PD168393 (PD-168393)

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 对接至 EGFR TK 的 ATP 结合袋中。 PD168393 在连续暴露期间完全抑制 A431 细胞中的 EGF 依赖性受体自身磷酸化，甚至在不含化合物的培养基中 8 小时后也能持续抑制。 PD168393 抑制 MDA-MB-453 细胞中调蛋白诱导的酪氨酸磷酸化，IC50 为 5.7 nM。 PD168393 对胰岛素、PDGF 和基本 FGFR TK 以及 PKC 无活性。 PD168393 抑制 HS-27 人成纤维细胞中 EGF 介导的酪氨酸磷酸化，IC50 为 1-6 nM，但对 FGF 或 PDGF 介导的酪氨酸磷酸化几乎没有影响。 PD168393 在 3T3-Her2 细胞中显示出对 Her2 诱导的酪氨酸磷酸化的快速有效抑制，IC50 约为 100 nM。 D168393 还抑制 3T3-Her2 细胞中 PLCγ1/Stat1/Dok1/δ-catenin 的磷酸化（Fyb 除外）。激酶测定：PD168393 是一种有效的、细胞渗透性的、不可逆的 EGFR 抑制剂，IC50 为 0.70 nM，不可逆地烷基化 Cys-773，对胰岛素、PDGFR、FGFR 和 PKC 无活性。目标：EGFR IC 50：0.7 nM (1) PD 168393 抑制 A431 人表皮样癌细胞中的 EGFr 自身磷酸化，其效力比 PD 174265 高 9 倍以上。(2) PD 168393 减少 TNF-α 的产生和 ERK1/磷酸化LPS 在心肌细胞中诱导 2 和 p38。 (3) PD168393 在低至 0.03 umol/L 的浓度下即可完全抑制 AKT 和 ERK 磷酸化。 (4) PD168393可以诱导ErbB2阳性肺癌和乳腺癌细胞系凋亡并抑制细胞生长。 (5) 通过抑制磷酸化 p44/42 ERK 来确定，PD168393 会破坏 HaCaT 细胞中的 MEK1/p44/42 ERK 信号传导。细胞测定：(1) PD 168393 抑制 A431 人表皮样癌细胞中的 EGFr 自磷酸化，其效力比 PD 174265 高 9 倍。(2) PD 168393 减少 TNF-α 的产生以及由 TNF-α 诱导的 ERK1/2 和 p38 磷酸化心肌细胞中的脂多糖。 (3) PD168393 在低至 0.03 umol/L 的浓度下即可完全抑制 AKT 和 ERK 磷酸化。 (4) PD168393可以诱导ErbB2阳性肺癌和乳腺癌细胞系凋亡并抑制细胞生长。

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PD168393（PD-168393）剂量依赖性抑制EGFR过表达的肿瘤细胞系增殖，包括A431表皮样癌细胞（IC₅₀=0.04μM）和MDA-MB-468乳腺癌细胞（IC₅₀=0.06μM）。浓度≥0.1μM时，可阻断这些细胞中表皮生长因子（EGF）诱导的EGFR磷酸化及下游ERK1/2信号通路[1]
PD168393（PD-168393）在0.2μM浓度下，可分别抑制A431细胞的迁移和侵袭约70%和65%，其机制与下调基质金属蛋白酶-9（MMP-9）表达有关[2]
在原代皮质神经元中，PD168393（PD-168393）（1μM）通过阻断EGFR磷酸化及下游PI3K/Akt信号，抑制EGFR介导的神经突生长[3]

PD 168393 在裸鼠 A431 人表皮样癌异种移植物中产生 115% 的肿瘤生长抑制，同时 EGFR 磷酸酪氨酸含量降低 50%。 PD 168393 还显示出低血浆浓度。

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在本研究中，研究人员评估了EGFR抑制剂PD168393（PD）对小鼠挫伤性SCI模型髓鞘形成的影响。研究发现，经PD168393处理的小鼠脊髓损伤后，髓鞘碱性蛋白（MBP）的表达显著升高。PD168393处理后，病变中的胶质前体细胞和少突胶质细胞（OLs）密度增加，细胞凋亡减弱。此外，PD168393治疗降低了脊髓受损区域OX42+小胶质细胞和胶质纤维酸性蛋白+星形胶质细胞的数量。因此，他们得出结论，SCI后EGFR抑制的治疗作用包括促进受损脊髓的髓鞘再生，增加少突胶质前体细胞和OLs，以及抑制星形胶质细胞和小胶质细胞/巨噬细胞的活化[3]。

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PD168393（PD-168393）以15mg/kg/天的剂量腹腔注射给药21天，可抑制裸鼠A431异种移植瘤的生长。与对照组相比，肿瘤体积减少约68%，瘤内EGFR磷酸化水平显著下调[1]
在7,12-二甲基苯并(a)蒽（DMBA）和12-O-十四酰佛波醇-13-乙酸酯（TPA）诱导的小鼠皮肤癌发生模型中，PD168393（PD-168393）（10mg/kg/天，腹腔注射，持续14周）使皮肤乳头状瘤数量减少约55%[2]

PD168393 是一种有效的、细胞渗透性的、不可逆的 EGFR 抑制剂，可不可逆地烷基化 Cys-773。它对 PDGFR、FGFR、PKC 和胰岛素无活性。其 IC50 为 0.70 nM。目标：EGFR IC 50 = 0.7 nM (1) PD 168393 在抑制 A431 人表皮样癌细胞中 EGFr 自磷酸化方面的效力比 PD 174265 高 9 倍以上。 (2) 在脂多糖 (LPS) 刺激的心肌细胞中，PD 168393 减少 TNF-α 的产生以及 ERK1/2 和 p38 的磷酸化。 (3) 在低至0.03 umol/L的浓度下，PD168393完全抑制AKT和ERK的磷酸化。 (4) 在ErbB2阳性肺癌和乳腺癌细胞系中，PD168393可能导致细胞凋亡并抑制细胞生长。 (5)磷酸化p44/42 ERK的抑制表明PD168393干扰HaCaT细胞中的MEK1/p44/42 ERK信号传导。

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将重组人EGFR激酶结构域与ATP及特异性多肽底物在系列稀释的PD168393（PD-168393）存在下孵育，反应在37°C下进行60分钟，采用放射免疫法检测磷酸化底物。通过与溶媒对照组的放射性对比计算抑制率，从量效曲线中得出IC₅₀值[1]
采用相同方案检测PD168393（PD-168393）对重组胰岛素受体、PDGFR和c-Src激酶的抑制活性以评估选择性，反应条件保持一致，通过确定IC₅₀值证实其对EGFR的选择性靶向[2]

(1) 在 A431 人表皮样癌细胞中，PD 168393 抑制 EGFr 自磷酸化的效力比 PD 174265 高 9 倍以上。 (2) 在脂多糖 (LPS) 刺激的心肌细胞中，PD 168393 减少 TNF-α 的产生以及 ERK1/2 和 p38 的磷酸化。 (3) 在低至0.03 umol/L的浓度下，PD168393完全抑制AKT和ERK的磷酸化。 (4) 在ErbB2阳性肺癌和乳腺癌细胞系中，PD168393可能导致细胞凋亡并抑制细胞生长。

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将A431和MDA-MB-468细胞以5×10³个细胞/孔接种到96孔板中，用PD168393（PD-168393）（0.01-1μM）处理72小时，采用四唑盐法检测细胞活性并计算IC₅₀值。蛋白质印迹分析中，用0.1-0.5μM药物处理细胞并加入EGF刺激，裂解后与抗磷酸化EGFR、ERK1/2和GAPDH的抗体孵育[1]
用PD168393（PD-168393）（0.1-0.5μM）处理A431细胞24小时，采用Boyden小室进行迁移和侵袭实验，通过逆转录-聚合酶链反应（RT-PCR）定量MMP-9 mRNA的表达[2]
分离原代皮质神经元并接种到24孔板中，用PD168393（PD-168393）（0.5-2μM）预处理1小时后，用EGF刺激细胞。48小时后在显微镜下观察神经突生长，通过蛋白质印迹法检测磷酸化EGFR和Akt[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]

PD168393 (PD-168393) had an oral bioavailability of ~28% in mice after a single dose of 15 mg/kg. The plasma half-life was approximately 3.5 hours, and the maximum plasma concentration (Cmax) was 1.8 μg/mL achieved at 1 hour post-administration [1]
In rats, intraperitoneal administration of PD168393 (PD-168393) at 10 mg/kg resulted in an AUC₀-24h of 12.6 μg·h/mL. The drug was primarily distributed in the liver and tumor tissues, with a tumor-to-plasma concentration ratio of ~2.1 [2]

Mice treated with PD168393 (PD-168393) at 15 mg/kg/day (i.p.) for 21 days showed mild weight loss (~6%) but no significant liver or kidney toxicity. Serum ALT, AST, and creatinine levels were within normal ranges [1]
In long-term toxicity studies (14 weeks, 10 mg/kg/day, i.p.), rats showed no hematological abnormalities or gastrointestinal side effects. The plasma protein binding rate of PD168393 (PD-168393) was ~90% in human plasma as determined by equilibrium 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 member of the class of quinazolines carrying bromoanilino and acrylamido substituents at positions 4 and 6 respectively. It has a role as an epidermal growth factor receptor antagonist. It is a member of quinazolines, a member of acrylamides, a substituted aniline, a member of bromobenzenes and a secondary carboxamide.
PD168393 is an epidermal growth factor receptor inhibitor.
EGFR Inhibitor PD-168393 is a quinazolone compound with anti-tumor activity. PD-168393 is a cell-permeable, irreversible, and selective inhibitor of ligand-dependent epidermal growth factor (EGF) receptor (EGFR). This agent binds to the catalytic domain of EGFR with a 1:1 stoichiometry and inactivates the EGFR tyrosine kinase activity through alkylation of a cystine residue (Cys-773) within the ATP-binding pocket, thereby inhibiting proliferation of EGFR-expressing tumor cells.

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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 PD168393 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]

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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 sought to characterize Her2 signal transduction pathways further by using MS-based quantitative proteomics. Stably transfected cell lines overexpressing Her2 or empty vector were generated, and the effect of an EGFR and Her2 selective tyrosine kinase inhibitor, PD168393, on these cells was characterized. Quantitative measurements were obtained on 462 proteins by using the SILAC (stable isotope labeling with amino acids in cell culture) method to monitor three conditions simultaneously. Of these proteins, 198 showed a significant increase in tyrosine phosphorylation in Her2-overexpressing cells, and 81 showed a significant decrease in phosphorylation. Treatment of Her2-overexpressing cells with PD168393 showed rapid reversibility of the majority of the Her2-triggered phosphorylation events. Phosphoproteins that were identified included many known Her2 signaling molecules as well as known EGFR signaling proteins that had not been previously linked to Her2, such as Stat1, Dok1, and delta-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site in Her2, Y877, which is located in the activation loop of the kinase domain, is distinct from the known C-terminal tail autophosphorylation sites, and may have important implications for regulation of Her2 signaling. Network modeling, which combined phosphoproteomic results with literature-curated protein-protein interaction data, was used to suggest roles for some of the previously unidentified Her2 signaling proteins.[2]

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Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD 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. We 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) is a reversible small-molecule inhibitor that binds to the ATP-binding site of EGFR tyrosine kinase, blocking EGF-mediated signaling pathways involved in cell proliferation, migration, and survival [1]
Beyond antitumor activity, PD168393 (PD-168393) exhibits potential in studying EGFR-mediated neurite outgrowth and may provide insights into the role of EGFR in neurodevelopmental and neurodegenerative diseases [3]
The drug is widely used as a tool compound in preclinical research to investigate EGFR signaling, but it has not been advanced to clinical trials due to suboptimal pharmacokinetic properties [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

70.4

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.77 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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