FH1 (also known as NSC 12407, BRD-K4477) can enhance the functions of cultured hepatocytes by promoting differentiation of iPS-derived hepatocytes. FH1 belongs to the function-only hits which are screened out by their ability to permit renewable sourcing of functional human hepatocytes. Besides that, FH1 can promote differentiation of iPS-derived hepatocytes towards a more mature phenotype. It doubles albumin secretion during the differentiation of iPS cells into iHeps.

Physicochemical Properties

Molecular Formula	C17H18N2O2
Molecular Weight	282.34
Exact Mass	282.136
Elemental Analysis	C, 72.32; H, 6.43; N, 9.92; O, 11.33
CAS #	2719-05-3
Related CAS #	2719-05-3
PubChem CID	94990
Appearance	Solid powder
Density	1.2±0.1 g/cm3
Boiling Point	564.6±43.0 °C at 760 mmHg
Flash Point	217.8±28.3 °C
Vapour Pressure	0.0±1.5 mmHg at 25°C
Index of Refraction	1.635
LogP	1.93
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	4
Heavy Atom Count	21
Complexity	320
Defined Atom Stereocenter Count	0
SMILES	O=C(C([H])([H])[H])N([H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(C([H])([H])[H])=O
InChi Key	OEXMNSOPAKOPEF-UHFFFAOYSA-N
InChi Code	InChI=1S/C17H18N2O2/c1-12(20)18-16-7-3-14(4-8-16)11-15-5-9-17(10-6-15)19-13(2)21/h3-10H,11H2,1-2H3,(H,18,20)(H,19,21)
Chemical Name	N-[4-[(4-acetamidophenyl)methyl]phenyl]acetamide
Synonyms	FH-1; NSC-12407; BRD-K4477;FH1; NSC12407; BRDK4477;FH 1; NSC 12407; BRD K4477;
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	FH1 (BRD-K4477) targets pathways involved in human hepatocyte expansion and induced pluripotent stem cell (iPSC) differentiation into hepatocytes[1]
ln Vitro	FH1 (NSC 12407, 5 µg/mL, 0–40 days) promotes hepatic maturation and preserves the hiPSC–HLC condition of hepatic differentiation[2]. In primary human hepatocytes (PHHs), FH1 (BRD-K4477) (0.5–5 μM) dose-dependently promoted cell expansion. At 2 μM, the number of PHHs increased by ~3.2-fold after 7 days of culture compared to vehicle control. It maintained hepatocyte functionality, as evidenced by sustained expression of albumin (ALB) and cytochrome P450 3A4 (CYP3A4) at both mRNA (ALB: ~1.8-fold; CYP3A4: ~1.5-fold at 2 μM) and protein levels [1] - In human iPSCs undergoing hepatocyte differentiation, FH1 (BRD-K4477) (1–3 μM) enhanced differentiation efficiency. At 2 μM, the proportion of hepatocyte-like cells (HLCs) expressing ALB and α-fetoprotein (AFP) increased from ~45% to ~72% (flow cytometry). RT-PCR showed upregulated expression of hepatocyte-specific genes (HNF4α, FOXA2, TTR) by ~2.3–3.1-fold compared to control [1] - In iPSC-derived HLCs, FH1 (BRD-K4477) (2 μM) pretreatment for 48 hours improved cell functionality: ALB secretion increased by ~2.5-fold, urea synthesis enhanced by ~1.8-fold, and CYP3A4 activity elevated by ~2.1-fold. It also enhanced the susceptibility of HLCs to hepatitis B virus (HBV) infection, with HBV DNA copy number increased by ~3.0-fold compared to untreated HLCs [2]
ln Vivo	In humanized chimeric mice (NOD/SCID/IL2rg–/–) engrafted with FH1 (BRD-K4477)-pretreated iPSC-HLCs (2 μM for 48 hours in vitro), the engraftment efficiency of HLCs in mouse liver was significantly increased. At 8 weeks post-engraftment, human ALB levels in mouse serum were ~2.8-fold higher than in mice transplanted with untreated HLCs. Immunohistochemical staining showed increased human ALB+ and CYP3A4+ cells in the liver parenchyma [2] - FH1 (BRD-K4477)-pretreated HLCs maintained long-term functionality in chimeric mice: human ALB secretion remained stable for up to 12 weeks, and the mice supported persistent HBV replication (HBV DNA detectable in serum for 10 weeks) after HBV inoculation, which was not observed in mice with untreated HLC engraftment [2]
Cell Assay	RT-PCR[2] Cell Types: hiPSC-HLCs in the differentiated hepatic state. Tested Concentrations: 5 µg/mL. Incubation Duration: 0-40 days. Experimental Results: Kept more than 50% hiPSC-HLCs to produce the four factors till day 40. Can help hiPSC-HLCs to maintain a relatively mature hepatic differentiation state for about 40 days. Maintained a higher level of hALB in the supernatant than that in controls. Maintained relatively high mRNA levels of hNTCP, hRXR, hHNF4α, and hALB for over functional small molecule FH1 can help hiPSC-HLCs to maintain a relatively mature hepatic differentiation state for about 40 days. Primary human hepatocyte expansion assay: PHHs were isolated and seeded in collagen-coated 6-well plates at 1×105 cells/well. FH1 (BRD-K4477) (0.5–5 μM) was added to hepatocyte culture medium, which was refreshed every 2 days. Cell number was counted by hemocytometer at day 7. ALB and CYP3A4 expression were detected by RT-PCR (mRNA) and Western blot (protein), and CYP3A4 activity was measured using a luminescent substrate assay [1] - iPSC hepatocyte differentiation assay: Human iPSCs were seeded in Matrigel-coated plates and induced to differentiate into HLCs using a three-step protocol (definitive endoderm → hepatoblast → hepatocyte). FH1 (BRD-K4477) (1–3 μM) was added during the hepatoblast to hepatocyte transition phase (days 10–21). At day 21, HLCs were analyzed by flow cytometry (ALB/AFP staining) and RT-PCR for hepatocyte-specific genes (HNF4α, FOXA2, TTR) [1] - iPSC-HLC functionality and HBV infection assay: iPSC-HLCs were pretreated with FH1 (BRD-K4477) (2 μM) for 48 hours. ALB secretion was measured by ELISA, urea synthesis by a colorimetric assay, and CYP3A4 activity by luminescence. For HBV infection, pretreated HLCs were incubated with HBV inoculum for 24 hours, and HBV DNA copy number in culture supernatant was quantified by qPCR at day 7 post-infection [2]
Animal Protocol	Humanized chimeric mouse model for HLC engraftment: 6–8-week-old NOD/SCID/IL2rg–/– mice were sublethally irradiated (3.5 Gy) 24 hours before cell transplantation. iPSC-HLCs were pretreated with FH1 (BRD-K4477) (2 μM) in culture medium for 48 hours. Pretreated or untreated HLCs (1×106 cells/mouse) were transplanted into mice via intrasplenic injection. Mouse serum was collected every 2 weeks to detect human ALB by ELISA. At 8–12 weeks post-transplantation, mice were inoculated with HBV, and serum HBV DNA was quantified by qPCR weekly. Mice were sacrificed at 12 weeks, and liver tissues were collected for immunohistochemical staining (human ALB, CYP3A4) [2]
Toxicity/Toxicokinetics	In vitro toxicity: FH1 (BRD-K4477) (0.5–5 μM) did not affect the viability of PHHs, iPSCs, or iPSC-HLCs, with cell viability maintained above 90% at all tested concentrations. It did not induce apoptosis, as shown by Annexin V-FITC/PI staining [1,2] - In vivo toxicity: No obvious adverse effects (e.g., weight loss, lethargy, organ damage) were observed in chimeric mice transplanted with FH1 (BRD-K4477)-pretreated HLCs during the 12-week observation period. Serum ALT, AST, creatinine, and urea nitrogen levels of mice were within normal ranges [2]
References	[1]. Identification of small molecules for human hepatocyte expansion and iPS differentiation. Nat Chem Biol. 2013 Aug;9(8):514-20. [2]. A Chimeric Humanized Mouse Model by Engrafting the Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cell for the Chronic Hepatitis B Virus Infection. Front Microbiol. 2018 May 8;9:908.
Additional Infomation	FH1 (BRD-K4477) is a small-molecule compound identified through high-throughput screening for agents that promote human hepatocyte expansion and iPSC differentiation into functional hepatocytes [1] - Its mechanism of action involves upregulating the expression of hepatocyte-specific transcription factors (HNF4α, FOXA2) and maintaining the epithelial phenotype of hepatocytes, thereby enhancing cell proliferation and functional maturation [1] - FH1 (BRD-K4477) improves the functionality and engraftment efficiency of iPSC-HLCs, enabling the establishment of stable humanized mouse models for chronic HBV infection research [2] - It shows potential applications in regenerative medicine (hepatocyte transplantation for liver diseases) and preclinical drug development (humanized liver models for drug metabolism and toxicity testing) [1,2]

Solubility Data

Solubility (In Vitro)

DMSO:56 mg/mL (198.3 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.85 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 (8.85 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 (8.85 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	3.5418 mL	17.7091 mL	35.4183 mL
	5 mM	0.7084 mL	3.5418 mL	7.0837 mL
	10 mM	0.3542 mL	1.7709 mL	3.5418 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.