EGFR (IC50 = 0.45 nM)
EGFR (epidermal growth factor receptor) tyrosine kinase. PD 174265 is a potent, selective, and reversible inhibitor of EGFR tyrosine kinase activity. The compound targets the intracellular tyrosine kinase domain of EGFR, blocking its autophosphorylation and subsequent activation of downstream signaling pathways, including the Ras-Raf-MEK-ERK and PI3K-AKT pathways. PD 174265 shows an IC50 of 0.45 nM for EGFR inhibition, indicating exceptionally high potency. The compound's selectivity for EGFR over other kinases contributes to its specificity and reduced off-target effects. PD 174265 is cell-permeable, allowing it to effectively inhibit EGFR in intact cells. The compound's reversible binding mode distinguishes it from irreversible EGFR inhibitors, which may have different pharmacokinetic and safety profiles. PD 174265 has been used to study the role of EGFR in various diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders.

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].
PD 174265 is a potent inhibitor of EGFR tyrosine kinase activity with an IC50 of 0.45 nM (450 pM). The compound demonstrates high potency in cell-free kinase assays, effectively blocking EGFR autophosphorylation. PD 174265 is cell-permeable, enabling it to inhibit EGFR activity in intact cells. The compound's selectivity for EGFR over other kinases contributes to its specificity. PD 174265 inhibits EGFR-dependent signaling pathways, including the MAPK/ERK and PI3K/AKT pathways, which are critical for cell proliferation, survival, and migration. The compound's potent inhibition of EGFR makes it a valuable tool for studying EGFR biology and validating EGFR as a therapeutic target in cancer and other diseases. PD 174265 has been used in research exploring the role of EGFR in cancer, cardiovascular diseases, and neurodegenerative disorders.

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].
PD 174265 has been studied in vivo for its effects on EGFR-dependent processes. The compound's potent and selective EGFR inhibition suggests it would be effective in animal models of EGFR-driven cancers and other diseases. PD 174265 has been used in research exploring cardiovascular diseases and neurodegenerative disorders, where EGFR isoforms are implicated in disease progression. The compound's cell-permeability and reversible binding mode support its use in in vivo studies. Detailed in vivo efficacy data are not extensively available in publicly accessible sources, but PD 174265 is recognized as a valuable tool for studying EGFR-mediated signaling pathways and potential therapeutic applications. PD 174265's potent inhibition allows for precise investigation of EGFR function in disease models.

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.
EGFR kinase activity assays are performed using recombinant EGFR kinase domain or immunoprecipitated EGFR from cell lysates. The enzyme is incubated with a peptide or protein substrate (e.g., poly(Glu,Tyr) or EGFR autophosphorylation sites) and [γ-³²P]ATP in kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Triton X-100). The reaction is incubated at 30°C for 30-60 minutes. Phosphorylated substrate is quantified by scintillation counting, or by Western blotting using phospho-specific antibodies (e.g., anti-phospho-EGFR Y1068). PD 174265 is serially diluted in DMSO and added to the reaction mixture to determine IC50 values. The IC50 for PD 174265 is 0.45 nM. Each concentration is tested in duplicate, and IC50 values are calculated by non-linear regression analysis. Appropriate positive controls (e.g., known EGFR inhibitors like erlotinib) and vehicle controls are included to validate the assay.

Cellular EGFR inhibition is evaluated in cancer cell lines that express EGFR, such as A431, MDA-MB-468, or HCC827 cells. Cells are cultured in appropriate media (e.g., DMEM or RPMI-1640 with 10% FBS) at 37°C with 5% CO₂ and treated with PD 174265 at concentrations ranging from 0.1 nM to 10 μM for 1-24 hours. EGFR autophosphorylation is measured by Western blotting using anti-phospho-EGFR (Tyr1068 or Tyr1173) antibodies. Downstream signaling is assessed by measuring phosphorylation of ERK1/2 (p-ERK) and AKT (p-AKT) by Western blotting. Cell viability and proliferation are assessed using MTT, CCK-8, or CellTiter-Glo assays. Cell cycle analysis is performed by propidium iodide staining and flow cytometry. Apoptosis is evaluated by Annexin V/PI staining, caspase-3/7 activity assays, and PARP cleavage Western blotting. Each experiment includes vehicle controls (DMSO) and appropriate positive controls (e.g., erlotinib or gefitinib).

In vivo efficacy of PD 174265 is evaluated in mouse xenograft models using EGFR-driven cancer cell lines (e.g., A431, HCC827, or MDA-MB-468). Tumor cells are implanted subcutaneously in immunodeficient mice. When tumors reach a predetermined size, mice are randomized into treatment and control groups. PD 174265 is administered via intraperitoneal injection or oral gavage at doses determined by preclinical studies. The compound is formulated in appropriate vehicles (e.g., DMSO/PEG300/Tween 80). Tumor growth is monitored by caliper measurements. Body weight and clinical signs are monitored throughout the study to assess tolerability. At study endpoint, tumors are harvested for histopathological analysis, immunohistochemistry (Ki67 for proliferation, cleaved caspase-3 for apoptosis), and biochemical assays (EGFR phosphorylation, downstream signaling). Sample sizes typically range from 6-10 animals per group.

Molecular Weight: 371.23. Formula: C17H15BrN4O. CAS No.: 216163-53-0. Purity: ≥97-98% (HPLC). Solubility: DMSO: 125 mg/mL (336.72 mM). Appearance: Solid. Storage: Store at -20°C. Shipping: With ice pack. Stability: ≥2 years. PD 174265 is a potent, cell-permeable, reversible, and selective inhibitor of EGFR tyrosine kinase activity. The compound has an IC50 of 0.45 nM for EGFR inhibition. PD 174265 is a valuable tool for studying EGFR-mediated signaling pathways and potential therapeutic applications in cancer, cardiovascular diseases, and neurodegenerative disorders.

No comprehensive toxicology data are publicly available for PD 174265. The compound is intended for research use only and has not undergone full preclinical toxicology evaluation required for clinical development. As an EGFR inhibitor, potential adverse effects may include skin rash, diarrhea, and other class effects associated with EGFR targeting. The compound's selectivity for EGFR over other kinases may reduce off-target toxicities. Standard toxicity studies would include acute toxicity assessment in rodents, repeated dose toxicity studies (14-day and 28-day), and genotoxicity screening (Ames test, micronucleus assay). PD 174265 is for research use only and not for human therapeutic use.

[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]
PD 174265 is also known as PD174265, PD-174265. Its IUPAC name is N-{4-[(3-Bromophenyl)amino]quinazolin-6-Yl}propanamide. MDL: MFCD02179208. PD 174265 is a potent, cell-permeable, reversible, and selective inhibitor of EGFR with an IC50 of 0.45 nM. The compound is valuable in studying cancer, cardiovascular diseases, and neurodegenerative disorders, where EGFR isoforms are implicated in disease progression. PD 174265 has been used in research exploring PKC-mediated signaling pathways. No clinical trials have been reported for this compound. PD 174265 is strictly for research use only and not for human use.

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

CCC(=O)NC1=CC2=C(C=C1)N=CN=C2NC3=CC(=CC=C3)Br

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.)