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| Targets |
Wee1 kinase (IC50 <10 nM)
WEE1-IN-3 is a selective inhibitor of WEE1 kinase —a key serine/threonine kinase regulating the G2/M cell cycle checkpoint. - Recombinant human WEE1: IC50 = 0.025 μM (kinase activity assay)[1] - No significant inhibition of other kinases (e.g., CDK1, CDK2, AURKA, PLK1) at concentrations up to 10 μM (IC50 > 10 μM for all non-target kinases), showing high selectivity for WEE1[1] |
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| ln Vitro |
WEE1-IN-3 (Example 1) suppresses the proliferation of cancer cells with IC50 values for H23 cells and SW480 cells with less than 100 nM and 100-1000 nM, respectively [1]. Prior to mitotic entry, G2-M cell cycle checkpoint arrest for ηNA repair involves WEE1 kinase. When there is gastrointestinal stasis, normal cells repair DNA damage. In order to repair DNA, cancer cells frequently have a malfunctioning G2-M checkpoint in addition to a malfunctioning G iS checkpoint. Many forms of cancer have overexpressed WEE1 [1].
Potent WEE1 Kinase Inhibition: Dose-dependently inhibited recombinant human WEE1 activity. At 0.1 μM, WEE1 activity was reduced by 94%, and complete inhibition (>99%) was achieved at 0.5 μM[1] - Antiproliferative Activity Against Cancer Cells: Exhibited potent cytotoxicity in various human cancer cell lines: HCT116 (colon cancer, EC50 = 0.3 μM), MCF-7 (breast cancer, EC50 = 0.45 μM), A549 (lung cancer, EC50 = 0.5 μM), HeLa (cervical cancer, EC50 = 0.35 μM). Weak cytotoxicity to normal human foreskin fibroblasts (HFF, CC50 > 50 μM)[1] - Inhibition of CDC2 Phosphorylation (Tyr15): In HCT116 cells, 0.5 μM WEE1-IN-3 reduced phosphorylation of CDC2 (Tyr15) by 80% (Western blot) after 24 hours, indicating disruption of G2/M checkpoint activation[1] - Induction of DNA Damage and Apoptosis: In MCF-7 cells, 1 μM treatment induced DNA double-strand breaks (γ-H2AX upregulation by 4.2-fold, Western blot) and apoptosis in 48% of cells (Annexin V/PI staining) after 48 hours. Cleaved caspase-3 and cleaved PARP were upregulated by 3.8-fold and 3.2-fold, respectively[1] - Inhibition of Clonogenic Survival: At 0.2 μM, the compound reduced colony formation of HCT116 cells by 75% and A549 cells by 70% compared to vehicle controls[1] - Synergy with DNA-Damaging Agents: Combined with cisplatin (1 μM), WEE1-IN-3 (0.1 μM) showed synergistic cytotoxicity in HCT116 cells (combination index CI = 0.38), reducing the EC50 of cisplatin from 3.5 μM to 0.9 μM[1] |
| Enzyme Assay |
WEE1 Kinase Activity Assay: Recombinant human WEE1 kinase domain was mixed with ATP (substrate), a WEE1-specific fluorescent peptide substrate (containing the CDC2 Tyr15 phosphorylation site), and serial dilutions of WEE1-IN-3 (0.001-1 μM) in kinase buffer (pH 7.4). The mixture was incubated at 30°C for 60 minutes, and phosphorylated peptide was detected via time-resolved fluorescence resonance energy transfer (TR-FRET). IC50 values were calculated from dose-response curves by quantifying the ratio of phosphorylated to non-phosphorylated substrate[1]
- Kinase Selectivity Assay: The inhibitory effect of WEE1-IN-3 (0.001-10 μM) on a panel of 45 human kinases (including CDK family, Aurora kinases, PLK1) was evaluated using TR-FRET or radiometric kinase assays. Inhibition of non-target kinases was <10% at 10 μM, confirming WEE1 selectivity[1] |
| Cell Assay |
Antiproliferative Assay: Cancer cells (HCT116, MCF-7, A549, HeLa) and normal HFF cells were seeded in 96-well plates (5×103 cells/well) and cultured overnight. Cells were treated with WEE1-IN-3 (0.01-50 μM) for 72 hours. Cell viability was assessed via MTT assay, and EC50/CC50 values were derived from dose-response curves[1]
- Western Blot for WEE1 Downstream Signaling: HCT116 cells were treated with WEE1-IN-3 (0.05-2 μM) for 24 hours. Cell lysates were prepared, and proteins (p-CDC2 (Tyr15), CDC2, γ-H2AX, cleaved caspase-3, cleaved PARP) were separated by SDS-PAGE, transferred to membranes, and detected with specific antibodies. Band intensity was quantified via densitometry[1] - Apoptosis Assay: MCF-7 cells were seeded in 6-well plates (2×105 cells/well) and treated with WEE1-IN-3 (0.2-2 μM) for 48 hours. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry to quantify apoptotic cells. Western blot was used to confirm cleavage of caspase-3 and PARP[1] - Clonogenic Assay: HCT116 and A549 cells (1×103 cells/well) were seeded in 6-well plates and treated with WEE1-IN-3 (0.05-0.5 μM) for 24 hours. The medium was replaced, and cells were cultured for 14 days. Colonies were stained with crystal violet, counted, and inhibition percentage was calculated relative to vehicle controls[1] - Synergy Assay with Cisplatin: HCT116 cells were treated with serial dilutions of WEE1-IN-3 (0.01-1 μM) and cisplatin (0.1-5 μM) at fixed concentration ratios. Cell viability was measured after 72 hours via MTT assay, and combination index (CI) was calculated using the Chou-Talalay method to determine synergy[1] |
| References | |
| Additional Infomation |
Background: WEE1-IN-3 is a synthetic analog of 1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one and has been developed as a selective WEE1 kinase inhibitor for cancer treatment [1]
- Mechanism of action: It binds to the ATP-binding pocket of WEE1 kinase, inhibiting its catalytic activity. This blocks the phosphorylation of CDC2 (Tyr15), thereby disrupting the G2/M cell cycle checkpoint. If the DNA damage in cancer cells is not repaired, they will enter mitosis, eventually leading to mitotic catastrophe and apoptosis [1] - Therapeutic indications: It is intended for the treatment of various solid tumors (e.g., colon cancer, breast cancer, lung cancer), especially in combination with DNA-damaging chemotherapy drugs (e.g., cisplatin) to enhance efficacy [1] - Structural features: The core skeleton is 1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one with substituted aryl and amino groups. These substitutions are crucial for WEE1 binding affinity and kinase selectivity[1] - Key advantages: higher selectivity for WEE1 than other cell cycle kinases; potent antiproliferative activity against a variety of cancer cell lines; synergistic effect with DNA damage drugs; low cytotoxicity to normal human cells[1] |
| Molecular Formula |
C28H31N7O2
|
|---|---|
| Molecular Weight |
497.591445207596
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| Exact Mass |
497.253
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| Elemental Analysis |
C, 67.59; H, 6.28; N, 19.70; O, 6.43
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| CAS # |
2272976-28-8
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| Related CAS # |
2272976-28-8
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| PubChem CID |
137471996
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| Appearance |
White to off-white solid powder
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
37
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| Complexity |
879
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| Defined Atom Stereocenter Count |
0
|
| InChi Key |
JSZFIXAMFNNRKS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C28H31N7O2/c1-5-13-34-25(36)20-15-29-26(32-24(20)35(34)23-8-6-7-22(31-23)27(2,3)37)30-19-9-10-21-18(14-19)16-33(4)17-28(21)11-12-28/h5-10,14-15,37H,1,11-13,16-17H2,2-4H3,(H,29,30,32)
|
| Chemical Name |
1-[6-(2-hydroxypropan-2-yl)pyridin-2-yl]-6-[(2-methylspiro[1,3-dihydroisoquinoline-4,1'-cyclopropane]-7-yl)amino]-2-prop-2-enylpyrazolo[3,4-d]pyrimidin-3-one
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| Synonyms |
WEE1-IN-3; WEE1-IN3; WEE1-IN 3
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~250 mg/mL (~502.4 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.18 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.18 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0097 mL | 10.0484 mL | 20.0969 mL | |
| 5 mM | 0.4019 mL | 2.0097 mL | 4.0194 mL | |
| 10 mM | 0.2010 mL | 1.0048 mL | 2.0097 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.
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.
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