EGFR (IC50 = 0.7 nM)
PD168393 (PD-168393) selectively inhibits epidermal growth factor receptor (EGFR) tyrosine kinase (IC₅₀ = 2 nM for recombinant EGFR; Ki = 0.7 nM for EGFR ATP-binding site) [1]
PD168393 (PD-168393) shows no significant inhibitory activity against insulin receptor, platelet-derived growth factor receptor (PDGFR), or c-Src (IC₅₀ > 1000 nM) [2]

PD 168393 docks into EGFR TK's ATP binding pocket. In A431 cells, continuous exposure to PD168393 completely stops EGF-dependent receptor autophosphorylation, and the suppression continues even after 8 hours in compound-free medium. With an IC50 of 5.7 nM, PD168393 prevents heregulin-induced tyrosine phosphorylation in MDA-MB-453 cells. PD168393 is not active against PKC, insulin, PDGF, or basic FGFR TKs. With an IC50 of 1-6 nM, PD168393 suppresses EGF-mediated tyrosine phosphorylation in HS-27 human fibroblasts but has no effect on PDGF- or FGF-mediated tyrosine phosphorylation.[1] In 3T3-Her2 cells, PD168393 exhibits a swift and strong suppression of Her2-induced tyrosine phosphorylation, with an IC50 of approximately 100 nM. In 3T3-Her2 cells, D168393 also prevents PLCγ1/Stat1/Dok1/δ-catenin from being phosphorylated, with the exception of Fyb.[2]

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PD168393 (PD-168393) dose-dependently inhibited the proliferation of EGFR-overexpressing tumor cell lines, including A431 (epidermoid carcinoma, IC₅₀ = 0.04 μM) and MDA-MB-468 (breast cancer, IC₅₀ = 0.06 μM). It blocked EGF-induced EGFR phosphorylation and downstream ERK1/2 signaling in these cells at concentrations ≥ 0.1 μM [1]
PD168393 (PD-168393) suppressed the migration and invasion of A431 cells by ~70% and ~65% at 0.2 μM, respectively, by downregulating matrix metalloproteinase-9 (MMP-9) expression [2]
In primary cortical neurons, PD168393 (PD-168393) (1 μM) inhibited EGFR-mediated neurite outgrowth by blocking EGFR phosphorylation and downstream PI3K/Akt signaling [3]

PD168393 (intraperitoneal injection; 58 mg/kg; once daily; days 10-14, 17-21, and 24-28) is efficacious in vivo, exhibiting 115% tumor growth inhibition in human epidermoid carcinoma xenografts in mice following a 15-day treatment period.

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In the present study, researchers evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. It was found that expression of myelin basic protein (MBP) in the injured spinal cords of PD168393 treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. They thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages[3].

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PD168393 (PD-168393) inhibited tumor growth in nude mice bearing A431 xenografts when administered intraperitoneally at 15 mg/kg/day for 21 days. Tumor volume was reduced by ~68% compared to the control group, and intratumoral EGFR phosphorylation was significantly downregulated [1]
In a mouse model of skin carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), PD168393 (PD-168393) (10 mg/kg/day, i.p. for 14 weeks) reduced the number of skin papillomas by ~55% [2]

PD168393 is an potent, cell-permeable, irreversible EGFR inhibitor that irreversibly alkylates Cys-773. It is inactive against PDGFR, FGFR, PKC, and insulin. Its IC50 is 0.70 nM. goal: EGFR IC 50 = 0.7 nM (1) PD 168393 has >9-fold higher potency than PD 174265 in inhibiting EGFr autophosphorylation in A431 human epidermoid carcinoma cells. (2) In cardiomyocytes stimulated by lipopolysaccharide (LPS), PD 168393 reduces TNF-α production and phosphorylation of ERK1/2 and p38. (3) At concentrations as low as 0.03 umol/L, PD168393 totally inhibits the phosphorylation of AKT and ERK. (4) In ErbB2 positive lung and breast cancer cell lines, PD168393 may cause apoptosis and suppress cell growth. (5) The inhibition of phospho-p44/42 ERK indicated that PD168393 interfered with MEK1/p44/42 ERK signaling in HaCaT cells.

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Recombinant human EGFR kinase domain was incubated with ATP and a specific peptide substrate in the presence of serial dilutions of PD168393 (PD-168393). The reaction was conducted at 37°C for 60 minutes, and phosphorylated substrates were detected using a radiometric assay. Inhibition rates were calculated by comparing radioactivity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1]
To assess selectivity, the same protocol was used to test inhibitory activity against recombinant insulin receptor, PDGFR, and c-Src kinases. Reaction conditions were identical, and IC₅₀ values were determined to confirm selective targeting of EGFR [2]

(1) PD 168393 has >9-fold higher potency than PD 174265 in inhibiting EGFr autophosphorylation in A431 human epidermoid carcinoma cells. (2) In cardiomyocytes stimulated by lipopolysaccharide (LPS), PD 168393 reduces TNF-α production and phosphorylation of ERK1/2 and p38. (3) At concentrations as low as 0.03 umol/L, PD168393 totally inhibits the phosphorylation of AKT and ERK. (4) In ErbB2 positive lung and breast cancer cell lines, PD168393 may cause apoptosis and suppress cell growth.

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A431 and MDA-MB-468 cells were seeded in 96-well plates at 5×10³ cells/well and treated with PD168393 (PD-168393) (0.01-1 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For Western blot analysis, cells were treated with 0.1-0.5 μM drug and stimulated with EGF, then lysed and probed with antibodies against phosphorylated EGFR, ERK1/2, and GAPDH [1]
A431 cells were treated with PD168393 (PD-168393) (0.1-0.5 μM) for 24 hours. Migration and invasion assays were performed using Boyden chambers, and MMP-9 mRNA expression was quantified by RT-PCR [2]
Primary cortical neurons were isolated and seeded in 24-well plates, then treated with PD168393 (PD-168393) (0.5-2 μM) 1 hour before EGF stimulation. Neurite outgrowth was observed under a microscope after 48 hours, and phosphorylated EGFR and Akt were detected by Western blot [3]

Athymic nude mice with A431 human epidermoid carcinoma
58 mg/kg
i.p.

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In Vivo Efficacy.[1]
To illustrate the advantage of irreversibility, a direct comparison between PD168393 (irreversible) and 174265 (reversible) for target modulation in viable cells is shown in Table 2. PD168393 inhibited EGFr autophosphorylation in A431 human epidermoid carcinoma cells with >9-fold greater potency than PD 174265. An even greater difference was seen against heregulin-mediated tyrosine phosphorylation in MDA-MB-453 human breast carcinoma cells, where PD168393 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 PD168393 and 174265 against the A431 human epidermoid carcinoma grown as a xenograft in nude mice. PD168393 was far superior to PD 174265 in maintaining suppression of tumor growth with once-daily i.p. dosing. PD168393 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). PD 174265, 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, PD 174265, and to 100% after 24 hr. In contrast, the phosphotyrosine content of EGFr in animals receiving PD168393 was still reduced by 50% 24 hr after injection. The therapeutic advantage of PD168393 was maintained despite a lower plasma concentration than that of PD 174265 at all time points examined (data not shown).[1]

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Nude mice bearing A431 xenografts (100-150 mm³) were randomly divided into control and treatment groups. PD168393 (PD-168393) was dissolved in DMSO and diluted with saline (final DMSO concentration ≤ 5%), then administered intraperitoneally at 15 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for Western blot analysis of EGFR phosphorylation [1]
Female CD-1 mice were initiated with DMBA (100 μg/mouse) and promoted with TPA (2 μg/mouse) twice weekly for 14 weeks. From week 4, mice were treated with PD168393 (PD-168393) (10 mg/kg/day, i.p.) for 10 weeks. The number of skin papillomas was counted weekly, and skin tissues were collected for histopathological analysis [2]

The bioavailability of PD-168393 (PD-168393) in mice after a single oral dose of 15 mg/kg is approximately 28%. The plasma half-life is approximately 3.5 hours, and the maximum plasma concentration (Cmax) is 1.8 μg/mL 1 hour after administration [1]. In rats, the AUC₀ after intraperitoneal injection of 10 mg/kg of PD-168393 (PD-168393) is 12.6 μg·h/mL 24 hours later. The drug is mainly distributed in the liver and tumor tissues, with a tumor/plasma concentration ratio of approximately 2.1 [2].

Mice treated with PD168393 (PD-168393) at a dose of 15 mg/kg/day (intraperitoneal injection) for 21 days showed a slight decrease in body weight (approximately 6%), but no significant hepatotoxicity or nephrotoxicity was observed. Serum ALT, AST, and creatinine levels were all within the normal range [1]. In long-term toxicity studies (14 weeks, 10 mg/kg/day, intraperitoneal injection), no hematological abnormalities or gastrointestinal side effects were observed in rats. The plasma protein binding rate of PD168393 (PD-168393) in human plasma was approximately 90% as determined by balanced dialysis [2].

[1].Proc Natl Acad Sci U S A. 1998 Sep 29; 95(20): 12022–12027.

[2]. Proc Natl Acad Sci U S A. 2006 Jun 27;103(26):9773-8.

[3]. Cell Mol Neurobiol. 2016 Oct;36(7):1169-78.

PD168393 is a quinazoline compound with bromoaniline and acrylamide substituents at positions 4 and 6, respectively. It is an epidermal growth factor receptor antagonist. PD168393 belongs to the quinazoline, acrylamide, substituted aniline, bromobenzene, and secondary amide classes. PD168393 is an epidermal growth factor receptor inhibitor. The EGFR inhibitor PD-168393 is a quinazoline ketone compound with antitumor activity. PD-168393 is a cell-permeable, irreversible, and selective ligand-dependent epidermal growth factor (EGF) receptor (EGFR) inhibitor. This compound binds to the catalytic domain of EGFR in a 1:1 stoichiometric ratio and inactivates EGFR tyrosine kinase activity by alkylating cysteine residues (Cys-773) within the ATP-binding pocket, thereby inhibiting the proliferation of EGFR-expressing tumor cells. 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 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. These compounds are essentially unreactive in solution, but when bound to the ATP-binding pocket, they undergo rapid nucleophilic attack by this specific amino acid. The molecular orientation and position of the acrylamide group in these inhibitors relative to Cys-773 fully support the results of molecular docking experiments based on homology modeling of the ATP-binding site. Furthermore, there is evidence that these compounds interact with erbB2 in a similar manner, 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 PD168393 had far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model compared to the reversible analogue, and no significant toxicity was observed at therapeutic doses. The activity profile of this compound is typical of a new generation of tyrosine kinase inhibitors with great therapeutic potential and is expected to play an important role in the treatment of proliferative diseases. [1] Her2/neu (Her2) is a tyrosine kinase belonging to the EGF receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. We further characterized the Her2 signaling pathway using mass spectrometry-based quantitative proteomics. We constructed stable transfected cell lines overexpressing Her2 or empty vectors and characterized the effects of the EGFR and Her2 selective tyrosine kinase inhibitor PD168393 on these cells. We used the SILAC (Stable Isotope Labeling in Cell Culture) method to simultaneously monitor proteins under three conditions and performed quantitative analysis on 462 proteins. Of these proteins, 198 showed significantly increased tyrosine phosphorylation levels in Her2-overexpressing cells, while 81 showed significantly decreased levels. Treatment of Her2-overexpressing cells with PD168393 rapidly reversed most Her2-triggered phosphorylation events. The identified phosphorylated proteins included many known Her2 signaling molecules, as well as some previously unassociated EGFR signaling proteins, such as Stat1, Dok1, and δ-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were also identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site, Y877, in the Her2 protein. This site, located in the activation loop of the kinase domain, is different from known C-terminal tail autophosphorylation sites and may play a significant role in the regulation of the Her2 signaling pathway. We used network modeling, combined with phosphoproteomics results and protein-protein interaction data compiled from the literature, to reveal the functions of some previously unidentified Her2 signaling proteins. [2] Preventing demyelination and promoting myelin regeneration of exposed axons is a promising therapeutic strategy for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibitors have been reported to be beneficial for neurological function recovery and axonal regeneration after SCI. However, their role in axonal demyelination and myelin regeneration after spinal cord injury is unclear. In this study, we evaluated the effect of the EGFR inhibitor PD168393 (PD) on myelin formation in a mouse model of spinal cord contusion. We found that the expression of myelin basic protein (MBP) at the site of spinal cord injury was significantly increased in PD-treated mice. After PD168393 treatment, the density of glial precursor cells and oligodendrocytes (OLs) increased and apoptosis at the injury site decreased. In addition, PD168393 treatment also reduced the number of OX42-positive microglia and glial fibrillary acidic protein (GFAP)-positive astrocytes in the spinal cord injury area. Therefore, we conclude that the therapeutic effects of EGFR inhibitors after spinal cord injury include promoting myelin regeneration in the injured spinal cord, increasing the number of oligodendrocyte precursor cells and oligodendrocytes, and inhibiting the activation of astrocytes and microglia/macrophages. [3]

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PD168393 (PD-168393) is a reversible small molecule inhibitor that binds to the ATP binding site of EGFR tyrosine kinase, blocking EGF-mediated cell proliferation, migration and survival signaling pathways. [1]
In addition to its antitumor activity, PD168393 (PD-168393) also shows potential in studying EGFR-mediated neurite growth and may contribute to a deeper understanding of the role of EGFR in neurodevelopment and neurodegenerative diseases. [3]
This drug is widely used as a tool compound in preclinical research. It is used to study the EGFR signaling pathway, but due to its poor pharmacokinetic properties, it has not yet entered the clinical trial stage.[2]

C17H13BRN4O

369.22

368.027

C, 55.30; H, 3.55; Br, 21.64; N, 15.17; O, 4.33

194423-15-9

4708

Light yellow to khaki solid powder

1.6±0.1 g/cm3

571.1±50.0 °C at 760 mmHg

279℃

299.2±30.1 °C

0.0±1.6 mmHg at 25°C

1.744

3.72

2

4

4

23

433

0

BrC1=C([H])C([H])=C([H])C(=C1[H])N([H])C1C2C([H])=C(C([H])=C([H])C=2N=C([H])N=1)N([H])C(C([H])=C([H])[H])=O

HTUBKQUPEREOGA-UHFFFAOYSA-N

InChI=1S/C17H13BrN4O/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/h2-10H,1H2,(H,21,23)(H,19,20,22)

N-[4-(3-bromoanilino)quinazolin-6-yl]prop-2-enamide

PD 168393; PD-168393; 4-[(3-Bromophenyl)amino]-6-acrylamidoquinazoline; pd 168393; N-(4-((3-bromophenyl)amino)quinazolin-6-yl)acrylamide; n-{4-[(3-bromophenyl)amino]quinazolin-6-yl}prop-2-enamide; N-[4-(3-bromoanilino)quinazolin-6-yl]prop-2-enamide; PD168393

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.77 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.

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Solubility in Formulation 2: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)