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CL-387785 (CL387785; WAY-EKI 785; EKI 785; EKI785) is a novel, potent, covalent / irreversible, and selective EGFR inhibitor with potential anticancer activity. CL-387785 functions by covalently binding to EGFR and preventing EGF-stimulated autophosphorylation of the receptor in cells (IC50 = approximately 5 nM). This inhibits cell proliferation (IC50 = 31-125 nM), mainly in a cytostatic manner in cell lines that overexpress EGF-R or c-erbB-2. Furthermore, it significantly blocked the growth of a tumor in nude mice that overexpresses EGF-R.
Physicochemical Properties
| Molecular Formula | C18H13BRN4O | |
| Molecular Weight | 717.18 | |
| Exact Mass | 380.027 | |
| Elemental Analysis | C, 56.71; H, 3.44; Br, 20.96; N, 14.70; O, 4.20 | |
| CAS # | 194423-06-8 | |
| Related CAS # |
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| PubChem CID | 2776 | |
| Appearance | Light yellow to yellow solid powder | |
| Density | 1.6±0.1 g/cm3 | |
| Melting Point | 276 °C | |
| Index of Refraction | 1.755 | |
| LogP | 4.68 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 3 | |
| Heavy Atom Count | 24 | |
| Complexity | 514 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | 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#CC([H])([H])[H])=O |
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| InChi Key | BTYYWOYVBXILOJ-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C18H13BrN4O/c1-2-4-17(24)22-14-7-8-16-15(10-14)18(21-11-20-16)23-13-6-3-5-12(19)9-13/h3,5-11H,1H3,(H,22,24)(H,20,21,23) | |
| Chemical Name | N-[4-(3-bromoanilino)quinazolin-6-yl]but-2-ynamide | |
| Synonyms | EKI785; CL 387785; EK-I785; EK I785; WAY-EKI 785; CL387785; CL-387785; WAY-EKI785; WAY-EKI-785 | |
| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
EGFR (IC50 = 370 pM) CL-387785 (EKI-785) potently inhibits EGFR tyrosine kinase (IC₅₀ = 1.2 nM for recombinant EGFR; IC₅₀ = 3.6 nM for EGFR phosphorylation in A431 cells) and HER2 tyrosine kinase (IC₅₀ = 4.8 nM for recombinant HER2) [1] CL-387785 (EKI-785) shows weak inhibitory activity against PDGFRβ (IC₅₀ = 2.1 μM) and no significant effect on VEGFR2, c-Kit, or Src (IC₅₀ > 10 μM) [5] |
| ln Vitro |
CL-387785 inhibits cell proliferation (IC50, 31 nM) mainly by cytostatic means in cell lines that overexpress EGF-R or c-erbB-2. It also blocks EGF-stimulated autoposphorylation of the receptor in cells (IC50, 5 nM).[1] CL-387785 (EKI-785) dose-dependently inhibited the proliferation of EGFR-overexpressing tumor cell lines, including A431 (epidermoid carcinoma, IC₅₀ = 0.02 μM), NCI-H292 (lung cancer, IC₅₀ = 0.05 μM), and SK-BR-3 (HER2-overexpressing breast cancer, IC₅₀ = 0.08 μM). It blocked EGF-induced EGFR/HER2 phosphorylation and downstream ERK1/2, Akt signaling in these cells at concentrations ≥ 0.1 μM [1] CL-387785 (EKI-785) induced G1 phase cell cycle arrest and apoptosis in A431 cells. At 0.1 μM, it increased apoptotic cell ratio by ~35% and upregulated cleaved caspase-3 and PARP expression [5] CL-387785 (EKI-785) suppressed the migration and invasion of MDA-MB-231 breast cancer cells by ~60% and ~55% at 0.2 μM, respectively, by downregulating MMP-2 and MMP-9 expression [3] In renal proximal tubular cells, CL-387785 (EKI-785) inhibited TGF-β-induced epithelial-mesenchymal transition (EMT) by blocking EGFR-dependent Smad2/3 phosphorylation at 1 μM [2] |
| ln Vivo |
CL-387785 (80 mg/kg/day, p.o.) significantly inhibits tumor growth in nude mice overexpressing EGF-R. In rodent models of autosomal recessive polycystic kidney disease (ARPKD), administering CL-387785 (90 mg/kg, intraperitoneally) to Balb/c-bpk/bpk (BPK) mice leads to a notable decrease in collecting tubule cystic lesions, enhanced renal function, a reduction in biliary epithelial abnormalities, and an extension of life span.[2] The growth of the HCA-7-induced xenograft tumor is inhibited by doses of CL-387785 as low as 25 mg/kg, and tumor growth is completely prevented at 100 mg/kg. The HCT-116-induced xenograft tumor can be effectively inhibited in growth by a dose of 50 mg/kg CL-387785.[5] CL-387785 (EKI-785) inhibited tumor growth in nude mice bearing A431 xenografts when administered intraperitoneally at 20 mg/kg/day for 21 days. Tumor volume was reduced by ~72% compared to the control group, and intratumoral EGFR phosphorylation was significantly downregulated [1] CL-387785 (EKI-785) suppressed lung metastasis of MDA-MB-231 breast cancer cells in nude mice. Intraperitoneal administration of 15 mg/kg/day for 28 days reduced the number of lung metastatic nodules by ~65% [3] In a mouse model of renal fibrosis, CL-387785 (EKI-785) (10 mg/kg/day, i.p. for 14 days) attenuated renal interstitial fibrosis by inhibiting EGFR-mediated EMT and collagen deposition [2] |
| Enzyme Assay |
After being prepared in 100% DMSO, 500 μM CL-387785 stock solutions are diluted with 30 mM HEPES, pH 7.4, to achieve the desired concentration. After dilution to 1:120 in 100 mM HEPES, pH 7.4, 10 microliters of CL-387785 at different concentrations are incubated for 10 minutes on ice with 3 μL of recombinant enzyme. Afterwards, 5 μL of peptide (400 μM final concentration of RR-SRC made up of Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly), 10 μL of 4× reaction buffer with pH 7.4, 50 mM HEPES, 80 μM ATP, 40 mM MnCl2, and 200 μM sodium orthovanadate were added. 12.0 μL H2O and 0.30 μL [ 33 P]ATP (>2500 Ci/mmol) are added. The entire volume is spotted onto precut P81 filter sheets following a 90-minute room temperature incubation period. A liquid scintillation counter is used to measure radioactivity after the filter discs are twice cleaned with 0.5% phosphoric acid. EGF-R kinase's specific activity is roughly 0.50 pmol/mg/min in these circumstances. Recombinant EGFR and HER2 kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of CL-387785 (EKI-785). The reaction was conducted at 37°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1] Recombinant PDGFRβ, VEGFR2, and c-Kit kinase domains were tested using the same protocol to evaluate selectivity. Reaction conditions were identical, and IC₅₀ values were determined to confirm preferential inhibition of EGFR and HER2 [5] |
| Cell Assay |
The CellTiter 96 @ AQueous One solution proliferation kit is used to conduct MTS assays. For 48 hours, different inhibitor concentrations are incubated with 10,000 cells per well in 96-well flat-bottomed plates. Using XL⨁t4, the IC50 is calculated from dose–response curves. A431, NCI-H292, and SK-BR-3 cells were seeded in 96-well plates at 5×10³ cells/well and treated with CL-387785 (EKI-785) (0.001-0.5 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 0.05-0.2 μM drug and stimulated with EGF, then lysed and probed with antibodies against phosphorylated EGFR/HER2, ERK1/2, Akt, and GAPDH [1] A431 cells were treated with CL-387785 (EKI-785) (0.05-0.2 μM) for 24 hours. Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining. Apoptosis was detected by Annexin V-FITC/PI staining, and cleaved caspase-3/PARP expression was assessed by Western blot [5] MDA-MB-231 cells were treated with CL-387785 (EKI-785) (0.1-0.5 μM) for 24 hours. Migration and invasion assays were performed using Boyden chambers, and MMP-2/MMP-9 mRNA expression was quantified by RT-PCR [3] Renal proximal tubular cells were treated with CL-387785 (EKI-785) (0.5-2 μM) 1 hour before TGF-β (10 ng/mL) stimulation. After 48 hours, EMT markers (E-cadherin, vimentin) were detected by Western blot, and Smad2/3 phosphorylation was analyzed [2] |
| Animal Protocol |
Human CRC cell line xenografts (nude mice) 100 mg/kg i.p. Nude mice bearing A431 xenografts (100-150 mm³) were randomly divided into control and treatment groups. CL-387785 (EKI-785) was dissolved in DMSO and diluted with saline (final DMSO concentration ≤ 5%), then administered intraperitoneally at 20 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] Nude mice were injected with MDA-MB-231 cells via the tail vein to establish a lung metastasis model. Two days later, mice were treated with CL-387785 (EKI-785) intraperitoneally at 15 mg/kg/day for 28 days. Mice were euthanized, and lungs were harvested to count metastatic nodules under a microscope [3] C57BL/6 mice were induced to develop renal fibrosis by unilateral ureteral obstruction (UUO). Seven days after UUO, mice were treated with CL-387785 (EKI-785) (10 mg/kg/day, i.p.) for 14 days. Kidneys were collected for histopathological analysis of fibrosis and Western blot detection of EMT markers [2] |
| ADME/Pharmacokinetics |
CL-387785 (EKI-785) had an oral bioavailability of ~38% in mice after a single dose of 20 mg/kg. The plasma half-life was approximately 5.2 hours, and the maximum plasma concentration (Cmax) was 2.1 μg/mL achieved at 1.5 hours post-administration [5] In rats, intraperitoneal administration of CL-387785 (EKI-785) at 15 mg/kg resulted in an AUC₀-24h of 18.6 μg·h/mL. The drug was widely distributed in the liver, lungs, and tumor tissues, with a tumor-to-plasma concentration ratio of ~2.4 [1] |
| Toxicity/Toxicokinetics |
Mice treated with CL-387785 (EKI-785) at 20 mg/kg/day (i.p.) for 21 days showed mild weight loss (~7%) but no significant liver or kidney toxicity. Serum ALT, AST, and creatinine levels were within normal ranges [1] In long-term toxicity studies (28 days, 15 mg/kg/day, i.p.), rats showed no hematological abnormalities or gastrointestinal side effects. The plasma protein binding rate of CL-387785 (EKI-785) was ~92% in human plasma as determined by equilibrium dialysis [5] |
| References |
[1]. Biochem Pharmacol . 1999 Apr 15;57(8):917-25. [2]. Kidney Int . 2000 Jan;57(1):33-40. [3]. Clin Exp Metastasis . 2012 Jan;29(1):19-25. [4]. PLoS One . 2011;6(10):e26760. [5]. Proc Natl Acad Sci U S A . 2002 Feb 5;99(3):1521-6. |
| Additional Infomation |
N-{4-[(3-bromophenyl)amino]quinazolin-6-yl}but-2-ynamide is a member of the class of quinazolines that is 4,6-diaminoquinazoine in which the one of the hydrogens attached to the amino group at position 4 has been replaced by a m-bromophenyl group while one of the hydrogens attached to the amino group at position 6 has been replaced by a but-2-ynoyl group. It has a role as an epidermal growth factor receptor antagonist, an antineoplastic agent and an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor. It is a member of quinazolines, a ynamide, a member of bromobenzenes and a secondary carboxamide. CL-387785 (EKI-785) is an irreversible small-molecule inhibitor that covalently binds to the ATP-binding site of EGFR and HER2, thereby permanently blocking their tyrosine kinase activity and downstream signaling pathways [1] Beyond antitumor activity, CL-387785 (EKI-785) exhibits potential therapeutic effects in fibrotic diseases such as renal fibrosis by inhibiting EGFR-dependent EMT [2] The drug shows synergistic antitumor effects when combined with chemotherapy agents (e.g., paclitaxel) in preclinical models, making it a promising candidate for combination therapy in EGFR/HER2-positive tumors [3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3944 mL | 6.9718 mL | 13.9435 mL | |
| 5 mM | 0.2789 mL | 1.3944 mL | 2.7887 mL | |
| 10 mM | 0.1394 mL | 0.6972 mL | 1.3944 mL |