = 2) under isoflurane anesthesia under nonfasted conditions. Blood samples (0.2 mL) were collected with 1 mL syringes containing anticoagulants (EDTA-2K and heparin) at 3, 10, 30, 60, 120, 240, and 360 min after dosing. Blood samples were centrifuged to obtain plasma samples, which were transferred to each tube and stored in a freezer until analysis. Plasma concentrations were determined by LC/MS/MS after protein precipitation with MeOH or MeCN. LC/MS/MS analysis was performed using a SCIEX Triple Quad 5500 or SCIEX API5000 or SCIEX Triple Quad 5500. PK parameters were calculated by noncompartmental analysis.

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Dog/Monkey PK Studies[3]
PK experiments in dogs and monkeys were conducted in accordance with the guidelines provided by AAALAC. The animal study protocol was approved by the director of the institute after reviewing the protocol by the Institutional Animal Care and Use Committee in terms of the 3R (Replacement/Reduction/Refinement) principles.
Dog and Monkey PK studies were done at Shionogi Aburahi Research Center. Male beagles were purchased from Marshall BioResources. Female cynomolgus monkeys were purchased from Shin Nippon Biomedical Laboratories, Ltd. or Hamri Co., Ltd. For oral administration, dosing vehicles were 0.5% methylcellulose (400 cP). The compound was orally administered at 3 mg/2 mL/kg (n = 3) under nonfasted conditions. Blood samples (0.3 mL) were collected with 1 mL syringes containing anticoagulants (EDTA-2K and heparin) at 0.25, 0.5, 1, 2, 4, 8, and 24 h after dosing. For intravenous administration, compounds were formulated as solutions in dimethyl acetamide/ethanol/20% HP-β-CD in carbonate buffer (pH 9.0) (2:3:5, by volume) and intravenously administered via a forelimb or hind limb vein at 0.1 mg/0.2 mL/kg (n = 2) under nonfasted conditions. Blood samples (0.2 mL) were collected with 1 mL syringes containing anticoagulants (EDTA-2K and heparin) at 2, 5, 15, 30, 60, 120, 240, 480, and 1440 min after dosing. Blood samples were centrifuged to obtain plasma samples, which were transferred to each tube and stored in a freezer until analysis. Plasma concentrations were determined by LC/MS/MS after protein precipitation with MeOH or MeCN. LC/MS/MS analysis was performed using a SCIEX API5000 or SCIEX Triple Quad 6500 or Triple Quad 6500+ (Sciex, Framingham, MA). PK parameters were calculated by noncompartmental analysis.

[1]. Discovery of S-217622, a Non-Covalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19. bioRxiv 2022.01.26.477782.
[2]. COVID-19, Influenza and RSV: Surveillance-informed prevention and treatment - Meeting report from an isirv-WHO virtual conference. Antiviral Res. 2022;197:105227.
[3]. Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19. J Med Chem. 2022 May 12;65(9):6499-6512.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.[3]

C22H17CLF3N9O2

531.884

531.11

C, 49.68; H, 3.22; Cl, 6.66; F, 10.72; N, 23.70; O, 6.02

2647530-73-0

2647530-73-0;2757470-18-9 (fumarate);

162533924

White to light yellow solid powder

2.5

114Ų

FC1=CC(CN(C(N2CC3=NN(C)C=N3)=O)/C(NC2=O)=N/C4=CC5=CN(C)N=C5C=C4Cl)=C(F)C=C1F

QMPBBNUOBOFBFS-UHFFFAOYSA-N

InChI=1S/C22H17ClF3N9O2/c1-32-7-12-4-18(13(23)5-17(12)30-32)28-20-29-21(36)35(9-19-27-10-33(2)31-19)22(37)34(20)8-11-3-15(25)16(26)6-14(11)24/h3-7,10H,8-9H2,1-2H3,(H,28,29,36)

(E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-methyl-1H-1,2,4-triazol-3-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazinane-2,4-dione

Ensitrelvir; S-217622; S 217622; S217622; Xocova; S-217622; Ensitrelvir [INN]; PX665RAA3H; Ensitrelvir (S-217622); (E)-6-((6-chloro-2-methyl-2H-indazol-5-yl)imino)-3-((1-methyl-1H-1,2,4-triazol-3-yl)methyl)-1-(2,4,5-trifluorobenzyl)-1,3,5-triazinane-2,4-dione; 1,3,5-Triazine-2,4(1H,3H)-dione, 6-[(6-chloro-2-methyl-2H-indazol-5-yl)imino]dihydro-3-[(1-methyl-1H-1,2,4-triazol-3-yl)methyl]-1-[(2,4,5-trifluorophenyl)methyl]-, (6E)-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~50 mg/mL (~94.01 mM）

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (4.70 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)