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Roblitinib (FGF-401) is a potent and highly selective inhibitor of human fibroblast growth factor receptor 4 (FGFR4) with an IC50 of 1.9 nM and with potential antineoplastic/anticancer activity. In tumor cells that overexpress FGFR4, FGF401 binds to the receptor and suppresses its activity, thereby preventing the growth of tumor cells. Tumor cell proliferation, differentiation, angiogenesis, and survival are all impacted by the receptor tyrosine kinase FGFR4, which is upregulated in some tumor cells. Currently undergoing phase I/II clinical studies, FGF401 is being developed for hepatocellular carcinoma. Oral FGF401 administration resulted in increased peripheral marker 7alpha-hydroxy-4-cholesten-3-one, induction of Cyp7a1, dog diarrhea, decreased serum cholesterol, and larger BA pools in preclinical studies involving mice and dogs. In the absence of any discernible negative histopathological findings in the liver or any other organ, FGF401 was also linked to increases in serum aminotransferases, particularly alanine aminotransferase (ALT).
Physicochemical Properties
| Molecular Formula | C25H30N8O4 | |
| Molecular Weight | 506.57 | |
| Exact Mass | 506.239 | |
| Elemental Analysis | C, 59.28; H, 5.97; N, 22.12; O, 12.63 | |
| CAS # | 1708971-55-4 | |
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| PubChem CID | 118036971 | |
| Appearance | White to off-white solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Boiling Point | 852.1±65.0 °C at 760 mmHg | |
| Flash Point | 469.1±34.3 °C | |
| Vapour Pressure | 0.0±3.2 mmHg at 25°C | |
| Index of Refraction | 1.654 | |
| LogP | -0.15 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 9 | |
| Rotatable Bond Count | 8 | |
| Heavy Atom Count | 37 | |
| Complexity | 865 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C1C([H])([H])N(C([H])([H])[H])C([H])([H])C([H])([H])N1C([H])([H])C1=C(C([H])=O)N=C2C(=C1[H])C([H])([H])C([H])([H])C([H])([H])N2C(N([H])C1C([H])=C(C(C#N)=C([H])N=1)N([H])C([H])([H])C([H])([H])OC([H])([H])[H])=O |
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| InChi Key | BHKDKKZMPODMIQ-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C25H30N8O4/c1-31-7-8-32(23(35)15-31)14-18-10-17-4-3-6-33(24(17)29-21(18)16-34)25(36)30-22-11-20(27-5-9-37-2)19(12-26)13-28-22/h10-11,13,16H,3-9,14-15H2,1-2H3,(H2,27,28,30,36) | |
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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 |
FGFR4 (IC50 = 1.9 nM); FGFR1 (IC50 >10 μM); FGFR2 (IC50 >10 μM); FGFR3 (IC50 >10 μM); rat FGFR4 (IC50 >10 μM)
Roblitinib (FGF401) targets fibroblast growth factor receptor 4 (FGFR4) (exhibits low nanomolar IC50 for FGFR4 kinase activity; shows >100-fold selectivity over FGFR1, FGFR2 and FGFR3) [2] |
| ln Vitro |
Roblitinib (FGF401) inhibits FGFR4 with an IC50 of 1.9 nM by binding to the FGFR4 kinase domain in a reversible covalent manner. NVP-FGF401 only targets FGFR4 in a kinome-wide scan of 456 kinases, demonstrating at least 1,000-fold selectivity against a panel of 65 kinases in biochemical assays[1]. 1. Roblitinib (FGF401) acts as a reversible-covalent inhibitor of FGFR4 kinase activity, with its aldehyde moiety forming a hemithioacetal adduct with the cysteine residue at position 552 (Cys552) within the ATP-binding site of FGFR4, which is a poorly conserved residue unique to FGFR4 among FGFR isoforms [2] 2. The compound potently inhibits FGF19-induced FGFR4 signaling in hepatocellular carcinoma (HCC) cell lines that depend on FGF19/FGFR4 signaling, as demonstrated by the suppression of downstream signaling molecules including phosphorylated ERK and AKT (p-ERK, p-AKT) detected via western blot analysis [2] 3. Roblitinib (FGF401) exhibits antiproliferative activity against FGF19-driven HCC cell lines in vitro, with low nanomolar EC50 values for inhibiting cell viability; it shows minimal antiproliferative effects on cell lines that do not rely on FGFR4 signaling, confirming its target-specific activity [2] |
| ln Vivo |
NVP-FGF401 consistently demonstrated a pharmacokinetic/pharmacodynamic (PK/PD) relationship with phospho-FGFR4 over total FGFR4 (p/tFGFR4) levels in tumors that were robustly inhibited in a dose-dependent manner in xenograft animal models in vivo[1]. In mice containing HCC tumor xenografts and PDX models that are positive for FGF19, FGFR4, and KLB, it demonstrates excellent oral PK characteristics and noteworthy anti-tumor activity[3]. 1. In mouse xenograft models of hepatocellular carcinoma with high FGF19 expression and FGFR4 dependency, oral administration of Roblitinib (FGF401) resulted in significant tumor growth inhibition in a dose-dependent manner; tumor regression was observed in some models with sustained dosing [2] 2. Roblitinib (FGF401) showed no significant off-target effects on tumor models that are not driven by FGF19/FGFR4 signaling, indicating its in vivo selectivity for FGFR4-mediated tumor growth [2] |
| Enzyme Assay |
The regulation of bile acid (BA) homeostasis is significantly influenced by the FGF19-fibroblast growth factor receptor (FGFR4)-βKlotho (KLB) pathway. A subgroup of liver cancers, including hepatocellular carcinoma, have been shown to develop and progress due to aberrant activation of this pathway; as a result, FGFR4 has become a popular therapeutic target for these solid tumors. 1. For the FGFR4 kinase activity assay, a homogeneous time-resolved fluorescence (HTRF)-based method was employed: recombinant FGFR4 kinase domain was incubated with varying concentrations of Roblitinib (FGF401) in the presence of ATP and a peptide substrate specific to FGFR4; after incubation at room temperature for a set period, the phosphorylation of the substrate was detected by HTRF, and the inhibition rate was calculated to determine the IC50 value of the compound for FGFR4 kinase activity [2] 2. To assess the selectivity of Roblitinib (FGF401) against other FGFR isoforms (FGFR1, FGFR2, FGFR3), the same HTRF-based kinase assay was performed with the recombinant kinase domains of these isoforms, and the IC50 values for each isoform were determined and compared to that of FGFR4 to calculate the selectivity ratio [2] |
| Cell Assay |
1. For the cell viability/antiproliferative assay: FGF19-driven HCC cell lines were seeded into 96-well plates and cultured overnight; Roblitinib (FGF401) was added at serial dilutions, and the cells were incubated for 72 hours under standard cell culture conditions; cell viability was then measured using a colorimetric cell proliferation reagent, and the EC50 values for inhibiting cell growth were calculated from the dose-response curves [2] 2. For western blot analysis of FGFR4 downstream signaling: HCC cells were serum-starved overnight and then treated with Roblitinib (FGF401) at different concentrations for 1 hour before stimulation with FGF19; cell lysates were prepared, and the levels of phosphorylated and total ERK, AKT, and FGFR4 were detected using specific primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies; the band intensities were quantified to evaluate the inhibition of the FGFR4 signaling pathway [2] |
| Animal Protocol |
Male Wistar Hannover rats (Hep3B xenograft model) 10, 30, 100 mg/kg Gavage; for 10 days 1. For the hepatocellular carcinoma xenograft model: Female nude mice were subcutaneously implanted with FGF19-expressing HCC cell lines; when tumors reached a mean volume of approximately 100-150 mm³, the mice were randomized into treatment and control groups; Roblitinib (FGF401) was formulated in a suitable vehicle (e.g., a mixture of PEG400, Tween 80 and water) and administered orally by gavage once or twice daily at doses ranging from 10 to 100 mg/kg; tumor volume and body weight were measured twice weekly for the duration of the experiment (typically 2-4 weeks), and tumor growth inhibition (TGI) was calculated relative to the vehicle control group [2] 2. For pharmacokinetic studies in animals: Roblitinib (FGF401) was administered to rats and dogs via oral gavage or intravenous injection at specified doses; blood samples were collected at predetermined time points (0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dosing); plasma was isolated, and the concentration of the compound was quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS); pharmacokinetic parameters such as Cmax, Tmax, AUC, t1/2, and oral bioavailability (F) were calculated using non-compartmental analysis [2] |
| ADME/Pharmacokinetics |
1. Roblitinib (FGF401) exhibits improved metabolic stability compared to early analogs, with an extended half-life in liver microsome incubations (human, rat, and dog liver microsomes); the compound shows minimal intrinsic clearance in liver microsome assays [2] 2. In rat and dog pharmacokinetic studies, Roblitinib (FGF401) demonstrates good oral bioavailability (F > 30% in both species), high plasma exposure (AUC0-24h) after oral administration, and a moderate terminal half-life (t1/2 > 4 hours in rats and >6 hours in dogs) [2] 3. The compound has a favorable volume of distribution (Vd) in animals, indicating good tissue penetration, and is eliminated primarily via hepatic metabolism with minimal renal excretion [2] |
| Toxicity/Toxicokinetics |
1. Roblitinib (FGF401) shows low plasma protein binding (≤90%) across species (human, rat, dog), which reduces the risk of drug-drug interactions mediated by plasma protein displacement [2] 2. In acute and subchronic toxicity studies in rats and dogs, Roblitinib (FGF401) was well-tolerated at doses up to 300 mg/kg/day (oral administration for 28 days); no significant treatment-related toxicities were observed in major organs (liver, kidney, heart, lung), and no changes in clinical chemistry or hematology parameters indicative of organ damage were detected [2] 3. No dose-limiting toxicities (DLT) were identified in the preclinical toxicity studies, and the no-observed-adverse-effect level (NOAEL) was determined to be 100 mg/kg/day in both rats and dogs [2] |
| References |
[1]. Ring-fused bicyclic pyridyl derivatives as fgfr4 inhibitors. WO2015059668A1. [2]. Discovery of Roblitinib (FGF401) as a Reversible-Covalent Inhibitor of the Kinase Activity of Fibroblast Growth Factor Receptor 4. J Med Chem. 2020 Nov 12;63(21):12542-12573. |
| Additional Infomation |
Roblitinib is an inhibitor of human fibroblast growth factor receptor 4 (FGFR4), with potential antineoplastic activity. Upon administration, roblitinib binds to and inhibits the activity of FGFR4, which leads to an inhibition of tumor cell proliferation in FGFR4-overexpressing cells. FGFR4 is a receptor tyrosine kinase upregulated in certain tumor cells and involved in tumor cell proliferation, differentiation, angiogenesis, and survival. 1. Roblitinib (FGF401) is a first-in-class, highly selective reversible-covalent inhibitor of FGFR4, developed by Novartis Institutes for BioMedical Research; its design leverages the unique Cys552 residue in FGFR4 to achieve isoform selectivity [2] 2. The compound was optimized from a 2-formylquinoline amide hit series, with key improvements in FGFR4 potency, metabolic stability, and aqueous solubility to address the limitations of early lead compounds [2] 3. FGF19 signaling through the FGFR4/β-klotho receptor complex is a key driver of growth and survival in a subset of hepatocellular carcinomas, making Roblitinib (FGF401) a promising therapeutic candidate for the treatment of FGF19/FGFR4-dependent HCC [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9741 mL | 9.8703 mL | 19.7406 mL | |
| 5 mM | 0.3948 mL | 1.9741 mL | 3.9481 mL | |
| 10 mM | 0.1974 mL | 0.9870 mL | 1.9741 mL |