EGFR (IC50 = 0.45 nM)

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].

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].

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.

[1]. Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):12022-7.

This invention discloses a class of high-affinity inhibitors that selectively and irreversibly inactivate epidermal growth factor receptor tyrosine kinases by specifically covalently modifying cysteine residues present in the ATP-binding pocket. A series of experiments using mass spectrometry, molecular modeling, site-directed mutagenesis, and live-cell 14C labeling clearly demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor in a 1:1 stoichiometric ratio and alkylate Cys-773. Although these compounds are essentially inactive in solution, they are subjected to rapid nucleophilic attack by this specific amino acid when bound to the ATP-binding pocket. The molecular orientation and position of the acrylamide group in these inhibitors relative to Cys-773 fully support the results determined by molecular docking experiments based on homology modeling of the ATP-binding site. Evidence also indicates that these compounds interact similarly with erbB2, but are inactive against other receptor tyrosine kinases or intracellular tyrosine kinases tested in this study. Finally, a direct comparison between 6-acrylamido-4-aniline-quinazoline and a potent but reversible analogue showed that the irreversible inhibitor had far superior in vivo antitumor activity compared to the reversible analogue in a human epidermoid carcinoma xenograft model, with no significant toxicity observed at therapeutic doses. The activity profile of this compound is typical of a new generation of tyrosine kinase inhibitors and has great potential for application in the treatment of proliferative diseases. [1]

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The irreversibility of these compounds may bring other potential advantages in terms of target inhibition and in vivo pharmacokinetics. To achieve optimal antitumor activity, long-term inhibition of kinase targets may be required. Irreversible inhibitors have an advantage in this regard because they permanently eliminate existing kinase activity, which is only restored after the synthesis of new receptors. Current compounds only need to remain in plasma for a short enough time to expose the receptor to the drug briefly, thereby irreversibly inhibiting its kinase activity. Afterward, plasma concentrations can drop rapidly while kinase activity remains inactive. This has the potential advantages of reducing minimum effective plasma concentrations, reducing the need for multiple dosings, and not requiring a long plasma half-life without affecting efficacy. All of these factors contribute to reducing toxicity caused by nonspecific interactions that may occur at high or prolonged plasma concentrations. These pharmacokinetic factors may have some influence on the data in Figure 6, which clearly shows that PD 168393 is significantly superior to the reversible compound PD 174265 under the dosing regimen given in this experiment. [1]

C17H15BRN4O

371.24

370.043

C, 55.00; H, 4.07; Br, 21.52; N, 15.09; O, 4.31

216163-53-0

4709

Light yellow to yellow solid powder

4.63

2

4

4

23

408

0

WUPUZEMRHDROEO-UHFFFAOYSA-N

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)

N-[4-(3-bromoanilino)quinazolin-6-yl]propanamide

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

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~336.72 mM)

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.)