OTUB1/USP8-IN-1 is a potent dual OTUB1/USP8 inhibitor (antagonist) with IC50s of 0.17 and 0.28 nM for OTUB1 and USP8 inhibition, respectively. OTUB1/USP8-IN-1 may be utilized in cancer research.

Physicochemical Properties

Molecular Formula	C22H16CLFN2O4
Molecular Weight	426.824848175049
Exact Mass	426.078
CAS #	2858800-98-1
PubChem CID	165437860
Appearance	Light yellow to yellow solid powder
LogP	4.5
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	3
Heavy Atom Count	30
Complexity	696
Defined Atom Stereocenter Count	1
SMILES	CN1CC[C@H](C1)OC2=C(C(=C(C=C2)F)C3=NC4=C(O3)C(=O)C5=CC=CC=C5C4=O)Cl
InChi Key	HXIYHNFNCGXLAU-LLVKDONJSA-N
InChi Code	InChI=1S/C22H16ClFN2O4/c1-26-9-8-11(10-26)29-15-7-6-14(24)16(17(15)23)22-25-18-19(27)12-4-2-3-5-13(12)20(28)21(18)30-22/h2-7,11H,8-10H2,1H3/t11-/m1/s1
Chemical Name	2-[2-chloro-6-fluoro-3-[(3R)-1-methylpyrrolidin-3-yl]oxyphenyl]benzo[f][1,3]benzoxazole-4,9-dione
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

Targets	IC50: 0.17 nM (OTUB1) and 0.28 nM (USP8)[1]
ln Vitro	KRAS-WT (H1975, EBC-1, H1703) and KRAS-mutated (H23, A549) NSCLC cell lines have antiproliferative effects in response to OTUB1/USP8-IN-1 (compound 61; 10 nM-10 μM; 72 h)[1]. In H1975 cells, OTUB1/USP8-IN-1 (500 nM; 24 h) reduces the amounts of both UBE2N and EGFR protein[1].
ln Vivo	In the H1975 xenograft mouse model, OTUB1/USP8-IN-1 (compound 61; 10 nM-10 μM; 72 h) reduces the tumor burden[1].
Cell Assay	Cell Viability Assay[1] Cell Types: KRAS-WT (H1975, EBC-1, H1703) and KRAS-mutated (H23, A549) NSCLC cell lines Tested Concentrations: 10 nM-10 μM Incubation Duration: 72 hrs (hours) Experimental Results: Inhibited cell proliferative with IC50 values of 118, 145, 172, 431, and 1004 nM for H1975, H1703, EBC-1, H23, and A549 cells, respectively. Western Blot Analysis[1] Cell Types: H1975 cells Tested Concentrations: 500 nM Incubation Duration: 24 hrs (hours) Experimental Results: diminished the levels of both UBE2N and EGFR in a dose-dependent manner.
Animal Protocol	Animal/Disease Models: Female BALB/c nude mice with H1975 xenografts (5 weeks of age)[1] Doses: 5 mg/kg Route of Administration: intraperitoneal (ip)injection; QD and BID, for 2 weeks Experimental Results: diminished total tumor weight and average tumor volume in an over twofold with BID dosing. Animal/Disease Models: Female BALB/c nude mice with H1975 xenograft (5 weeks of age)[1] Doses: 1 and 10 mg/kg Route of Administration: intravenous (iv)injection (1 mg/kg) and oral administration (10 mg/kg) Experimental Results: 1.19 Administration iv (1 mg/kg) po (10 mg/kg) T1/2 (h) 0.83 1.75 Tmax (h) 0.33 Cmax (μg/L) 4274 AUC (μg·h/ L) 1345 3747 CL (L/h/kg) 44 Vdss (L/kg) 0.77
References	[1]. Discovery of Potent OTUB1/USP8 Dual Inhibitors Targeting Proteostasis in Non-Small-Cell Lung Cancer. J Med Chem. 2022 Oct 11.

Solubility Data

Solubility (In Vitro)

DMSO : 50 mg/mL (117.15 mM)

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	2.3429 mL	11.7145 mL	23.4291 mL
	5 mM	0.4686 mL	2.3429 mL	4.6858 mL
	10 mM	0.2343 mL	1.1715 mL	2.3429 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.