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C527

Cat No.:V12576 Purity: ≥98%
C527 is a pan-DUB enzyme inhibitor (antagonist) with high activity against USP1/UAF1 complex, with IC50 of 0.88 μM.
C527
C527 Chemical Structure CAS No.: 192718-06-2
Product category: New1
This product is for research use only, not for human use. We do not sell to patients.
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Product Description
C527 is a pan-DUB enzyme inhibitor (antagonist) with high activity against USP1/UAF1 complex, with IC50 of 0.88 μM.
Biological Activity I Assay Protocols (From Reference)
ln Vitro
Cutting USP1/UAF1 with C527 results in a 0.88±0.03 μM IC50, which decreases the enzyme's enzymatic activity. USP12/USP46 complex and other DUB enzymes' DUB activity is inhibited in vitro by C527. When compared to the USP1/UAF1 complex, C527's IC50 for these DUB enzymes is greater. A factor of less is used to block the distinct DUB enzyme subtypes UCH-L1 and UCH-L3. Ub-FANCD2 and Ub-FANCI levels rose after receiving C527 therapy. Mitomycin C and camptothecin cytotoxicity was increased when cells were pretreated with C527. Reduction of homologous recombination activity and purification of DNA damage-sensitive cells are caused by an increase in ubiquitinated versions of FANCD2 and FANCI following C527 therapy [1].
References

[1]. Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells. Mol Cancer Ther. 2013 Dec;12(12):2651-62.

These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C17H8NO3F
Molecular Weight
293.249
Exact Mass
293.048
CAS #
192718-06-2
PubChem CID
2307331
Appearance
Light yellow to yellow solid powder
Density
1.4±0.1 g/cm3
Boiling Point
488.6±47.0 °C at 760 mmHg
Flash Point
249.3±29.3 °C
Vapour Pressure
0.0±1.2 mmHg at 25°C
Index of Refraction
1.639
LogP
4.82
Hydrogen Bond Donor Count
0
Hydrogen Bond Acceptor Count
5
Rotatable Bond Count
1
Heavy Atom Count
22
Complexity
475
Defined Atom Stereocenter Count
0
InChi Key
ULJDFEYQOPCCPM-UHFFFAOYSA-N
InChi Code
InChI=1S/C17H8FNO3/c18-10-7-5-9(6-8-10)17-19-13-14(20)11-3-1-2-4-12(11)15(21)16(13)22-17/h1-8H
Chemical Name
2-(4-fluorophenyl)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

Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
H2O : ~1 mg/mL (~3.41 mM)
DMSO : ~1 mg/mL (~3.41 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 : PEG300Tween 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).
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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 : PEG300Tween 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).
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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 3.4101 mL 17.0503 mL 34.1006 mL
5 mM 0.6820 mL 3.4101 mL 6.8201 mL
10 mM 0.3410 mL 1.7050 mL 3.4101 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.

Calculator

Molarity Calculator allows you to calculate the mass, volume, and/or concentration required for a solution, as detailed below:

  • Calculate the Mass of a compound required to prepare a solution of known volume and concentration
  • Calculate the Volume of solution required to dissolve a compound of known mass to a desired concentration
  • Calculate the Concentration of a solution resulting from a known mass of compound in a specific volume
An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?
  • Enter 350.26 in the Molecular Weight (MW) box
  • Enter 10 in the Concentration box and choose the correct unit (mM)
  • Enter 5 in the Volume box and choose the correct unit (mL)
  • Click the “Calculate” button
  • The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:
  • Enter 10 into the Concentration (Start) box and choose the correct unit (mM)
  • Enter 25 into the Concentration (End) box and select the correct unit (mM)
  • Enter 25 into the Volume (End) box and choose the correct unit (mL)
  • Click the “Calculate” button
  • The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box
g/mol

Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4  c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:
  • To calculate molar mass of a chemical compound, please enter the chemical/molecular formula and click the “Calculate’ button.
Definitions of molecular mass, molecular weight, molar mass and molar weight:
  • Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
  • Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.
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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

  • Enter the mass of the reagent and the desired reconstitution concentration as well as the correct units
  • Click the “Calculate” button
  • The answer appears in the Volume (to add to vial) box
In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)
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Calculation results

Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
             (2) Be sure to add the solvent(s) in order.

Biological Data
  • (A) Schematic of the USP1/UAF1 constructs. (B) Coomassie blue staining of the purified USP1/UAF1 complex (C) A schematic of the Ub-Rhodamine based screening assay (D) Chemical structure of a parental USP1 inhibitor compound 527 (C527). (E) C527 inhibits USP1 activity in a time-dependent manner. Purified USP1/UAF1 complex was incubated with 1 μM C527 or DMSO for the indicated time, followed by the addition of Ub-AMC at a 0.5 μM final concentration. Fluorescence (RFU) at 535 nm was measured to indicate the enzymatic activity of USP1. (F) Dose-dependent inhibition of USP1 enzymatic activity by USP1 inhibitor C527. Purified USP1/UAF1 complex was incubated with C527 or DMSO for 3 hrs followed by the addition of Ub-AMC at 0.5 μM concentration. The fluorescence (RFU) at 535 nm was measured to indicate the enzymatic activity of USP1. (G) IC50 plot of C527 against USP/UAF1 complex in an enzymatic reaction, as described in panels E and F. (H,I) USP1 inhibitor SJB2-043 1 μM inhibits the activity of native USP1/UAF1. K562 cells were treated with DMSO or SJB2-043 for 24 hrs and cell extracts were incubated with 0.5 μM Ub-Vs (see structure in panel H) for 45 min. An aliquot of untreated cell extract was treated with Ubiquitin-aldehyde (Ub-Aldehyde, 5 μM) before adding Ub-Vs. The reactions were then subjected to immunoblotting with anti-HA or anti-USP1 antibodies.[1].Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells. Mol Cancer Ther. 2013 Dec;12(12):2651-62.
  • USP1 inhibitor increases the levels of Ub-FANCD2, decreases the HR activity, and sensitizes the cells to chemotherapy agents.(A) Western blots of the lysates from Hela cells treated with DMSO or USP1 inhibitor C527 for 8 hrs. (B) Increased cytotoxicity of C527 on Hela cells in presence of chemotherapy agents. Cells were treated with C527 (1 μM) for 24 hrs, followed by the treatment with Mitomycin C (MMC) (0.25 μM), Camptothecin (CPT) (0.1 μM) or Etoposide (0.5 μM) for 4 days, and cell survival was determined. (C) C527 inhibits DRGFP reporter for homologous recombination repair activity. U2OS-DRGFP cells were transfected with I-SCE-I reporter plasmid and then exposed to C527 at the indicated concentration for 24 h. Cells were then subjected to flow cytometry analysis. The percentage of GFP positive cells was normalized by solvent vehicle treated group are shown. (D) C527 inhibits camptothecin-induced RAD51 foci formation. HeLa cells were pre-treated with DMSO or C527 at the indicated concentration and then exposed to camptothecin (CPT) for 1 hr. RAD51 foci were detected using immunofluorescence. Data are represented as mean ± SD from three independent experiments.[1].Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells. Mol Cancer Ther. 2013 Dec;12(12):2651-62.
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