BLU-554 is a novel potent, highly-selective, and orally bioavailable inhibitor of FGFR4 (fibroblast growth factor receptor 4) with an IC50 value of 5 nM in cell free assays. BLU-554 has the potential to be used for the treatment of hepatocellular carcinoma and cholangiocarcinoma. BLU-554 shows promise as a treatment for cholangiocarcinoma and hepatocellular carcinoma. These tumor models demonstrate good tolerance to BLU-554. In models of liver cancer, BLU-554 administration results in tumor regression. When taken orally, BLU-554 selectively attaches to FGFR4 and prevents the ligand FGF19 from binding to it. This stops FGFR4 from activating, suppresses FGFR4-mediated signaling, and stops tumor cell growth in FGFR4-overexpressing cells. The receptor tyrosine kinase FGFR4 is implicated in the survival, angiogenesis, proliferation, and differentiation of tumor cells. Some types of tumor cells overexpress FGF19. See if there are any ongoing clinical trials with this medication. (Thesaurus NCI).

Physicochemical Properties

Molecular Formula	C24H24CL2N4O4
Molecular Weight	503.38
Exact Mass	502.117
Elemental Analysis	C, 57.27; H, 4.81; Cl, 14.08; N, 11.13; O, 12.71
CAS #	1707289-21-1
Related CAS #	1707289-21-1
PubChem CID	91885617
Appearance	White to light yellow solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.647
LogP	3.14
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	7
Heavy Atom Count	34
Complexity	688
Defined Atom Stereocenter Count	2
SMILES	ClC1C(=C([H])C(=C(C=1C1C([H])=C([H])C2C(C=1[H])=C([H])N=C(N=2)N([H])[C@]1([H])C([H])([H])OC([H])([H])C([H])([H])[C@]1([H])N([H])C(C([H])=C([H])[H])=O)Cl)OC([H])([H])[H])OC([H])([H])[H]
InChi Key	MGZKYOAQVGSSGC-DLBZAZTESA-N
InChi Code	InChI=1S/C24H24Cl2N4O4/c1-4-20(31)28-16-7-8-34-12-17(16)30-24-27-11-14-9-13(5-6-15(14)29-24)21-22(25)18(32-2)10-19(33-3)23(21)26/h4-6,9-11,16-17H,1,7-8,12H2,2-3H3,(H,28,31)(H,27,29,30)/t16-,17+/m0/s1
Chemical Name	N-[(3S,4S)-3-[[6-(2,6-dichloro-3,5-dimethoxyphenyl)quinazolin-2-yl]amino]oxan-4-yl]prop-2-enamide
Synonyms	BLU-554; BLU 554; BLU554
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	FGFR4 (IC50 = 5 nM) FGFR4 (IC50 = 0.4 nM); FGFR1 (IC50 = 1050 nM); FGFR2 (IC50 = 1200 nM); FGFR3 (IC50 = 980 nM) [1] FGFR4 (Ki = 0.3 nM) [2]
ln Vitro	BLU-554 confers potency and selectivity by taking advantage of a special interaction with FGFR4. BLU-554 potently inhibited recombinant human FGFR4 kinase activity with an IC50 of 0.4 nM and Ki of 0.3 nM, showing >2500-fold selectivity over FGFR1-3 [1][2] It suppressed proliferation of FGFR4-dependent hepatocellular carcinoma (HCC) cell lines: Hep3B (IC50 = 3.2 nM), HuH-7 (IC50 = 5.1 nM), and JHH-7 (IC50 = 4.7 nM) [2] Western blot analysis revealed that BLU-554 (10 nM) blocked FGF19-induced FGFR4 phosphorylation (Tyr642) and downstream ERK1/2, AKT phosphorylation in Hep3B cells [2] The compound induced caspase-dependent apoptosis in Hep3B cells, with 4.3-fold increase in Annexin V-positive cells at 50 nM [2] It inhibited colony formation of HuH-7 cells by 81% at 20 nM and reduced FGF19-mediated cell migration by 68% at 15 nM [1] No significant inhibition of other kinases (e.g., VEGFR2, PDGFRβ) was observed at concentrations up to 1000 nM [1]
ln Vivo	BLU-554 is well tolerated in these tumor models. In models of liver cancer, BLU-554 administration results in tumor regression[1]. Oral administration of BLU-554 at 10, 30, and 60 mg/kg once daily inhibited tumor growth in Hep3B (FGFR4-amplified) xenograft mice by 58%, 83%, and 91% respectively after 28 days of treatment [1] In a FGFR4-amplified HCC patient-derived xenograft (PDX) model, 30 mg/kg daily oral dosing reduced tumor volume by 65% compared to vehicle controls, accompanied by decreased phospho-FGFR4 and phospho-ERK levels in tumor tissues [2] Pharmacodynamic analysis in treated mice showed a 74% reduction in tumor FGFR4 signaling activity, confirming target engagement [2] In a murine orthotopic HCC model, BLU-554 (40 mg/kg, p.o., daily) prolonged median survival by 52% and reduced intrahepatic tumor burden [2]
Enzyme Assay	BLU-554 is a novel potent, highly-selective, and orally bioavailable inhibitor of FGFR4 (fibroblast growth factor receptor 4) with an IC50 value of 5 nM in cell free assays. Recombinant human FGFR4, FGFR1, FGFR2, and FGFR3 kinases were used to evaluate inhibitory activity. The assay was conducted in a buffer containing ATP, MgCl2, and a biotinylated peptide substrate specific for FGFR kinases. Serial dilutions of BLU-554 were incubated with enzyme, substrate, and ATP at 37°C for 60 minutes. The reaction was terminated with a stop buffer, and phosphorylated substrate was captured using streptavidin-coated plates. Detection was performed with a phosphospecific antibody, and absorbance was measured to calculate IC50 values [1] Surface Plasmon Resonance (SPR) assay: FGFR4 kinase domain was immobilized on a sensor chip, and serial dilutions of BLU-554 were injected. Binding kinetics (ka, kd, KD) were derived from sensorgrams, with a KD of 0.2 nM for FGFR4 [2]
Cell Assay	For four hours, either DMSO or BLU-554 at a concentration of one microgram per cell were applied to ST8814 resistant cells. HCC cell proliferation assay: Hep3B, HuH-7, and JHH-7 cells were seeded in 96-well plates at 2×103 cells/well and allowed to adhere overnight. Serial dilutions of BLU-554 were added, and cells were incubated for 72 hours at 37°C in 5% CO2. Cell viability was measured using a colorimetric assay to determine antiproliferative IC50 [2] FGFR4 signaling inhibition assay: Hep3B cells were starved for 12 hours, pretreated with BLU-554 (0.1–100 nM) for 1 hour, then stimulated with FGF19 (50 ng/mL) for 15 minutes. Cell lysates were analyzed by Western blot using anti-phospho-FGFR4, anti-phospho-ERK1/2, anti-phospho-AKT, and total protein antibodies [2] Apoptosis assay: Hep3B cells were treated with BLU-554 (0–100 nM) for 48 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry [2] Colony formation assay: HuH-7 cells were seeded in 6-well plates at 500 cells/well, treated with BLU-554 (0–50 nM), and incubated for 14 days. Colonies were stained with crystal violet and counted [1]
Animal Protocol	Male FVB/NRj mice 10 mg/kg p.o. Hep3B xenograft model: Female nude mice were subcutaneously implanted with 5×106 Hep3B cells. When tumors reached 150–200 mm3, mice were randomized into vehicle and treatment groups. BLU-554 was formulated in 0.5% hydroxypropyl cellulose + 0.1% Tween 80 and administered orally at 10, 30, 60 mg/kg once daily for 28 days. Tumor volume and body weight were measured twice weekly [1] HCC PDX model: Female NOD/SCID mice were implanted with patient-derived HCC tumor fragments. When tumors reached 250 mm3, BLU-554 (30 mg/kg) was administered orally once daily for 21 days. Tumor samples were collected at study end for phospho-FGFR4 and phospho-ERK immunohistochemical analysis [2] Orthotopic HCC model: Male BALB/c nude mice were injected intrahepatically with 1×106 FGFR4-amplified HCC cells. BLU-554 (40 mg/kg) was given orally once daily starting 7 days post-cell injection. Survival was monitored for 60 days, and intrahepatic tumor burden was assessed by bioluminescence imaging [2]
ADME/Pharmacokinetics	Oral bioavailability of BLU-554 in mice was 62% after a single 20 mg/kg dose [3] The compound had a plasma half-life (t1/2) of 4.8 hours in mice following intravenous administration at 10 mg/kg [3] In rats, oral bioavailability was 57% (20 mg/kg dose) with a plasma t1/2 of 5.5 hours [3] Plasma protein binding of BLU-554 was 95% in human plasma, 93% in mouse plasma, and 91% in rat plasma [3] Metabolic stability in human liver microsomes was high, with a half-life of 310 minutes [3] It showed good tumor penetration, with a tumor-to-plasma concentration ratio of 4.9 in Hep3B xenograft mice 4 hours after oral dosing [2]
Toxicity/Toxicokinetics	In a 28-day repeated-dose toxicity study in rats, oral doses of BLU-554 up to 100 mg/kg/day did not cause significant body weight loss, hematological abnormalities, or changes in liver/kidney function markers [2] Clinical adverse events associated with BLU-554 were mild to moderate, including diarrhea (18%), fatigue (12%), and nausea (9%) [2] No significant cardiotoxicity was observed in hERG channel assays (IC50 > 10 μM) [1]
References	[1]. BLU-554, A Novel, Potent and Selective Inhibitor of FGFR4 for the Treatment of Liver Cancer. [2]. Cancer Res. 2021 Feb 1;81(3):747-762. [3]. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Mar 15:1110-1111:116-123.
Additional Infomation	Fisogatinib is under investigation in clinical trial NCT04194801 (A Phase Ib/ii Study of Fisogatinib(blu-554) in Subjects With Hepatocellular Carcinoma). Fisogatinib is an orally bioavailable inhibitor of human fibroblast growth factor receptor 4 (FGFR4), with potential antineoplastic activity. Upon oral administration, fisogatinib specifically binds to and blocks the binding of the ligand FGF19 to FGFR4. This prevents the activation of FGFR4, inhibits FGFR4-mediated signaling and leads to an inhibition of tumor cell proliferation in FGFR4-overexpressing cells. FGFR4 is a receptor tyrosine kinase and is involved in tumor cell proliferation, differentiation, angiogenesis, and survival. FGF19 is overexpressed by certain tumor cell types. BLU-554 is a novel, potent, and highly selective FGFR4 inhibitor developed for the treatment of FGFR4-driven hepatocellular carcinoma (HCC) [1][2] Its mechanism of action involves binding to the ATP-binding pocket of FGFR4, inhibiting its catalytic activity and downstream MAPK/ERK and PI3K/AKT signaling pathways, thereby blocking tumor cell proliferation and survival [2] The compound targets HCC with FGFR4 amplification, FGFR4 mutations, or FGF19 overexpression, which are key drivers of HCC progression [1][2] It has advanced to Phase I/II clinical trials for the treatment of advanced or metastatic HCC with FGFR4 abnormalities [1]

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~198.7 mM) Water: <1 mg/mL Ethanol: ~2 mg/mL (~4 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.97 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 25.0 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 2: ≥ 2.5 mg/mL (4.97 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 25.0 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 3: ≥ 2.5 mg/mL (4.97 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.9866 mL	9.9329 mL	19.8657 mL
	5 mM	0.3973 mL	1.9866 mL	3.9731 mL
	10 mM	0.1987 mL	0.9933 mL	1.9866 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.