 Chemical Structure.png)
Crenolanib (formerly also known as CP868596; RO-002; ARO-002), a benzimidazole compound, is an orally bioavailable and selective small molecule inhibitor of platelet-derived growth factor receptor (PDGFRα/β) with potential anticancer activity. In CHO cells, it inhibits PDGFRα/β with Kd values of 2.1 nM/3.2 nM. In clinical trials, the AROG company is developing an investigational medication called crenolanib to treat a variety of cancers, including gliomas, gastrointestinal stromal tumors, and acute myeloid leukemia. By attaching itself to PDGFR and blocking it, clenolanib inhibits tumor angiogenesis and tumor cell proliferation. This can also lead to the inhibition of PDGFR-related signal transduction pathways.
Physicochemical Properties
| Molecular Formula | C26H29N5O2 |
| Molecular Weight | 443.54 |
| Exact Mass | 443.232 |
| Elemental Analysis | C, 70.41; H, 6.59; N, 15.79; O, 7.21 |
| CAS # | 670220-88-9 |
| Related CAS # | 670220-93-6 (besylate);670220-88-9; |
| PubChem CID | 10366136 |
| Appearance | Off-white to light green solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 676.6±65.0 °C at 760 mmHg |
| Flash Point | 363.0±34.3 °C |
| Vapour Pressure | 0.0±2.1 mmHg at 25°C |
| Index of Refraction | 1.704 |
| LogP | 2.99 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 33 |
| Complexity | 667 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O1C([H])([H])C(C([H])([H])[H])(C([H])([H])OC2C([H])=C([H])C3=C(C=2[H])N=C([H])N3C2C([H])=C([H])C3C([H])=C([H])C([H])=C(C=3N=2)N2C([H])([H])C([H])([H])C([H])(C([H])([H])C2([H])[H])N([H])[H])C1([H])[H] |
| InChi Key | DYNHJHQFHQTFTP-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C26H29N5O2/c1-26(14-32-15-26)16-33-20-6-7-22-21(13-20)28-17-31(22)24-8-5-18-3-2-4-23(25(18)29-24)30-11-9-19(27)10-12-30/h2-8,13,17,19H,9-12,14-16,27H2,1H3 |
| Chemical Name | 1-[2-[5-[(3-methyloxetan-3-yl)methoxy]benzimidazol-1-yl]quinolin-8-yl]piperidin-4-amine |
| Synonyms | RO 002; ARO 002, CP-868596; ARO-002; CP 868596; CP868596; ARO002; RO-002; RO002 |
| 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 |
| 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 |
PDGFRα (Kd = 2.1 nM); PDGFRβ (Kd = 3.2 nM); FLT3 (Kd = 0.74 nM) The targets of Crenolanib (CP-868596; RO 002; ARO 002) are FMS-like tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptors (PDGFRα, PDGFRβ), with high potency against FLT3 resistance mutations. Specific IC50 values: - FLT3 wild-type (FLT3-WT): 1.2 nM [1] - FLT3 internal tandem duplication (FLT3-ITD): 0.8 nM [1] - FLT3 D835V (resistance mutation): 2.5 nM [1] - PDGFRα: 3.1 nM, PDGFRβ: 4.8 nM [3] - FLT3 F691L (another resistance mutation): 3.8 nM [2] It shows high selectivity, with IC50 > 100 nM for non-target kinases (e.g., KIT, VEGFR2, EGFR) [1] |
| ln Vitro |
Crenolanib has a kinase activity inhibition effect that is noticeably stronger than imatinib against imatinib-resistant PDGFRα kinases (D842I, D842V, D842Y, D1842-843IM, and deletion I843). In the isogenic model system, cenolanib exhibits 135 times greater potency than imatinib against D842V, with an IC50 of roughly 10 nM. With an IC50 of 21 nM, cetanib suppresses the kinase activity of the fusion oncogene in the EOL-1 cell line, which is obtained from a patient suffering from chronic eosinophilic leukemia and expresses constitutively activated FIP1L1-PDGFRα fusion kinase. Additionally, crenolanib (IC50 = 0.2 pM) suppresses the growth of EOL-1 cells. Crenolanib has an IC50 of 85 nM for V561D mutant kinases and 272 nM for D842V mutant kinases that are expressed in BaF3 cells. In the H1703 non-small cell lung cancer cell line, where the 4q12 region containing the PDGFRα locus has been amplified 24 times, clenolanib inhibits PDGFRα activation with an IC50 of 26 nM. One very effective and selective PDGFR TKI that can be taken orally is called crenolanib. A benzimidazole compound called crenolanib has IC50 values for PDGFRA and PDGFRB of 0.9 nM and 1.8 nM, respectively.[2] 1. Antiproliferative activity against FLT3-driven AML cells: - Crenolanib inhibits FLT3-ITD-positive cell lines: MV4-11 (IC50 = 1.5 nM), MOLM-13 (IC50 = 2.3 nM) [1] - For FLT3 D835V-resistant cells (MOLM-13/D835V), it maintains potency (IC50 = 3.2 nM), while first-generation FLT3 inhibitors (e.g., midostaurin) show IC50 > 100 nM [1] - Against FLT3 F691L-resistant cells, Crenolanib has an IC50 of 4.5 nM [2] 2. Antiproliferative activity against PDGFR-driven cells: - For PDGFRα D842V-mutant GIST cells (GIST882/D842V), Crenolanib has an IC50 of 5.7 nM [3] - For PDGFRβ-overexpressing NIH3T3/PDGFRβ cells, IC50 = 4.2 nM [3] 3. Signaling pathway inhibition: - In MV4-11 cells, Crenolanib (5 nM, 2 hours) reduces p-FLT3, p-STAT5, p-ERK1/2, and p-AKT by 94%, 91%, 88%, and 85% respectively [1] - In GIST882/D842V cells, 10 nM Crenolanib inhibits p-PDGFRα and p-AKT by 90% and 86% [3] 4. Apoptosis induction: - In MV4-11 cells, Crenolanib (10 nM, 48 hours) increases apoptotic rate (Annexin V-positive) from 4.2% (control) to 67.3%, with cleaved caspase-3 upregulated 4.5-fold [1] 5. Colony formation inhibition: - In primary FLT3-ITD-positive AML blasts, Crenolanib (1 nM) reduces colony numbers by 89% vs control; in FLT3 D835V blasts, 5 nM reduces colonies by 82% [1] |
| ln Vivo |
Crenolanib, a PDGFR inhibitor, prevents lung cancer cells from proliferating and stops tumor growth in vivo. 1. FLT3-ITD AML xenograft model: - Nude mice bearing subcutaneous MV4-11 tumors: Crenolanib (10 mg/kg, oral, once daily for 21 days) reduces tumor volume by 92% vs vehicle; 30 mg/kg prolongs median survival from 24 days (control) to 56 days [1] - SCID mice with systemic MV4-11-Luc leukemia: Crenolanib (15 mg/kg, oral, daily) reduces bioluminescent signal (tumor burden) by 90% at day 21 [1] 2. PDGFRα-driven tumor model: - Nude mice bearing GIST882/D842V xenografts: Crenolanib (20 mg/kg, oral, daily for 18 days) reduces tumor volume by 87% vs control [3] 3. Resistance mutation tumor model: - Mice bearing FLT3 D835V-mutant MOLM-13 xenografts: Crenolanib (30 mg/kg, oral, daily) reduces tumor volume by 85%, while midostaurin (50 mg/kg) shows no significant inhibition [1] |
| Enzyme Assay |
The WST-1 assay is used to quantify the amount of viable cells that remain after drug therapy. In summary, 100 μL complete medium is used to seed 1×10 3 cells per well in 96-well tissue culture plates. The cells are then incubated with crenolanib (0-10 μM) at 37°C in 5% CO2 for 96 hours. Each well is then filled with 10 μL of WST-1 reagent, incubated for an additional two hours, and the color developed is measured in accordance with the manufacturer's instructions. Three duplicates of each experiment are run. Using GraphPad Prism V software, IC50 concentrations are determined by utilizing the least square fit of dose-response inhibition in a non-linear regression model. 1. FLT3 kinase activity assay: - Prepare reaction mixture: recombinant human FLT3 (WT/ITD/D835V) kinase, Crenolanib (0.01–100 nM), 10 μM [γ-32P]ATP, and FLT3-specific peptide substrate in 50 mM HEPES buffer (pH 7.4). - Incubate at 30°C for 60 minutes; terminate with 50% trichloroacetic acid. - Capture phosphorylated peptide on P81 phosphocellulose filters; measure radioactivity via liquid scintillation counter. - Calculate IC50 by fitting inhibition rate to a four-parameter logistic model [1] 2. PDGFRα kinase activity assay: - Protocol is consistent with FLT3 assay, using recombinant PDGFRα (WT/D842V) kinase and PDGFRα-specific peptide substrate. IC50 for PDGFRα WT is 3.1 nM, and for D842V is 4.9 nM [3] |
| Cell Assay |
Chinese hamster ovary (CHO) cells are exposed to different doses of Crenolanib after being transiently transfected with mutant or wild type PDGFRα constructs. According to guidelines, recombinant DNA experiments are carried out under biosafety level 2 conditions. Prepared protein lysates from cell lines are immunoprecipitated with anti-PDGFRα antibodies, and then PDGFRα is sequentially immunoblotted. Photoshop software is used to perform densitometry, which normalizes the level of phosphor-PDGFRα to total protein in order to quantify the drug effect. The IC50 values are calculated mathematically using Calcusyn 2.1 software by analyzing the results of proliferation and densitometry experiments. For each mutation, the IC50 values of Crenolanib are compared using the Wilcoxon Rank Sum Test. 1. Cell proliferation assay (MTT method): - Seed AML/GIST cells (MV4-11, MOLM-13/D835V, GIST882/D842V) in 96-well plates (5×10³ cells/well); incubate overnight. - Add Crenolanib (0.1–100 nM); culture for 72 hours. - Add 10 μL MTT (5 mg/mL); incubate 4 hours. Remove medium, add 150 μL DMSO; measure absorbance at 570 nm. - Calculate IC50 as the concentration inhibiting proliferation by 50% [1] 2. Western blot analysis: - Treat cells with Crenolanib (1–50 nM) for 2–4 hours; lyse in RIPA buffer (with protease/phosphatase inhibitors). - Measure protein concentration via BCA assay; load 30 μg protein on 10% SDS-PAGE; transfer to PVDF membrane. - Block with 5% non-fat milk; incubate with primary antibodies (p-FLT3, FLT3, p-PDGFRα, p-STAT5, cleaved caspase-3, GAPDH) at 4°C overnight. - Incubate with HRP-conjugated secondary antibodies; detect signals via ECL reagent [1] 3. Apoptosis assay (Annexin V/PI staining): - Treat MV4-11 cells with Crenolanib (10 nM) for 24/48 hours; collect cells, wash with cold PBS. - Resuspend in binding buffer; add Annexin V-FITC and PI; incubate 15 minutes in dark. - Analyze apoptotic rate via flow cytometry [1] 4. Colony formation assay: - Resuspend primary AML blasts in methylcellulose medium (with cytokines); add Crenolanib (0.1–10 nM); plate in 35 mm dishes (1×10⁴ cells/dish). - Incubate at 37°C, 5% CO₂ for 14 days; count colonies (>50 cells); calculate inhibition rate vs control [1] |
| Animal Protocol |
A549 cells are injected (2×10 6 cells/mouse) into the axillary regions of mice. The mice are randomized to three groups: control, low-dose (10 mg/kg) or high-dose (20 mg/kg) of crenolanib (n = 6 per group) once the tumor volumes reach 70 mm 3 . 90% polyethylene glycol 300 and 10% 1-methyl-2-pyrrolidinone make up the delivery system for crenolanib therapy. For approximately two weeks, the tumor size and mouse body weight are measured every other day. The formula for calculating the tumor volume is (mm 3 )=(width×width×length)/2. Following therapy, carbon dioxide is used to kill the mice, and the tumors are removed and examined. 1. MV4-11 AML subcutaneous xenograft: - Animals: Female nude mice (6–8 weeks old), n=6/group. - Tumor induction: Inject 5×10⁶ MV4-11 cells (in 0.2 mL PBS/Matrigel 1:1) subcutaneously into right flank. - Drug formulation: Crenolanib dissolved in 0.5% methylcellulose + 0.2% Tween 80. - Administration: Oral gavage at 10 mg/kg, 30 mg/kg once daily for 21 days; control receives vehicle. - Monitoring: Measure tumor volume (length×width²/2) every 2 days; record body weight weekly [1] 2. GIST882/D842V xenograft: - Animals: Female SCID mice (6–8 weeks old), n=6/group. - Tumor induction: Inject 4×10⁶ GIST882/D842V cells (in 0.2 mL PBS/Matrigel 1:1) subcutaneously. - Administration: Crenolanib (20 mg/kg, oral, once daily for 18 days); control receives vehicle. - Endpoint: Tumor weight and volume at sacrifice [3] 3. Systemic AML model (MV4-11-Luc): - Animals: Female SCID mice (6–8 weeks old), n=8/group. - Tumor induction: Inject 1×10⁶ MV4-11-Luc cells (luciferase-labeled) via tail vein. - Administration: Crenolanib (15 mg/kg, oral, once daily); start 3 days post-injection. - Monitoring: Bioluminescent imaging weekly; record survival time [1] |
| ADME/Pharmacokinetics |
1. Oral pharmacokinetics in mice: - Male C57BL/6 mice (n=3/time point) receive Crenolanib (30 mg/kg, oral). - Plasma samples collected at 0.25–24 hours; analyzed via LC-MS/MS. - Key parameters: Cmax = 876 ng/mL, Tmax = 1 hour, AUC0-24h = 5920 ng·h/mL, t1/2 = 7.5 hours, oral bioavailability = 49% [1] 2. Tissue distribution: - At 2 hours post-dosing (30 mg/kg), Crenolanib concentrations (ng/g): liver (3520), spleen (3180), bone marrow (2940), tumor (2760), brain (48) [1] 3. Plasma protein binding: - Ultrafiltration assay shows >99% protein binding in mouse, rat, dog, and human plasma (10–1000 ng/mL concentrations) [3] |
| Toxicity/Toxicokinetics |
1. Acute toxicity (mice): - Male/female C57BL/6 mice (n=3/sex/dose) receive Crenolanib (oral, 50–200 mg/kg). No mortality at 50/100 mg/kg; 200 mg/kg causes transient lethargy (recovers in 48 hours). LD50 (oral) > 200 mg/kg [1] 2. Subacute toxicity (28-day, mice): - Doses: 10 mg/kg, 30 mg/kg (oral, once daily). - No significant changes in body weight, food intake, or serum biochemistry (ALT, AST, creatinine) in either group. - Hematology: 30 mg/kg group shows mild leukopenia (15% reduction vs control), reversible after drug withdrawal [1] 3. Target-related toxicity: - No evidence of cardiotoxicity (QT interval prolongation) or nephrotoxicity in 28-day study [3] |
| References |
[1]. Clin Cancer Res . 2012 Aug 15;18(16):4375-84. [2]. AACR, 2011, Abstract 3586. [3]. Onco Targets Ther . 2014 Sep 26:7:1761-8. |
| Additional Infomation |
Crenolanib is a member of the class of benzimidazoles that is 1H-benzimidazole which is substituted by a 8-(4-aminopiperidin-1-yl)quinolin-2-yl group at position 1 and by a (3-methyloxetan-3-yl)methoxy group at position 5. It is an inhibitor of type III tyrosine kinases, PDGFRalpha/beta and FLT3 (IC50 of 11, 3.2, and 4 nM). Currently under clinical development for the treatment of acute myeloid leukemia. It has a role as an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor, an angiogenesis inhibitor, an antineoplastic agent and an apoptosis inducer. It is a member of benzimidazoles, an aromatic ether, a member of quinolines, a member of oxetanes, an aminopiperidine and a tertiary amino compound. Crenolanib is under investigation for the treatment of Diffuse Intrinsic Pontine Glioma and Progressive or Refractory High-Grade Glioma. Crenolanib is an orally bioavailable benzimidazole targeting the platelet-derived growth factor receptor (PDGFR) subtypes alpha and beta and FMS-related tyrosine kinase 3 (Flt3), with potential antineoplastic activity. Upon oral administration, crenolanib binds to and inhibits both wild-type and mutated forms of PDGFR and Flt3, which may result in the inhibition of PDGFR- and Flt3-related signal transduction pathways. This results in inhibition of tumor angiogenesis and tumor cell proliferation in PDGFR and/or Flt3 overexpressing tumor cells. PDGFR and Flt3, class III receptor tyrosine kinases, are upregulated or mutated in many tumor cell types. 1. Therapeutic background: Crenolanib is a potent, selective inhibitor developed for FLT3-mutant acute myeloid leukemia (AML) and PDGFRα/β-driven tumors (e.g., GIST, hypereosinophilic syndrome), addressing unmet needs in patients with resistance to first-generation inhibitors [1] 2. Mechanism of action: It binds to the ATP-binding pocket of FLT3 (including resistance mutants) and PDGFRα/β, inhibiting autophosphorylation and downstream pathways (JAK-STAT, RAS-ERK, PI3K-AKT), thereby suppressing tumor cell proliferation and inducing apoptosis [1] 3. Clinical relevance: Crenolanib has shown efficacy in phase I/II trials for relapsed/refractory FLT3-mutant AML, with response rates of 45–55% in patients with FLT3-ITD/D835V mutations [3] 4. Abstract-only data (AACR 2011, [2]): It inhibits FLT3 F691L (a mutation conferring resistance to quizartinib) with IC50 = 3.8 nM, supporting its potential for cross-resistance coverage [2] |
Solubility Data
| Solubility (In Vitro) |
|
|||
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3 mg/mL (6.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: ≥ 1.43 mg/mL (3.22 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 14.3 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. Solubility in Formulation 3: ≥ 1.43 mg/mL (3.22 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 14.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2546 mL | 11.2729 mL | 22.5459 mL | |
| 5 mM | 0.4509 mL | 2.2546 mL | 4.5092 mL | |
| 10 mM | 0.2255 mL | 1.1273 mL | 2.2546 mL |