PROTAC ATR degrader-3 (compound A12) efficiently and selectively inhibited ATR kinase in cell-free experiments with an IC50 value of 2.7 ± 0.5 nM and very low activity against DNA-PK, PI3Kα and ATM [1]. PROTAC ATR degrader-3 (31 nM-5 μM; 8-72 h) induced efficient, time- and concentration-dependent proteasome-mediated degradation of ATR in LoVo cells, with a DC50 value of 127 nM and a Dmax value of 72% after 72 h [1]. PROTAC ATR degrader-3 (72 h) induced efficient degradation of ATR in SW480 (DC50 = 140 nM, Dmax = 95%) and HCT116 (DC50 = 48 nM, Dmax = 77%) colorectal cancer cells [1]. PROTAC ATR degrader-3 (0-10 μM; 16-72 h) induced potent, concentration-dependent proteasome-mediated degradation of CHK1 in LoVo cells, with a DC50 of 135 nM and a Dmax of 70% after 72 h [1]. PROTAC ATR degrader-3 (72 h) induced the degradation of CHK1 in SW480 (DC50 = 426 nM, Dmax = 90%) and HCT116 (DC50 = 95 nM, Dmax = 74%) colorectal cancer cells [1]. PROTAC ATR degrader-3 (serial concentrations; 72 hours) effectively inhibited the proliferation of LoVo colorectal cancer cells, with an IC50 value of 0.055 ± 0.010 μM. Its activity against SW480 (IC50 = 0.37 μM), HCT116 (IC50 = 2.774 μM), and normal NCM460 (IC50 = 13.161 μM) cells was low [1]. PROTAC ATR degrader-3 (250 nM; 48 hours, co-treated with 2 μM MG132) enhanced the polyubiquitination of ATR in LoVo cells, confirming that it was degraded through the ubiquitin-dependent proteasome pathway [1]. PROTAC ATR degrader-3 (0.5-2.0 μM; 48 h) treatment for 48 h induced S-phase cell cycle arrest in LoVo cells, with the highest proportion of S-phase cells reaching 38.05% at a concentration of 1.0 μM [1]. PROTAC ATR degrader-3 (0.5–4.0 μM; 48 h) induces LoVo cell apoptosis in a dose-dependent manner by activating the caspase-mediated apoptosis pathway [1]. PROTAC ATR degrader-3 (0.5–2.0 μM; 10 days) potently and dose-dependently inhibits the colony formation of LoVo cells over 10 days [1]. PROTAC ATR degrader-3 (0.5–2.0 μM; 24–48 h) inhibits the migration and invasion of LoVo cells in a dose-dependent manner [1]. PROTAC ATR degrader-3 (0.5–4.0 μM; 24 h) induces dose-dependent accumulation of DNA damage in LoVo cells, accompanied by increased γH2AX expression [1].

PROTAC ATR degrader-3 (compound A12) (10-30 mg/kg; intraperitoneal injection; twice daily; 32 days) showed significant dose-dependent inhibition of LoVo colorectal cancer xenograft growth in nude mice, with a maximum TGI of 74% at 30 mg/kg, and no adverse effects on body weight [1].

Western Blot Analysis [1]
Cell Types: LoVo human colorectal cancer cells
Tested Concentrations: 31, 62, 125, 250, 500, 1000, 2000, 5000 nM
Incubation Duration: 8, 16, 24, 48, 72 hours
Experimental Results: After 72 hours, the ATR degradation rate was 60 ± 5% at 0.1 μM and 78 ± 3% at 0.5 μM. After 72 hours, the DC50 was 127 nM and the maximum degradation rate (Dmax) was 72%. At 250 nM, ATR degradation was significantly induced after 48 hours. ATR degradation was inhibited when cells were pretreated with MG132 ( ) or lenalidomide.

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Western Blot Analysis [1]
Cell Types: LoVo human colorectal cancer cells
Tested Concentrations: 0-10000 nM (72 hours); 1 μM (16 hours, pretreated with 2 μM MG132, 2 μM lenalidomide or 2 μM C1 for 2 hours); 0.5-4 μM (24 hours)
Incubation Duration: 16 hours (pretreatment); 24 hours; 72 hours
Experimental Results: After 72 hours, CHK1 degradation was concentration-dependent, with DC50 at 135 nM and Dmax at 70%. Pretreatment of cells with MG132 or lenalidomide blocked CHK1 degradation. At concentrations ≥0.5 μM, CHK1 protein levels decreased after 24 hours.

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Western Blot Analysis [1]
Cell Types: LoVo human colorectal cancer cells
Tested Concentrations: 250 nM (co-treated with 2 μM MG132)
Incubation Duration: 48 hours
Experimental Results: As indicated by the increased ubiquitin signal in the immunoprecipitated ATR samples, the polyubiquitination of ATR protein was significantly enhanced.

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Cell cycle analysis [1]
Cell Types: LoVo human colorectal cancer cells
Tested Concentrations: 0.5, 1.0, 2.0, 4.0 μM
Incubation Duration: 48 hours
Experimental Results: Induction of S phase arrest: The proportion of S phase cells was 35.30% at 0.5 μM, 38.05% at 1.0 μM, and 37.65% at 2.0 μM, while the control group was 29.11%. The proportion of G0/G1 phase cells decreased to 53.69% (0.5 μM), 50.79% (1.0 μM), and 51.80% (2.0 μM), while the control group was 58.66%.

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Apoptosis analysis [1]
Cell Types: LoVo human colorectal cancer cells
Tested Concentrations: 0.5-2.0 μM
Incubation Duration: 48 hours
Experimental Results: Dose-dependent apoptosis was induced. PARP1 and Caspase-3 cleavage levels increased in a concentration-dependent manner. Bcl-2 levels decreased.

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Animal/Disease Models:Nude mice (female, 7-8 weeks old) carrying LoVo colorectal cancer xenografts [1]
Doses: 10, 20, 30 mg/kg; 10 mg/kg (in combination with cetuximab)
Route of Administration: Intraperitoneal injection; twice daily for 32 days; intravenous injection (cetuximab, once every 4 days)
Experimental Results: Tumor growth inhibition rate (TGI) was 43% with 10 mg/kg monotherapy. TGI was 57% with 20 mg/kg monotherapy. TGI was 74% with 30 mg/kg monotherapy. TGI was 81% with 10 mg/kg in combination with cetuximab. No adverse effects were observed on mouse body weight. The expression of ATR, p-ATR, CHK1, and p-CHK1 was downregulated in tumor tissue. The expression of p-p53, p21, and γH2AX was upregulated in tumor tissue.

[1]. Design and Synthesis of Pyrrolo [3, 4-d] pyrimidine-Based ATR Degraders for Effective Treatment of Colorectal Cancer in Mouse Model[J]. Journal of Medicinal Chemistry, 2026.

C41H38N8O6

738.79

Typically exists as solids at room temperature

C[C@@H]1COCCN1C2=C3C(CN(C3)C(C4=CC=C(C=C4)C(NCC5=CC=C6C(CN(C6=O)C7C(NC(CC7)=O)=O)=C5)=O)=O)=NC(C8=CC=CC9=C8C=CN9)=N2

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.)