Ensitrelvir fumarate (S-217622 fumarate; Xocova) is a novel and orally bioavailable SARS-CoV-2 3CL protease inhibitor (IC50=13 nM) approval in Japan under the emergency regulatory approval system (EUA) for COVID-19 treatment. It is an antiviral drug developed by Shionogi in partnership with Hokkaido University. It became the first Japanese domestic pill to treat COVID-19, third to be regulatorally approved in Japan; in February 2022.

Physicochemical Properties

Molecular Formula	C26H21CLF3N9O6
Molecular Weight	647.949853658676
Exact Mass	647.125
Elemental Analysis	C, 48.20; H, 3.27; Cl, 5.47; F, 8.80; N, 19.46; O, 14.81
CAS #	2757470-18-9
Related CAS #	2647530-73-0;2757470-18-9 (fumarate);
PubChem CID	162623410
Appearance	White to off-white solid powder
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	12
Rotatable Bond Count	8
Heavy Atom Count	45
Complexity	1040
Defined Atom Stereocenter Count	0
SMILES	CN1C=C2C=C(C(=CC2=N1)Cl)NC3=NC(=O)N(C(=O)N3CC4=CC(=C(C=C4F)F)F)CC5=NN(C=N5)C.C(=C/C(=O)O)\C(=O)O
InChi Key	FBOCUALVLIWPNQ-WLHGVMLRSA-N
InChi Code	InChI=1S/C22H17ClF3N9O2.C4H4O4/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)245-3(6)1-2-4(7)8/h3-7,10H,8-9H2,1-2H3,(H,28,29,36)1-2H,(H,5,6)(H,7,8)/b2-1+
Chemical Name	(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 fumaric acid
Synonyms	S 217622; S217622; Xocova;S-217622
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	SARS-CoV-2 3CL protease
ln Vitro	In a cytopathic effect (cpe)-inhibition assay of SARS-CoV-2 infected VeroE6/TMPRSS2 cells, Ensitrelvir shows the EC50 values are approximately 0.4 μM for both wild-type virus and Alpha, Beta, Gamma and Delta variants. EC50 values for SARS-CoV and MERS-CoV were 0.21 and 1.4 μM respectively[1]. The antiviral activities were evaluated as per their inhibitory ability of the cytopathic effects elicited in SARS-CoV-2-infected VeroE6/TMPRSS2 cells. S-217622 exhibited similar antiviral activities against all tested SARS-CoV-2 variants, including the Omicron strain, which is responsible for the current wave of the pandemic, indicating its potential broad usability as a therapeutic agent for treating COVID-19 (half-maximal effective concentration [EC50] = 0.29–0.50 μM. Antiviral activity of S-217622 against SARS-CoV (EC50 = 0.21 μM). was also comparable to that against SARS-CoV-2, where the sequence homology of 3CLpro between SARS-CoV-2 and SARS-CoV was well-conserved. S-217622 also exhibited potent antiviral activity against MERS-CoV (EC50 = 1.4 μM), HCoV-OC43 (EC90 = 0.074 μM), and HCoV-229E (EC50 = 5.5 μM). S-217622 showed no inhibitory activity against host-cell proteases, such as caspase-2, chymotrypsin, cathepsin B/D/G/L, and thrombin at up to 100 μM, suggesting its high selectivity for coronavirus proteases. S-217622 exhibited no safety concerns in vitro in studies involving ether-a-go-go-related gene inhibition, mutagenicity/clastogenicity, and phototoxicity. [3]
ln Vivo	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]
Enzyme Assay	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.
Cell Assay	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.
Animal Protocol	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.
References	[1]. COVID-19, Influenza and RSV: Surveillance-informed prevention and treatment - Meeting report from an isirv-WHO virtual conference. Antiviral Res. 2022;197:105227. [2]. 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.
Additional Infomation	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]

Solubility Data

Solubility (In Vitro)

DMSO : ~50 mg/mL (~77.17 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.86 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. Solubility in Formulation 2: 2.5 mg/mL (3.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (3.86 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.5433 mL	7.7166 mL	15.4333 mL
	5 mM	0.3087 mL	1.5433 mL	3.0867 mL
	10 mM	0.1543 mL	0.7717 mL	1.5433 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.