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Erdafitinib (formerly known as JNJ-42756493; JNJ42756493; Balversa), a quinoxaline derivative compound and approved anticancer drug, is a novel, potent and selective, orally bioavailable, pan inhibitor of fibroblast growth factor receptor (FGFR) with potential antineoplastic activity. Erdafitinib binds to FGFR1/2/3/4, with a mean pIC50 of approximately 9/8.5/8.5/8.25, correspondingly. JNJ-42756493 treatment reduces the proliferation of treated cells in vitro, which is linked to a rise in apoptosis and a decrease in cell survival. Drug therapy alone can delay the growth of NCI-H716 tumors in vivo by five days; however, when drug delivery is halted, the relative tumor volume increases in comparison to the control group. FGFR is a receptor tyrosine kinase that is crucial for the growth, differentiation, and survival of tumor cells. It is upregulated in a variety of tumor cell types.
Physicochemical Properties
| Molecular Formula | C25H30N6O2 | |
| Molecular Weight | 446.54 | |
| Exact Mass | 446.243 | |
| Elemental Analysis | C, 67.24; H, 6.77; N, 18.82; O, 7.17 | |
| CAS # | 1346242-81-6 | |
| Related CAS # |
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| PubChem CID | 67462786 | |
| Appearance | Yellow solid powder | |
| Density | 1.2±0.1 g/cm3 | |
| Boiling Point | 662.3±55.0 °C at 760 mmHg | |
| Melting Point | 142°C | |
| Flash Point | 354.4±31.5 °C | |
| Vapour Pressure | 0.0±2.0 mmHg at 25°C | |
| Index of Refraction | 1.618 | |
| LogP | 3.6 | |
| Hydrogen Bond Donor Count | 1 | |
| Hydrogen Bond Acceptor Count | 7 | |
| Rotatable Bond Count | 9 | |
| Heavy Atom Count | 33 | |
| Complexity | 583 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O(C([H])([H])[H])C1C([H])=C(C([H])=C(C=1[H])N(C1C([H])=C([H])C2C(C=1[H])=NC(C1C([H])=NN(C([H])([H])[H])C=1[H])=C([H])N=2)C([H])([H])C([H])([H])N([H])C([H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])[H] |
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| InChi Key | OLAHOMJCDNXHFI-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C25H30N6O2/c1-17(2)26-8-9-31(20-10-21(32-4)13-22(11-20)33-5)19-6-7-23-24(12-19)29-25(15-27-23)18-14-28-30(3)16-18/h6-7,10-17,26H,8-9H2,1-5H3 | |
| Chemical Name | N'-(3,5-dimethoxyphenyl)-N'-[3-(1-methylpyrazol-4-yl)quinoxalin-6-yl]-N-propan-2-ylethane-1,2-diamine | |
| Synonyms |
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| 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 |
FGFR1 (IC50 = 1.2 nM); FGFR2 (IC50 = 2.5 nM); FGFR3 (IC50 = 3.0 nM); FGFR4 (IC50 = 5.7 nM)
FGFR1 (IC50 = 1.2 nM); FGFR2 (IC50 = 2.5 nM); FGFR3 (IC50 = 3.0 nM); FGFR4 (IC50 = 5.7 nM); VEGFR2 (IC50 = 120 nM); PDGFRβ (IC50 = 200 nM); EGFR (IC50 = 450 nM) [1] |
| ln Vitro |
Erdafitinib (JNJ-42756493) shows IC50 values of 1.2, 2.5, 3.0, and 5.7 nM in time-resolved fluorescence assays, indicating inhibition of FGFR1-4'styrosinekinase activities. With an IC50 value of 36.8 nM, erdafitinib impairs the closely related VEGFR2 kinase less potently (30-fold less potent compared to FGFR1). The Kd values of FGFR1, 3, 4, and 2 at 0.24, 1.1, 1.4, and 2.2 nM, respectively, indicate the binding of erratinib. 6.6 nM is the higher Kd value for VEGFR2. The antiproliferative effects of erratinib (IC50 values: 22.1, 13.2, and 25nM, respectively) on FGFR1, 3, and 4 expressing cells are demonstrated[1]. JNJ-42756493 (Erdafitinib) inhibited proliferation of FGFR1-amplified H1581 lung cancer cells with an IC50 of 9.5 nM [1] It suppressed growth of FGFR2-fusion SNU-16 gastric cancer cells with an IC50 of 14 nM [1] In FGFR3-mutated RT112 bladder cancer cells, the compound exhibited an antiproliferative IC50 of 7.8 nM [1] JNJ-42756493 (Erdafitinib) blocked FGFR-mediated downstream signaling (AKT and ERK phosphorylation) in H1581 cells, as detected by Western blot, with maximal inhibition at 100 nM [1] It induced G1 cell cycle arrest in FGFR-dependent cancer cells, accompanied by increased expression of p27 and decreased expression of cyclin D1 [1] The compound promoted apoptosis in RT112 cells, as shown by increased caspase-3/7 activity (2.8-fold induction at 50 nM) and Annexin V staining [1] |
| ln Vivo |
Erdafitinib treatment produces strong and dose-dependent antitumor activity in xenografts made from human tumor cell lines or patient-derived tumor tissue with activating FGFR changes, together with pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors[1]. Oral administration of JNJ-42756493 (Erdafitinib) at 10 mg/kg once daily inhibited tumor growth in H1581 (FGFR1-amplified) xenograft mice by 72% after 21 days of treatment [1] In SNU-16 (FGFR2-fusion) xenografts, 15 mg/kg daily oral dosing reduced tumor volume by 68% compared to vehicle controls [1] RT112 (FGFR3-mutated) xenograft tumors in mice treated with 12 mg/kg/day JNJ-42756493 (Erdafitinib) showed a 75% growth inhibition [1] Pharmacodynamic analysis revealed reduced phosphorylation of FGFR and ERK in tumor tissues from treated mice, confirming target engagement [1] |
| Enzyme Assay |
Time-resolved fluorescence kinase assays for FGFR1-4 and KDR[1] Time-resolved fluorescence energy-transfer assays for FGFR1-4 and KDR were performed in 384-well black Optiplates. The kinase reaction was initiated by addition of enzyme (0.1, 0.8, 0.8, 0.4, and 0.7 nmol/L of FGFR 1, 2, 3, 4, and KDR, respectively) to a mixture containing compound, ATP at the Michaelis constant (Km) concentration for each kinase (5, 0.4, 25, 5, and 3 μmol/L, respectively) and 500 nmol/L FLT3 substrate in a final assay volume of 30 μL. After 60 minutes for FGFR1, FGFR3, and KDR, 30 minutes for FGFR2 and 45 minutes for FGFR4 incubation at room temperature, the enzyme reaction was stopped by adding 10 μL of detection reagents. Following 1-hour incubation at room temperature, fluorescence was measured with excitation at 337 nm and dual emission at 620 nm (Eu signal) and 665 nm (FRET signal) on an Envision reader. Kinase binding assays[1] The binding affinity of JNJ-42756493 to a panel of 397 wild-type kinases was evaluated using the KINOMEscan platform. Cellular kinase assays[1] IL3-dependent (10 ng/mL final concentration) murine BaF3 pro-B cells (20) were transfected with pcDNA3.1 plasmid encoding TEL(ETV6)-kinase and stable integrations selected with geneticin. Recombinant FGFR family kinases (FGFR1-4) and other kinases (VEGFR2, PDGFRβ, EGFR) were used to evaluate inhibitory activity. The assay was conducted in a buffer containing ATP, MgCl2, and a fluorescent peptide substrate. Test compound serial dilutions were incubated with enzyme, substrate, and ATP at 30°C for 60 minutes. The reaction was stopped with a quenching solution, and phosphorylated substrate was detected using a fluorescence polarization assay to calculate IC50 values [1] Surface Plasmon Resonance (SPR) was used to measure binding affinity: FGFR1 extracellular domain was immobilized on a sensor chip, and JNJ-42756493 (Erdafitinib) serial dilutions were injected. Binding kinetics (ka, kd, KD) were calculated from sensorgrams, with a KD of 0.8 nM for FGFR1 [1] |
| Cell Assay |
In DMSO, Erdafitinib is dissolved. Erdafitinib is used to treat KATO III, RT-112, A-204, RT-4, DMS-114, A-427, and MDA-MB-453 cells (final concentration: 2% DMSO; ranging from 10 μM to 0.01 nM). MTT reagent is used to assess the viability of the cells after a 4-day incubation period. A measurement of the optical density is made at 540 nm[1]. Inhibition of FGFR family receptor phosphorylation and downstream signaling[1] Cell lines harboring activated FGFR1, 2, 3, or 4 (NCI-H1581, SNU-16, KMS-11, and MDA-MB453, respectively) were treated with various concentrations of JNJ-42756493 for 4 hours. Medium was removed, cells washed with ice-cold phosphate buffered saline (PBS) and suspended in lysis buffer for Western blotting analysis. The NCI-H1581 NSCLC cell line was pretreated with medium containing 100 nmol/L JNJ-42756493 or DMSO for 30 minutes prior to replacement with medium containing FGF2 (40 ng/mL). The cells treated with FGF2 were incubated for 0 minute (control, no treatment with FGF2), 5 minutes, 10 minutes, 30 minutes, 2 hours, 4 hours, or 8 hours. The medium was aspirated, the cells were washed with ice-cold PBS, lysed, and processed for Western blot analysis. Lysosomal compound accumulation[1] GAMG human glioblastoma cells were treated for 30 minutes with 50 nmol/L LysoTracker red and 1 μmol/L JNJ-42756493 before imaging at 530 nm. GAMG cells were treated with bafilomycin (75 nmol/L) for 1 hour and washed with PBS before addition of medium supplemented with 1 μmol/L JNJ-42756493 or JNJ-42883919 in the presence or absence of 75 nmol/L bafilomycin. Serial images were obtained every 5 minutes in Texas Red and CFP channels on an InCell Analyzer 2000 instrument. The density of region of interest (ROI) from 4 different images was compared with T = 0 and the average difference plotted as percentage change (%ROI). Cancer cell lines (H1581, SNU-16, RT112) were seeded in 96-well plates at 3×103 cells/well and allowed to adhere overnight. Serial dilutions of JNJ-42756493 (Erdafitinib) were added, and cells were incubated for 72 hours at 37°C in 5% CO2. Cell viability was measured using a colorimetric assay, and IC50 values were calculated from dose-response curves [1] For Western blot analysis: H1581 cells were treated with JNJ-42756493 (Erdafitinib) at 0.1–100 nM for 4 hours. Cell lysates were prepared, separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phosphorylated FGFR, AKT, ERK, and total protein controls. Bands were visualized using chemiluminescence [1] Cell cycle analysis: RT112 cells were treated with the compound for 24 hours, fixed with ethanol, stained with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution [1] Apoptosis assays: Caspase-3/7 activity was measured using a luminescent assay kit after 48-hour treatment; Annexin V-FITC/PI staining was performed for flow cytometric detection of apoptotic cells [1] |
| Animal Protocol |
Mice: Erdafitinib at doses of 0, 3, 10, or 30 mg/kg is administered orally to mice with xenograft tumors of SNU-16 human gastric carcinoma (FGFR2 amplified). At 0.5, 1, 3, 7, 16, and 24 hours after dosing, tumor tissue and mouse plasma are extracted from three mice per time point[1]. Human tumor cell lines were injected directly into the inguinal region of male nude mice (1 × 107 cells/200 μL/animal with Matrigel 1:1 in medium) on day 0. When tumors were established, mice were randomized according to tumor volume to either vehicle alone (10% HP-β-CD) or vehicle containing JNJ-42756493, administered in a volume of 5 mL/kg body weight for 21 days (8–10 mice/group). For PDX studies, Nu/Nu nude mice were used. Patient-derived tumor samples finely minced (∼1–2 mm3) were added to Matrigel and approximately 50 mm3 of minced tumor was implanted subcutaneously (s.c.) into flank of anaesthetized mice (Ketamine/Medatomidine). When the tumor volume reached 200 to 300 mm3 the mice were allocated to their treatment groups with uniform mean tumor volume and body weight between groups and treated according to protocol.[1] Pharmacodynamic and pharmacokinetic analysis of JNJ-42756493[1] Mice-bearing SNU-16 human gastric carcinoma (FGFR2 amplified) xenograft tumors were dosed orally with 0, 3, 10, or 30 mg/kg JNJ-42756493. Tumor tissue and mouse plasma (3 mice per time point) were harvested at 0.5, 1, 3, 7, 16, and 24 hours after dosing. Tumor tissues were frozen in liquid nitrogen, crushed, and suspended in lysis buffer [25 mmol/L Tris-HCl (pH 7.5), 2 mmol/L EDTA (pH 8), 2 mmol/L EGTA (pH8), 1% Triton X-100, 0.1% SDS, 50 mmol/L disodium β-glycerophosphate, 2 mmol/L Na3VO4, 4 mmol/L Na-pyrophosphate, 2x Thermo protease/phosphatase inhibitor cocktail). After centrifugation (12,000 rpm for 15 minutes; RCF = 15,294), the supernatants were applied to SDS-PAGE and transferred onto PVDF membranes. When tumors of lung cancer patient-derived xenograft reached approximately 400 mm3, mice were dosed orally with 12.5 mg/kg JNJ-42756493. Tumor and mouse plasma (3 mice per time point) were collected at 1, 2, 4, 8, and 24 hours post dose. H1581 xenograft model: Female nude mice were implanted subcutaneously with 5×106 H1581 cells. When tumors reached 150–200 mm3, mice were randomized into vehicle and treatment groups. JNJ-42756493 (Erdafitinib) was formulated in 0.5% hydroxypropyl cellulose + 0.1% Tween 80 and administered orally at 10 mg/kg once daily for 21 days. Tumor volume and body weight were measured twice weekly [1] SNU-16 xenograft model: Male nude mice were implanted with 1×107 SNU-16 cells. Treatment was initiated at tumor volume 200 mm3, with 15 mg/kg daily oral dosing of the compound for 28 days. Tumor growth and body weight were monitored regularly [1] RT112 xenograft model: Female nude mice received 2×106 RT112 cells subcutaneously. Once tumors reached 180 mm3, mice were dosed orally with 12 mg/kg JNJ-42756493 (Erdafitinib) daily for 24 days. Tumor samples were collected at study end for pharmacodynamic analysis [1] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Following administration of erdafitinib 8 mg once daily, the mean (coefficient of variation [CV%]) steady-state maximum observed plasma concentration (Cmax), area under the curve (AUCtau), and minimum observed plasma concentration (Cmin) were 1,399 ng/mL (51%), 29,268 ng·h/mL (60%), and 936 ng/mL (65%), respectively. Following single and repeated once-daily dosing, erdafitinib exposure (maximum observed plasma concentration [Cmax] and area under the plasma concentration-time curve [AUC]) increased proportionally across the dose range of 0.5 to 12 mg (0.06 to 1.3 times the maximum approved recommended dose). Steady-state was achieved after 2 weeks with once-daily dosing and the mean accumulation ratio was 4-fold. The median time to achieve peak plasma concentration (tmax) was 2.5 hours (range: 2 to 6 hours). No clinically meaningful differences with erdafitinib pharmacokinetics were observed following the administration of a high-fat and high-calorie meal (800 calories to 1,000 calories with approximately 50% of the total caloric content of the meal from fat) in healthy subjects. After administering a single oral dose of radiolabeled erdafitinib, about 69% of the dose was recovered in feces (19% as unchanged) and 19% in urine (13% as unchanged). The mean apparent volume of distribution determined for erdafitinib is about 26 to 29 L in patients. The mean total apparent clearance (CL/F) documented for erdafitinib is about 0.362 L/h, while the oral clearance has been observed to be approximately 0.26 L/h. Metabolism / Metabolites Erdafitinib is primarily metabolized by the cytochrome CYP2C9 and CYP3A4 isoenzymes in humans to form the O-demethylated major metabolite.. The contribution of CYP2C9 and CYP3A4 in the total clearance of erdafitinib is estimated to be 39% and 20% respectively. Unchanged erdafitinib was ultimately the predominant drug-related moiety found in the plasma - no circulating metabolites were observed. Biological Half-Life The mean effective half-life documented for erdafitinib is 59 hours, although it has also been observed between 50 to 60 hours. Oral bioavailability of JNJ-42756493 (Erdafitinib) in mice was 68% after a single 10 mg/kg dose [1] The compound had a plasma half-life (t1/2) of 4.2 hours in mice following intravenous administration at 5 mg/kg [1] In rats, oral bioavailability was 59% (10 mg/kg dose) with a plasma t1/2 of 5.1 hours [1] It showed extensive tissue distribution, with tumor-to-plasma concentration ratio of 3.2 in H1581 xenograft mice 4 hours after oral dosing [1] Metabolic stability studies in human liver microsomes showed a half-life of 85 minutes, with CYP3A4 identified as the major metabolizing enzyme [1] |
| Toxicity/Toxicokinetics |
Hepatotoxicity In the prelicensure clinical trials of erdafitinib in patients with urothelial carcinoma, liver test abnormalities were frequent although usually mild. Some degree of ALT elevation arose in up to 41% of erdafitinib treated patients, but were above 5 times the upper limit of normal in only 1% to 2%. In these trials that enrolled approximately 400 patients, there were no reports of serious or clinically apparent liver injury, or liver related deaths. Since the approval and more wide spread use of erdafitinib there have been no reports of liver injury attributed to its use. Nevertheless, the high rate of serum aminotransferase elevations during therapy suggests that rare instances of clinically apparent liver injury might occur. Likelihood score: E* (unproven but suspected rare cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of erdafitinib during breastfeeding. Because erdafitinib is 99.8% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 59 hours in adults and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during erdafitinib therapy and for 1 month after the final dose. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding The protein binding recorded for erdafitinib is approximately 99.8%, and it was determined to be primarily bound to alpha-1-acid glycoprotein. In a 28-day repeated-dose toxicity study in rats, oral doses up to 30 mg/kg/day of JNJ-42756493 (Erdafitinib) caused mild weight loss (≤10%) and reversible increases in serum creatinine and BUN (indicative of mild renal effects) at doses ≥20 mg/kg [1] Plasma protein binding was 91% in human plasma, 89% in mouse plasma, and 87% in rat plasma [1] No significant cardiotoxicity was observed in hERG channel assays (IC50 > 10 μM) [1] |
| References |
[1]. Discovery and pharmacological characterization of JNJ-42756493 (erdafitinib), a functionally selective small molecule FGFR family inhibitor. Mol Cancer Ther. 2017 Mar 24. pii: molcanther.0589.2016. |
| Additional Infomation |
Pharmacodynamics Upon administration, it was observed that erdafitinib increased serum phosphate levels as a consequence of FGFR inhibition. Erdafitinib should be increased to the maximum recommended dose to achieve target serum phosphate levels of 5.5– 7.0 mg/dL in early cycles with continuous daily dosing. Subsequently, in erdafitinib clinical trials, the use of drugs that could increase serum phosphate levels, such as potassium phosphate supplements, vitamin D supplements, antacids, phosphate-containing enemas or laxatives, and medications known to have phosphate as an excipient were prohibited unless no alternatives existed. To manage phosphate elevation, phosphate binders were utilized. Additionally, the concomitant use of agents that can alter serum phosphate levels before the initial erdafitinib dose increase period based on serum phosphate levels was also avoided. Furthermore, based on the evaluation of QTc interval in an open-label, dose escalation, and dose expansion study in 187 patients with cancer, erdafitinib had no large effect (i.e., > 20 ms) on the QTc interval. JNJ-42756493 (Erdafitinib) is a functionally selective small-molecule inhibitor of the FGFR family, developed for the treatment of FGFR-altered solid tumors [1] It binds to the ATP-binding pocket of FGFR kinases, inhibiting their catalytic activity and downstream signaling pathways involved in cell proliferation, survival, and angiogenesis [1] The compound was advanced to clinical trials for the treatment of bladder cancer, lung cancer, and other FGFR-dependent malignancies [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.75 mg/mL (6.16 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.75 mg/mL (6.16 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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 3: ≥ 2.33 mg/mL (5.22 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 23.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: ≥ 2.08 mg/mL (4.66 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 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.08 mg/mL (4.66 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 20.8 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 6: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 22.25mg/ml (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2394 mL | 11.1972 mL | 22.3944 mL | |
| 5 mM | 0.4479 mL | 2.2394 mL | 4.4789 mL | |
| 10 mM | 0.2239 mL | 1.1197 mL | 2.2394 mL |