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Purity: ≥98%
NVP-AEW541 cis-isomer (also known as AEW541 cis-isomer), the cis-isomer of AEW541 is a novel and potent inhibitor of IGF-1R/InsR with IC50 of 150 nM/140 nM in cell-free assays, it has greater potency and selectivity for IGF-1R in a cell-based assay. In soft agar, NVP-AEW541 blocks IGF-I-mediated survival and colony formation at levels that are compatible with suppressing IGF-IR autophosphorylation. This oral bioavailable substance considerably slows the growth of IGF-IR-driven fibrosarcomas and inhibits IGF-IR signaling in tumor xenografts in vivo. As a result, NVP-AEW541 is a member of a class of small, selective IGF-IR kinase inhibitors that have demonstrated in vivo antitumor activity and may find use in therapeutic settings.
| Targets |
IGF-IR (IC50 = 0.15 ±0.036 μM); InsR (IC50 = 0.14±0.039 μM); Flt-3 (IC50 = 0.42±0.11 μM); PDGFR (IC50 = 2±0.61 μM); c-Src (IC50 = 2.4±0.38 μM); c-Kit (IC50 = 3.3±1.4 μM)
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| ln Vitro |
NVP-AEW541 is equipotent against the recombinant InsR kinase domain and inhibits the in vitro kinase activity of the recombinant IGF-IR kinase domain with an IC50 value of 0.15 μM. At the cellular level, NVP-AEW541 has demonstrated selectivity and is confirmed to be active against IGF-IR kinase (IC50=86 nM). When compared to the structurally related native InsR (IC50=2.3 μM), NVP-AEW541 is found to be 27-fold more potent toward the native IGF-IR. With IC50 values of 0.162 μM, 0.105 μM, and 1.64 μM, respectively, NVP-AEW541 inhibits the IGF-I-mediated survival, soft agar, and proliferation of MCF-7 cells[1].
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| ln Vivo |
NVP-AEW541 (20, 30, or 50 mg/kg) administered orally causes the NWT-21 tumor xenograft to lose its basal and IGF-I-induced receptors, as well as its phosphorylation of PKB and MAPK[1]. Using oral gavage, NVP-AEW541 is given twice a day for 14 days in a row at a dose of 50 mg/kg in 0.2 mL of 25 mM L-(+)-tartaric acid. A 0.2 mL carrier [25 mM L-(+)-tartaric acid] administered twice a day is the same treatment given to the control group. Measurements of tumor volume and animal weight are made three times a week until the course of treatment is completed. Tumors are gathered, formalin-fixed, and analyzed histologically and immunohistochemically at that point after the animals have been sacrificed. Tissue shrinkage resulting from NVP-AEW541 treatment reaches statistical significance in both cases (P=0.0156 and P=0.0111 for HTLA-230 and SK-N-BE2c, respectively)[2].
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| Enzyme Assay |
The incorporation of 33P from [γ33P]ATP (1000 Ci/mmol) into suitable substrates is used to measure the activities of protein kinases in the presence or absence of inhibitors. The protein kinase assays are conducted in 96-well plates at room temperature (RT) using the parameters outlined below. The assays are concluded with the addition of 20 μL of 125 mM EDTA. The reaction mixture is then transferred onto Immobilon-PVDF, which has been pre-soaked for five minutes with methanol, rinsed with water, and soaked for five minutes with 0.5% H3PO4 before being mounted on a vacuum manifold in 30 μL (c-Abl, c-Src, and IGF-1R) or 40 μL (all other kinases). Once every sample has been spotted, the vacuum is connected, and 200 μL of 0.5% H3PO4 is rinsed through each well. Membranes are taken out and given a 4× shake on 1% H3PO4 after being once again cleaned with ethanol. Membranes are counted after drying, mounting in a Packard TopCount 96-well frame, and adding 10 μL of Microscint per well. By performing a linear regression analysis on the percentage inhibition of each compound in duplicate at four different concentrations (typically 0.01, 0.1, 1, and 10 μM), IC50 values are determined. A single unit of protein kinase activity is measured as one nanomole of 33P transferred per minute per milligram of protein at 37C from [γ33P]ATP to the substrate protein[1].
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| Cell Assay |
In 96-well plates, 3000–6000 cells are seeded per well, with a 100 μL media volume per well overall. Twenty-four hours later, the compound is added in quadruplicate at increasing concentrations. After 72 hours, cells are fixed by adding 25 μL/well of 20% glutaraldehyde and letting them sit at room temperature for 10 minutes. After two rounds of washing with 200μL/well H2O, 100μL of Methylene Blue (0.05%) is added to the cells. Following a 10-minute RT incubation period, cells are triple-washed using 200 μL/well H2O. After adding 200 μL/well of HCl (3%) and incubating on a plate shaker for 30 minutes at room temperature, absorbance at 650 nm is measured[1].
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| Animal Protocol |
Mice: The Harlan athymic female nude mice are utilized. DMEM (high glucose, 4.5 g/L), 10% FCS, 1% L-glutamine, and 1% Na-pyruvate are used to cultivate NWT-21 cells. Five mice are first given a subcutaneous injection of 5×106 cells/animal into their right flank. Tumors ranging in size from 500 to 800 mm3 are removed, and non-crotic regions are divided into 3 x 3 x 3 mm pieces for the in vivo effectiveness trial. One tumor fragment per animal is transplanted s.c. into the right flank after being cleaned in sterile PBS. Body weights and tumor volumes (length × width × height ×π/6) are calculated three times a week. The therapy group (NVP-AEW541) and the control group (vehicle only) are chosen by stratification on the first day of treatment (day 0) (8 animals per group, average tumor volume of about 95 mm3 per group). The animals receive oral treatment twice a day, seven days a week, using either 25 mM L(+)-tartaric acid (control group) or NVP-AEW541 (20, 30, or 50 mg/kg; 10 mL/kg dissolved in 25 mM L(+)-tartaric acid, therapy group). T/C%, or the mean increase in tumor volumes of treated animals divided by the mean increase in tumor volumes of control animals multiplied by 100, is the expression used to express antitumor activity. When the mean tumor volume approaches 1500 mm3, the experiment comes to an end.
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| References |
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| Molecular Formula |
C27H29N5O
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|---|---|
| Molecular Weight |
439.55
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| Exact Mass |
439.237
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| Elemental Analysis |
C, 73.78; H, 6.65; N, 15.93; O, 3.64
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| CAS # |
475489-16-8
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| Related CAS # | |
| PubChem CID |
11476171
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| Appearance |
white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
669.0±55.0 °C at 760 mmHg
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| Melting Point |
145℃
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| Flash Point |
358.4±31.5 °C
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| Vapour Pressure |
0.0±2.0 mmHg at 25°C
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| Index of Refraction |
1.710
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| LogP |
4.66
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
33
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| Complexity |
632
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| Defined Atom Stereocenter Count |
0
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| SMILES |
NC1=C2C(N([C@@H]3C[C@@H](C3)CN4CCC4)C=C2C5=CC=CC(OCC6=CC=CC=C6)=C5)=NC=N1
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| InChi Key |
AECDBHGVIIRMOI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H29N5O/c28-26-25-24(21-8-4-9-23(14-21)33-17-19-6-2-1-3-7-19)16-32(27(25)30-18-29-26)22-12-20(13-22)15-31-10-5-11-31/h1-4,6-9,14,16,18,20,22H,5,10-13,15,17H2,(H2,28,29,30)
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| Chemical Name |
7-[3-(azetidin-1-ylmethyl)cyclobutyl]-5-(3-phenylmethoxyphenyl)pyrrolo[2,3-d]pyrimidin-4-amine
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| Synonyms |
AEW541 cis-isomer; NVP-AEW541; NVP-AEW 541; NVP-AEW-541; AEW-541; AEW 541; AEW541
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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: 50~88 mg/mL(113.8~200.2 mM)
Ethanol: ~24 mg/mL (~54.6 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.69 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 25.0 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: 2.5 mg/mL (5.69 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 20 mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2751 mL | 11.3753 mL | 22.7505 mL | |
| 5 mM | 0.4550 mL | 2.2751 mL | 4.5501 mL | |
| 10 mM | 0.2275 mL | 1.1375 mL | 2.2751 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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