ONO-5334 is a potent, selective and orally bioactive cathepsin K inhibitor (antagonist) with potency values of 0.10 nM, 0.049 nM and 0.85 nM for human, rabbit and rat cathepsin K respectively. ONO-5334 is a potent antiviral compound with activity against SAR-COV-2 virus with EC50 of 500 nM. ONO-5334 may be used to be used in osteoporosis and COVID-19 related research.

Physicochemical Properties

Molecular Formula	C21H34N4O4S
Molecular Weight	438.59
Exact Mass	438.23
CAS #	868273-90-9
PubChem CID	11582982
Appearance	Off-white to light yellow solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.644
LogP	1.34
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	6
Heavy Atom Count	30
Complexity	657
Defined Atom Stereocenter Count	2
SMILES	C[C@@H]1CS/C(=N\NC(=O)C(=O)[C@H](C2CCOCC2)NC(=O)C3CCCCCC3)/N1C
InChi Key	BTZCSXIUAFVRTE-CHGLIHOBSA-N
InChi Code	InChI=1S/C21H34N4O4S/c1-14-13-30-21(25(14)2)24-23-20(28)18(26)17(15-9-11-29-12-10-15)22-19(27)16-7-5-3-4-6-8-16/h14-17H,3-13H2,1-2H3,(H,22,27)(H,23,28)/b24-21-/t14-,17+/m1/s1
Chemical Name	N-[(1S)-3-[(2Z)-2-[(4R)-3,4-dimethyl-1,3-thiazolidin-2-ylidene]hydrazinyl]-1-(oxan-4-yl)-2,3-dioxopropyl]cycloheptanecarboxamide
Synonyms	ONO-5334 ONO5334 ONO 5334
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
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

ln Vitro	Ki values of 0.83 nM, 1.7 nM, 32 nM, 82 nM, and 69 nM for ONO-5334 indicate that it inhibits human cathepsin S, human cathepsin L, human cathepsin B, and porcine calpain I and II, respectively. [1]. ONO-5334 (0.1–1 μM; 24 hours) prevents bone resorption caused by human osteoclasts. It efficiently and dose-dependently lowers the release of CTX from bone slices mediated by osteoclasts [1]. By being designed to capture numerous cycles of replication, ONO-5334 (0-10 μM; 16 hours pre-treatment) suppresses antiviral activity in Vero E6 cells in a noticeable dose-dependent manner, demonstrating viability at 0.5 μM[2]/cell EC50 value detection [2].
ln Vivo	ONO-5334 (oral; 0.12–15 mg/kg; single dose) significantly (96% reduction) at 15 mg/kg reduces PTHrP-induced increases in plasma CTX levels in TPTX rats. ONO-5334 at doses of 3 mg/kg or 30 mg/kg (oral; 0.3–30 mg/kg; for 7 days) significantly reduces CTX (a marker of bone resorption) concentrations. On day 7, ONO-5334 at 3 mg/kg and 30 mg/kg reduced serum CTX concentrations by 62% and 79%, respectively [1].
Cell Assay	Cell Viability Assay[2] Cell Types: Vero E6 Cell Tested Concentrations: 0.001 μM, 0.003 μM, 0.1 μM, 0.3 μM, 1 μM, 2.5 μM Incubation Duration: Pretreatment for 16 hrs (hours), then culture for 24 hrs (hours) Experimental Results: Inhibition of SARS-COV - 2 Virus replication proceeds in a dose-dependent manner.
Animal Protocol	Animal/Disease Models: Monkey[1] Doses: 0.3 mg/kg; 3 mg/kg Route of Administration: oral administration; 7 days. Experimental Results: Bone resorption markers were diminished in normal monkeys, but bone formation markers were not diminished.
References	[1]. Effects of ONO-5334, a novel orally-active inhibitor of cathepsin K, on bone metabolism. Bone. 2011 Dec;49(6):1351-6. [2]. A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals. bioRxiv. 2020 Apr 17;2020.04.16.044016.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2800 mL	11.4002 mL	22.8003 mL
	5 mM	0.4560 mL	2.2800 mL	4.5601 mL
	10 mM	0.2280 mL	1.1400 mL	2.2800 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.