DC-U4106 is a novel USP8 inhibitor with anticancer activity. It inhibits USP8 with a Kdvalue of 4.7 μM and an IC50 value of 1.2 μM.

Physicochemical Properties

Molecular Formula	C29H27N5O5
Molecular Weight	525.56
Exact Mass	525.201
CAS #	2410534-62-0
PubChem CID	155301443
Appearance	Yellow to brown solid powder
LogP	4.6
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	5
Heavy Atom Count	39
Complexity	889
Defined Atom Stereocenter Count	0
SMILES	C1(C2=CC=C(N3CCN(C4=NC=CC=C4)CC3)C=C2)OC2=C(OC3=C(C)NN=C3C)C(O)=CC(O)=C2C(=O)C=1
InChi Key	VNWOJNLRTJFWRQ-UHFFFAOYSA-N
InChi Code	InChI=1S/C29H27N5O5/c1-17-27(18(2)32-31-17)39-28-23(37)15-21(35)26-22(36)16-24(38-29(26)28)19-6-8-20(9-7-19)33-11-13-34(14-12-33)25-5-3-4-10-30-25/h3-10,15-16,35,37H,11-14H2,1-2H3,(H,31,32)
Chemical Name	8-[(3,5-dimethyl-1H-pyrazol-4-yl)oxy]-5,7-dihydroxy-2-[4-(4-pyridin-2-ylpiperazin-1-yl)phenyl]chromen-4-one
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 (e.g. under nitrogen), avoid exposure to moisture and light.
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	DC-U4106 (1.2-45.2 μM) exhibits little activity against USP7 but inhibits USP8 and USP2 with IC50 values of 1.2 μM and 58.4 μM, respectively [1]. ERα and PR mRNA levels are decreased by DC-U4106 (0–7 μM, 24 hours) [1]. In addition to ERα and PR proteins, DC-U4106 (0-5 μM, 24 hours) can control the expression of proteins associated to the RTK pathway [1]. Cell growth can be inhibited and apoptosis can be induced by DC-U4106 (0-5 μM, 12 hours) [1].
ln Vivo	DC-U4106 (intraperitoneal injection, 5 mg/kg or 20 mg/kg, every 2 days for 14 days) suppresses tumor development in BALB/c nude mice and has no significant effect on body weight, organ morphology and structure [1].
Cell Assay	Western Blot Analysis[1] Cell Types: USP8 positive cell line MCF-7 Tested Concentrations: 0-5 μM Incubation Duration: 24 hrs (hours) Experimental Results: As the concentration increases, the expression of EGFR, ErbB2 and ErbB3 proteins decreases, and causes ERα and ErbB3 Degradation of proteins. PR proteins. RT-PCR[1] Cell Types: USP8 positive cell line MCF-7 Tested Concentrations: 0-7 μM Incubation Duration: 24 hrs (hours) Experimental Results: The mRNA levels of ERα and PR were diminished. Cell proliferation assay[1] Cell Types: USP8 positive cell line MCF-7 Tested Concentrations: 0-5 μM Incubation Duration: Experimental Results: Inhibition of cell growth in a dose-dependent manner. Apoptosis analysis [1] Cell Types: USP8 positive cell line MCF-7 Tested Concentrations: 0-5 μM Incubation Duration: 12 hrs (hours) Experimental Results: As the concentration increases, the proportion of apoptotic cells increases.
Animal Protocol	Animal/Disease Models: BALB/c nude mice [1] Doses: 5mg/kg, 20mg/kg Route of Administration: intraperitoneal (ip) injection, once every 2 days for 14 days. Experimental Results: Tumor growth was Dramatically inhibited at the concentration of 20mg/kg.
References	[1]. Discovery of Potent Small-Molecule USP8 Inhibitors for the Treatment of Breast Cancer through Regulating ERα Expression. J Med Chem. 2022 Jul 5.

Solubility Data

Solubility (In Vitro)

THF : 10 mg/mL (~19.03 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	1.9027 mL	9.5137 mL	19.0273 mL
	5 mM	0.3805 mL	1.9027 mL	3.8055 mL
	10 mM	0.1903 mL	0.9514 mL	1.9027 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.