Size | Price | Stock | Qty |
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10mg |
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50mg |
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100mg |
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250mg |
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Other Sizes |
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Targets |
EGFR (IC50 = 0.45 nM)
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ln Vitro |
When the protein was incubated with PD174265, no increase in mass occurred, indicating the lack of a covalent modification. Trypsin digestion of the drug-bound protein and analyses by liquid chromatography-ESI tandem MS (MS/MS) identified Cys-773 as the predominant site of interaction (Fig. 3b). The only other modified residue detected was Cys-926; however, this represented a relatively minor component. No other residues, including the four remaining cysteines, were found to be altered. As additional evidence that Cys-773 is the specific amino acid that combines with PD 168393, recombinant wild-type full cytoplasmic domain of human EGFr and a point mutant, in which Cys-773 was replaced with Ser (C773S), were expressed in baculovirus and purified from infected insect cells. Both wild-type and mutant protein exhibited comparable TK activity and were inhibited by both PD 168393 and PD174265 at similar doses[1].
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ln Vivo |
To illustrate the advantage of irreversibility, a direct comparison between PD 168393 (irreversible) and PD174265 (reversible) for target modulation in viable cells is shown in Table 2. PD 168393 inhibited EGFr autophosphorylation in A431 human epidermoid carcinoma cells with >9-fold greater potency than PD174265. An even greater difference was seen against heregulin-mediated tyrosine phosphorylation in MDA-MB-453 human breast carcinoma cells, where PD 168393 was >30-fold more potent. The therapeutic advantage of irreversible inhibition is illustrated quite dramatically in Fig. 6a, which shows a head-to-head comparison of in vivo activity for PD 168393 and PD174265 against the A431 human epidermoid carcinoma grown as a xenograft in nude mice. PD 168393 was far superior to PD174265 in maintaining suppression of tumor growth with once-daily i.p. dosing. PD 168393 produced tumor growth inhibition of 115%, which for this experiment is defined as the median time for treated tumors to reach three volume doublings minus the median time for control tumors to reach three volume doublings, expressed as a percent of treatment duration (15 days). PD174265, in contrast, produced a tumor growth inhibition of only 13%. The antitumor activity of these two compounds correlated with their ability to suppress the phosphotyrosine content of the EGFr. Both compounds had reduced the phosphorylation status by ≈80%, 4 hr after injection (Fig. 6b). However, by 8 hr, phosphorylation had returned to 75% of controls in mice treated with the reversible compound, PD174265, and to 100% after 24 hr. In contrast, the phosphotyrosine content of EGFr in animals receiving PD 168393 was still reduced by 50% 24 hr after injection. The therapeutic advantage of PD 168393 was maintained despite a lower plasma concentration than that of PD174265 at all time points examined (data not shown)[1].
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Enzyme Assay |
A solution of PD 168393 or PD174265 (in dimethyl sulfoxide) was added to 50 μg of EGFr TK catalytic domain in 20 mM Tris (pH 8.0), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 1 μg/ml of the protease inhibitors (aprotinin and leupeptin), and diluted with 75 mM ammonium bicarbonate (pH 7.5). After 90 min, the reaction was quenched by addition of 5% (vol/vol) acetic acid. An aliquot of protein was analyzed by electrospray ionization (ESI)-MS on a Finnigan MAT 900Q mass spectrometer, equipped with a low-flow micro-ESI source operating at 150 nl/min. The remaining protein was reduced, alkylated, and digested with 0.5 μg of trypsin as described (40). Lyophilized tryptic peptides were suspended in 0.1% trifluoroacetic acid/2% CH3CN and analyzed by liquid chromatography-ESI-MS on a Michrom BioResources Magic 2002 HPLC equipped with a 0.3 × 15 mm Vydac C18 column and coupled to a Finnigan LCQ quadruple ion trap mass spectrometer. Data were collected using the Finnigan navigator 1.0.1 data acquisition software set to the following default values for the automatic gain control for full scan MS, zoom scan MS, and MS/MS, respectively: 5 × 107, 1 × 107, and 2 × 107. A maximum injection time of 200 ms and three microscans were used to collect data in all modes. A relative collision energy of 35% was used for MS/MS fragmentation spectra.
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References | |
Additional Infomation |
A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.[1]
The irreversible nature of these compounds may offer other potential advantages in terms of target suppression and in vivo pharmacokinetics. Prolonged suppression of the kinase target(s) will likely be necessary for maximum antitumor activity, and an irreversible inhibitor will provide an advantage in this respect by permanently eliminating existing kinase activity, which will return only when new receptor is synthesized. The present compounds would require that plasma concentrations be attained only long enough to briefly expose the receptors to drug, which would irreversibly suppress their kinase activity. Plasma levels could then rapidly decline while kinase activity would remain inactivated. This has the potential advantage of lowering the minimal plasma concentration at which activity occurs, minimizing multiple dosing requirements and eliminating the requirement for long plasma half-lives without compromising efficacy. All of these considerations could reduce toxicity due to any nonspecific interactions that may occur at high or prolonged plasma levels. These pharmacokinetic factors may be contributing in part to the data in Fig. 6, where it is clear that under the dosing schedule given in this experiment, PD 168393 was markedly superior to the reversible compound, PD 174265.[1] |
Molecular Formula |
C17H15BRN4O
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Molecular Weight |
371.24
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Exact Mass |
370.043
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Elemental Analysis |
C, 55.00; H, 4.07; Br, 21.52; N, 15.09; O, 4.31
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CAS # |
216163-53-0
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PubChem CID |
4709
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Appearance |
Light yellow to yellow solid powder
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LogP |
4.63
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Hydrogen Bond Donor Count |
2
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Hydrogen Bond Acceptor Count |
4
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Rotatable Bond Count |
4
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Heavy Atom Count |
23
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Complexity |
408
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Defined Atom Stereocenter Count |
0
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InChi Key |
WUPUZEMRHDROEO-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C17H15BrN4O/c1-2-16(23)21-13-6-7-15-14(9-13)17(20-10-19-15)22-12-5-3-4-11(18)8-12/h3-10H,2H2,1H3,(H,21,23)(H,19,20,22)
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Chemical Name |
N-[4-(3-bromoanilino)quinazolin-6-yl]propanamide
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Synonyms |
PD-174265; PD174265; pd 174265; 216163-53-0; N-{4-[(3-Bromophenyl)amino]quinazolin-6-Yl}propanamide; 4-[(3-Bromophenyl)amino]-6-propionylamidoquinazoline; 4-aminoquinazoline, 2a; PD-174265; CHEMBL188762; N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)propionamide; PD 174265
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
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 : ~125 mg/mL (~336.72 mM)
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Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80: Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400  (Please use freshly prepared in vivo formulations for optimal results.) |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 2.6937 mL | 13.4684 mL | 26.9368 mL | |
5 mM | 0.5387 mL | 2.6937 mL | 5.3874 mL | |
10 mM | 0.2694 mL | 1.3468 mL | 2.6937 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.