Cat. No.: HY-183581

USP1-IN-18

USP1-IN-18 is an orally active USP1 inhibitor with a human IC₅₀ of 17.0 nM. USP1-IN-18 inhibits USP1-UAF1 deubiquitinase activity and drives ubiquitinated PCNA accumulation. USP1-IN-18 induces DNA damage, replication stress, and G2-M phase cell cycle arrest. USP1-IN-18 can be used for the research of triple-negative breast cancer.

For research use only. We do not sell to patients.

CAS No.: 3064481-29-1
Formula: C₂₆H₂₂F₃N₇O₂
Molecular Weight:521.49

Storage:

Please store the product under the recommended conditions in the Certificate of Analysis.

USP1-IN-18 Chemical Structure

CAS No.: 3064481-29-1

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Use of MCE Products

Strong Fluorescence Signal / Multi-Disciplinary Applications / High Fluorescence Specificity

Biological Activity
- Description
- IC₅₀ & Target
- In Vitro
- Parmacokinetics
- In Vivo
Chemical Information
Protocols (7)
Purity & Documentation
References
Calculators
Help & FAQs
Classifications
Bioz

Biological Activity

Description

IC₅₀ & Target

^[1]

USP1

All Deubiquitinase Isoforms :

All DNA/RNA Synthesis Isoforms :

In Vitro

USP1-IN-18 (Compound 57) potently inhibits the purified USP1-UAF1 complex with an IC₅₀ of 17 nM via stable binding to a hydrophobic tunnel and induction of a key β-turn conformational shift^[1].
USP1-IN-18 (10-10000 nM; 24 h-12 days) inhibits MDA-MB-436 triple-negative breast cancer cell proliferation with an IC₅₀ of 173 nM, induces G2-M phase arrest^[1].
USP1-IN-18 exhibits favorable metabolic stability in human, monkey, rat, mouse, and dog liver microsomes, with a half-life >120 min and intrinsic clearance <12 mL/min/kg in human liver microsomes^[1].
USP1-IN-18 (7 days) shows minimal cytotoxicity in nonmalignant LX-2, Vero, and H9C2 cell lines, with IC₅₀ values >30 μM after 7 days of treatment^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.

Cell Proliferation Assay^[1]

View Protocol

Cell Line:

MDA-MB-436 triple-negative breast cancer cells
Concentration:

10 nM; 30 nM; 100 nM; 300 nM; 1000 nM; 3000 nM; 10000 nM
Incubation Time:

12 days
Result:

Inhibited MDA-MB-436 cell proliferation with an IC₅₀ of 173 nM.
Dose-dependently suppressed colony formation, with significant inhibition observed at concentrations ≥30 nM.
Induced G2-M phase arrest: at 300 nM, the proportion of cells in G2-M phase increased from 19.4% to 30.5%.
Caused dose-dependent accumulation of ubiquitinated PCNA after 4 h treatment, confirming USP1 inhibition.
Upregulated P27 protein levels after 24 h treatment.
Showed minimal inhibition of other tested USP family members at concentrations exceeding those required for USP1 inhibition, demonstrating target selectivity.

Cell Cycle Analysis^[1]

View Protocol

Cell Line:

MDA-MB-436 triple-negative breast cancer cells
Concentration:

30 nM; 300 nM; 1000 nM
Incubation Time:

24 h
Result:

Induced G2-M phase arrest.

Parmacokinetics

Species	Dose	Route	AUC_0-t	T_1/2	T_max	C_max	MRT_0-t	V_z	CL_z/F	F
Rat^[1]	10 mg/kg	i.v.	2598300 μg·h/mL	5.7 h	0.08 h	1598.5 μg/L	3.2 h	33.9 L/kg	4.6 L/h/kg	/
Rat^[1]	10 mg/kg	p.o.	1963000 μg·h/mL	7.5 h	0.33 h	956.7 μg/L	5.2 h	49.9 L/kg	4.8 L/h/kg	76 %

In Vivo

USP1-IN-18 (Compound 57) (100 mg/kg; p.o.; daily; 34 consecutive days) achieves 42% tumor growth inhibition in MDA-MB-436 xenografts^[1].
USP1-IN-18 (100-300 mg/kg; p.o.; daily; 30 days) is well tolerated in female Balb/c mice at doses up to 300 mg/kg administered daily for 30 days, with no evident subacute toxicity^[1].
USP1-IN-18 (400-800 mg/kg; p.o.; single dose) is well tolerated in female Balb/c mice at single oral doses up to 800 mg/kg, with no evident acute toxicity^[1].
USP1-IN-18 (10 mg/kg; p.o.; single dose) exhibits favorable oral pharmacokinetic properties in rats, with 76% oral bioavailability following a single 10 mg/kg dose^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Model:

NOD-SCID mice^[1]
Dosage:

100 mg/kg
Administration:

p.o.; daily; 34 consecutive days
Result:

Achieved a tumor growth inhibition (TGI) of 42%.
Markedly increased ubiquitinated PCNA (ub-PCNA) and γ-H2AX levels in xenograft tumor tissues.

Animal Model:

Balb/c mice (female, 6−8 weeks old, 18−22 g)^[1]
Dosage:

100 mg/kg; 300 mg/kg
Administration:

p.o.; daily; 30 days
Result:

Caused no significant alterations in general health status, mortality, or body weight.
Revealed no organ damage via histopathological assessment (H&E staining).
Restored hematological parameters (neutrophils, lymphocytes) to normal after the 14-day drug-free period.

Animal Model:

Balb/c mice (female, 6−8 weeks old, 18−22 g)^[1]
Dosage:

400 mg/kg; 600 mg/kg; 800 mg/kg
Administration:

p.o.; single dose
Result:

Caused no significant tissue damage or pathological alterations after the 14-day drug-free period.
Showed a slight increase in platelet (PLT) levels, but no statistically significant changes in other hematological or serum biochemical parameters.

Animal Model:

Sprague-Dawley rats^[1]
Dosage:

10 mg/kg
Administration:

p.o.; single dose
Result:

Resulted in an area under the concentration-time curve (AUC_0-t) of 1963.0 μg·h/L, an elimination half-life (T_1/2) of 7.5 h, a maximum plasma concentration (C_max) of 956.7 μg/L, and an oral bioavailability (F) of 76%.

Chemical Information

CAS No. 3064481-29-1
Molecular Weight 521.49
Formula C₂₆H₂₂F₃N₇O₂
SMILES

COC1=NC=NC(C2CC2)=C1C3=CC(N(CC4=CC=C(C5=NC(C(F)(F)F)=CN5C)C=C4)C(N6)=O)=C6C=N3
Shipping

Room temperature in continental US; may vary elsewhere.
Storage

Please store the product under the recommended conditions in the Certificate of Analysis.

Protocols

All Cell Biology Animals and Diseases

Cell Cytotoxicity Assay
Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.
PCNA Immunodetection Proliferation Assay
PCNA immunodetection measures proliferative activity by detecting proliferating cell nuclear antigen, a nuclear protein associated with DNA polymerase δ function and DNA replication. The assay readout is the proportion of PCNA-positive nuclei among total counted cells, but PCNA labeling is not identical to BrdU labeling because PCNA can mark late G1/early S-associated replication competence and may persist beyond active DNA synthesis depending on fixation and extraction conditions.
Flow cytometric DNA-content cell-cycle staining
Flow cytometric DNA-content cell-cycle staining measures the fluorescence intensity of DNA-bound fluorochromes in single cells or nuclei to estimate DNA content distributions, allowing assignment of populations to G0/G1, S, and G2/M phases by DNA histogram deconvolution. Propidium iodide (PI) intercalates into DNA, and PI fluorescence is proportional to cellular DNA content when staining is performed under conditions that make DNA accessible and minimize non-DNA signal. Cells with G2/M DNA content are expected to show approximately twice the fluorescence intensity of G0/G1 cells, while S-phase cells occupy intermediate fluorescence values. PI-based DNA-content analysis can also detect cells with fractional DNA content, often reported as sub-G1, when DNA fragmentation and extraction during staining reduce retained DNA signal in apoptotic cells. DAPI is an alternative DNA fluorochrome for univariate DNA-content analysis, while bivariate approaches combining DNA content with proliferation
BrdU Incorporation Assay
Bromodeoxyuridine (BrdU) incorporation assay is based on the principle that BrdU, a thymidine analog, is incorporated into newly synthesized DNA during the S phase of the cell cycle, thereby serving as a marker of DNA replication and cellular proliferation. Incorporated BrdU can be detected using anti-BrdU antibodies following DNA denaturation, enabling visualization or quantification of proliferating cells through immunochemical detection methods such as immunofluorescence or immunohistochemistry.
Protocol for Cell Cycle
Cell-cycle analysis by flow cytometry measures DNA content in single cells to estimate the fraction of cells in G0/G1, S, and G2/M phases. Propidium iodide intercalates into DNA, and after RNA removal with RNase, fluorescence intensity reflects cellular DNA content: 2N cells are assigned to G0/G1, cells between 2N and 4N to S phase, and 4N cells to G2/M. DNA-content analysis alone cannot reliably separate G0 from G1 or G2 from M. Ki-67 can distinguish quiescent G0 cells from cycling cells, EdU or BrdU incorporation marks active DNA synthesis in S phase, and phospho-histone H3 staining identifies mitotic cells within the 4N population.
Breast Cancer Modeling
Breast cancer is a heterogeneous cancer, and it has been distinguished into four subtypes: luminal A, luminal B, HER2-positive and basal-like. Molecular mutations, epigenetic alterations, hormone exposure and immune microenvironment are related to the progression of breast cancer.
Genotoxicity/Mutagenicity Study
The bacterial reverse mutation assay detects point mutations that restore amino-acid prototrophy in auxotrophic Salmonella typhimurium or Escherichia coli tester strains; after exposure to a test article, mutagenic activity is read out as an increased number of revertant colonies on minimal agar compared with the vehicle control. The assay uses tester strains with different mutation targets so that base-substitution and frameshift mutagens can be detected, and testing is performed with and without exogenous mammalian metabolic activation because some chemicals require biotransformation to become mutagenic.

Purity & Documentation

Handling Instructions (2659 KB)

References

[1]. Teng Y, et al. Design and Synthesis of USP1 Inhibitors: Synergistic Antitumor Activity with PARP Inhibitors in Triple-Negative Breast Cancer. J Med Chem. 2026;69(9):10594-10620. [Content Brief]

Calculators

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Help & FAQs

How should lyophilized recombinant proteins be reconstituted and stored?

1. Before opening the cap, centrifuge the vial at 13000 rpm for 20-30 seconds. This step will ensure that any lyophilized powder that may have adhered to the cap or walls is collected at the bottom of the vial, minimizing the risk of product loss.

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USP1-IN-18

All Deubiquitinase Isoforms

All DNA/RNA Synthesis Isoforms