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Purity: =99.47%
Adavosertib (formerly known as AZD-1775; MK-1775; AZD1775; MK1775) is a novel potent and selective small molecule Wee1 tyrosine kinase inhibitor with potential anticancer activity. Adavosertib inhibits G2 DNA damage checkpoint; it inhibits Wee1 with an IC50 of 5.2 nM in a cell-free assay. A small molecule inhibitor of the tyrosine kinase WEE1, MK-1775 may have antineoplastic sensitizing properties. Cyclin-dependent kinase 1 (CDC2) is phosphorylated by WEE1, a tyrosine kinase that MK-1775 specifically targets and inhibits in order to inactivate the CDC2/cyclin B complex. MK-1775 enhanced, at tolerable doses, the antitumor efficaciousness of 5-FU or its prodrug, capecitabine, in vivo. The CDC2 phosphorylation inhibition and the induction of Histone H3 phosphorylation in tumors were well correlated with these enhancements. Furthermore, MK-1775 enhanced the in vitro cytotoxic effects of pemetrexed, doxorubicin, camptothecin, and mitomycin C. These findings validate the need to test MK-1775 in combination with different agents that damage DNA in cancer patients.
| Targets |
Wee1 (IC50 = 5.2 nM)
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| ln Vitro |
MK-1775 blocks Wee1 kinase in a way that is competitive with ATP. In contrast to Wee1, MK-1775 exhibits >100-fold selectivity over human Myt 1, another kinase that phosphorylates cyclin-dependent kinase 1 (CDC2) at a different site (Thr14), 2- to 3-fold less potency against Yes with an IC50 of 14 nM, and 10-fold less potency against seven other kinases with >80% inhibition at 1 μM. MK-1775 treatment inhibits the basal phosphorylation of CDC2 at Tyr15 (CDC2Y15) with an EC50 of 49 nM and suppresses gemcitabine-, carboplatin-, or cisplatin-induced phosphorylation of CDC2 and cell cycle arrest in a dose-dependent manner, with EC50 of 82 nM and 81 nM, 180 nM and 163 nM, as well as 159 nM and 160 nM, respectively. These effects are achieved by disrupting the DNA damage checkpoint in WiDr cells bearing mutated p53. While MK-1775 treatment at 300 nM, which is sufficient to inhibit Wee1 by >80%, exhibits moderate but significant antiproliferative effects by 34.1% in WiDr cells and 28.4% in H1299 cells, MK-1775 treatment at 30-100 nM has no significant antiproliferative effect in WiDr and H1299 cells.[1]
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| ln Vivo |
In rats with a T/C of 69% on day 3, MK-1775 treatment alone at ~20 mg/kg shows negligible antitumor effects against WiDr xenografts. In the TOV21G-shp53 and nude rat HeLa-luc xenograft models, MK-1775 alone has a moderate antitumor efficacy. (Source: )
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| Enzyme Assay |
Wee1 is a human recombinant protein. A kinase reaction is carried out using 10 μM ATP, 1.0 μCi of [γ-33P]ATP, and 2.5 μg of poly(Lys, Tyr) as a substrate, all in the presence of increasing MK-1775 concentrations, at 30°C for 30 minutes. Radioactivity that has been mixed into the substrate is trapped on MultiScreen-PH plates and quantified using a liquid scintillation counter.
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| Cell Assay |
The entire protein is extracted from the cell pellet using a lysis solution that contains 0.4 mol/L NaCl, 1 mM EDTA, and 50 mM HEPES (pH 7.9). The protein is then fortified with protease inhibitor, 1% NP-40, and 10 µL/mL phosphatase inhibitor cocktail 1 and 2. The Bio-Rad protein assay yields the protein concentration of the lysates. On an Immobilon membrane, equal volumes of protein are separated using 12% SDS-PAGE. Buffered saline (150 mM, pH 7.4) with 0.1% Tween (TBS-T) and 5% nonfat dry milk block nonspecific binding sites on the membrane. Protein signals are identified by soaking the membrane in 5% nonfat dry milk with a primary antibody overnight at 4°C. This is followed by 45 minutes of incubation in the corresponding secondary antibody that has been peroxidase-conjugated. After that, the membrane is developed using a Typhoon 9400 scanner and enhanced chemiluminescence with ECL and Western Blotting Detection Reagents.
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| Animal Protocol |
Inoculation of 1×106 Calu-6 cells in 10 µL results in the production of tumor xenografts in the leg. Tumors with a diameter of 8 mm are treated with radiation and Adavosertib (MK-1775) for 5 days. Unanesthetized mice are given gamma-rays locally at a dose rate of 5 Gy/min via a small-animal irradiator that consists of two parallel-opposed 137Cs sources for their tumor-bearing legs. Tumors are exposed to radiation twice a day, six hours apart. Give adavosertib (MK-1775) by gavage in volumes of 0.1 mL one hour prior to and two hours following the initial daily radiation dosage.
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| References |
>
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| Additional Infomation |
1-[6-(2-hydroxypropan-2-yl)-2-pyridinyl]-6-[4-(4-methyl-1-piperazinyl)anilino]-2-prop-2-enyl-3-pyrazolo[3,4-d]pyrimidinone is a member of piperazines.
MK-1775 has been used in trials studying the treatment of LYMPHOMA, Neoplasms, Ovarian Cancer, Tongue Carcinoma, and Adult Glioblastoma, among others. Adavosertib is a small molecule inhibitor of the tyrosine kinase WEE1 with potential antineoplastic sensitizing activity. Adavosertib selectively targets and inhibits WEE1, a tyrosine kinase that phosphorylates cyclin-dependent kinase 1 (CDK1, CDC2) to inactivate the CDC2/cyclin B complex. Inhibition of WEE1 activity prevents the phosphorylation of CDC2 and impairs the G2 DNA damage checkpoint. This may lead to apoptosis upon treatment with DNA damaging chemotherapeutic agents. Unlike normal cells, most p53 deficient or mutated human cancers lack the G1 checkpoint as p53 is the key regulator of the G1 checkpoint and these cells rely on the G2 checkpoint for DNA repair to damaged cells. Annulment of the G2 checkpoint may therefore make p53 deficient tumor cells more vulnerable to antineoplastic agents and enhance their cytotoxic effect. Drug Indication Treatment of malignant endometrial neoplasms, Treatment of pancreatic cancer |
| Molecular Formula |
C27H32N8O2
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| Molecular Weight |
500.6
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| Exact Mass |
500.264
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| Elemental Analysis |
C, 64.78; H, 6.44; N, 22.38; O, 6.39
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| CAS # |
955365-80-7
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| Related CAS # |
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| PubChem CID |
24856436
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| Appearance |
Yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
723.8±70.0 °C at 760 mmHg
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| Flash Point |
391.5±35.7 °C
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| Vapour Pressure |
0.0±2.5 mmHg at 25°C
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| Index of Refraction |
1.655
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| LogP |
0.5
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
37
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| Complexity |
795
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1N(CC=C)N(C2C=CC=C(C(C)(C)O)N=2)C2C1=CN=C(NC1C=CC(N3CCN(C)CC3)=CC=1)N=2
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| InChi Key |
BKWJAKQVGHWELA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H32N8O2/c1-5-13-34-25(36)21-18-28-26(29-19-9-11-20(12-10-19)33-16-14-32(4)15-17-33)31-24(21)35(34)23-8-6-7-22(30-23)27(2,3)37/h5-12,18,37H,1,13-17H2,2-4H3,(H,28,29,31)
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| Chemical Name |
1-[6-(2-hydroxypropan-2-yl)pyridin-2-yl]-6-[4-(4-methylpiperazin-1-yl)anilino]-2-prop-2-enylpyrazolo[3,4-d]pyrimidin-3-one
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.87 mg/mL (5.73 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (4.16 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 2.08 mg/mL (4.16 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 corn oil and mix evenly. Solubility in Formulation 5: 2% DMSO +30% PEG 300 +5% Tween+ddH2O: 5 mg/mL Solubility in Formulation 6: 5 mg/mL (9.99 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), Suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9976 mL | 9.9880 mL | 19.9760 mL | |
| 5 mM | 0.3995 mL | 1.9976 mL | 3.9952 mL | |
| 10 mM | 0.1998 mL | 0.9988 mL | 1.9976 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01849146 | Active Recruiting |
Drug: Adavosertib Drug: Temozolomide |
Glioblastoma Recurrent Glioblastoma |
National Cancer Institute (NCI) |
August 19, 2013 | Phase 1 |
| NCT02617277 | Active Recruiting |
Drug: AZD1775 Drug: MEDI4736 |
Advanced Solid Tumours | AstraZeneca | December 28, 2015 | Phase 1 |
| NCT02659241 | Active Recruiting |
Other: Laboratory Biomarker Analysis Drug: Adavosertib |
Advanced Ovarian Carcinoma Carcinomatosis |
M.D. Anderson Cancer Center | February 4, 2016 | Early Phase 1 |
| NCT01922076 | Active Recruiting |
Other: Pharmacological Study Drug: Adavosertib |
Glioblastoma Gliosarcoma |
National Cancer Institute (NCI) |
September 3, 2013 | Phase 1 |
| NCT04439227 | Active Recruiting |
Drug: Adavosertib | Advanced Lymphoma Refractory Lymphoma |
National Cancer Institute (NCI) |
March 13, 2017 | Phase 2 |
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