ULK1-IN-4 (compound 12i) (concentration used to determine IC50; 60 min) effectively inhibited ULK1 kinase activity in vitro with an IC50 of 4.7 μM, and also inhibited ULK2, and showed high selectivity for Aurora A/Aurora B kinases [1]. ULK1-IN-4 (1-100 μM; binding 60 s, dissociation 120 s) formed an irreversible covalent bond with ULK1 kinase, which was confirmed by BLI binding kinase assays [1]. ULK1-IN-4 (200 μM; 24 h) covalently bound to the Cys182 residue of human ULK1 kinase [1]. ULK1-IN-4 (IC50 determined using gradient concentrations; 48 h) inhibited the proliferation of SW620, HCT116 and CT26 CRC cells with IC50 values of 13.31 μM and 9.76 μM, respectively [1]. ULK1-IN-4 (5-10 μM; 2 weeks) inhibited long-term colony formation of SW620, HCT116, and CT26 CRC cells in a dose-dependent manner [1]. ULK1-IN-4 (10-20 μM; 48 hours) treatment for 48 hours induced cell cycle arrest in the S or G2/M phase of SW620, HCT116, and CT26 CRC cells [1]. ULK1-IN-4 (2.5-10 μM; 48 hours) treatment for 48 hours inhibited autophagy in SW620, HCT116, and CT26 colorectal cancer cells, manifested as altered expression of autophagy-related proteins [1]. ULK1-IN-4 (2.5-10 μM; 48 hours) treatment for 48 hours activated apoptosis pathways in SW620, HCT116, and CT26 colorectal cancer cells, manifested as altered expression of apoptosis-related proteins [1]. Treatment with ULK1-IN-4 (10 μM; 24 h) for 24 hours inhibited early autophagy flux in HCT116 colorectal cancer cells under basal autophagy and rapamycin-induced autophagy conditions [1]. Treatment with ULK1-IN-4 (10-20 μM; 48 h) for 48 hours induced apoptosis in SW620, HCT116, and CT26 colorectal cancer cells in a dose-dependent manner [1]. Treatment with ULK1-IN-4 (5-10 μM; 24-48 h) for 24 hours and 48 hours inhibited the migration ability of SW620, HCT116, and CT26 colorectal cancer cells in a dose-dependent manner [1]. Treatment with ULK1-IN-4 (5-10 μM; 24 h) for 24 hours inhibited the migration ability of SW620, HCT116, and CT26 colorectal cancer cells and increased the expression of E-cadherin [1]. ULK1-IN-4 (10 μM; 48 h) can exert pharmacological effects, including cell cycle arrest, migration inhibition, autophagy inhibition and apoptosis induction, which depend on ULK1 targeting in SW620 CRC cells [1].

ULK1-IN-4 (20-40 mg/kg; intraperitoneal injection; every two days; 14 days) inhibited the growth of colorectal tumors in Balb/c mice in a dose-dependent manner, achieving a tumor growth inhibition rate of 70.4% at a dose of 40 mg/kg, and no systemic toxicity was observed [1].

Animal/Disease Models:Balb/c (6-7 week old females, subcutaneously injected with 1×10⁶ CT26 cells)[1]
Doses: 20 mg/kg; 40 mg/kg
Route of Administration: Intraperitoneal injection; every two days; for 14 days
Experimental Results: The tumor growth inhibition rate (TGI) in the 20 mg/kg dose group was 19.2%, and the mean tumor weight was 0.873 g. The TGI in the 40 mg/kg dose group was 70.4%, and the mean tumor weight was 0.327 g. No significant weight loss or behavioral abnormalities were observed during treatment. No significant changes were observed in the histopathology of the heart, liver, spleen, lungs and kidneys compared with the control group. At a dose of 40 mg/kg, the expression of ULK1, p-Beclin-1 (Ser15) and Beclin-1 was downregulated, p62 expression was upregulated, TUNEL-positive apoptotic cells increased, cleavage caspase 3 levels increased, and Ki67-positive proliferating cells decreased.

[1]. Discovery of indolinone-based covalent ULK1 inhibitors that suppressed autophagy and induced apoptosis against colorectal carcinoma. Eur J Med Chem. 2026;307:118633.

C29H29CLN4O4

533.02

Typically exists as solids at room temperature

COC(C=C1)=CC=C1N/C(C2=CC=CC=C2)=C3C(C=C(NC(CCCCNC(CCl)=O)=O)C=C4)=C4NC/3=O

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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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).

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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 : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)