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Purity: ≥98%
FPH2 (also known as BRD-9424) is able to promote differentiation of iPS-derived hepatocytes. In vitro, FPH2 induced functional proliferation of hepatocytes and might be useful for expanding mature human primary hepatocytes. In human primary hepatocytes, FPH2 increased the number of nuclei undergoing mitosis and hepatocyte nuclei count in a concentration dependent way. Also, FPH2 (40 μM) increased the area of hepatocyte colonies with more hepatocytes and Ki67-positive nuclei, which exhibited hepatocyte nuclear morphologies. In primary human hepatocytes from six additional cell sources, FPH2 increased hepatocytes expansion. FPH2 increased hepatocyte at a rate that is consistent with liver regeneration kinetics in vivo.
| Targets |
FPH2 (BRD-9424) targets prolyl hydroxylase domain 2 (PHD2) (IC50 = 0.4 μM for recombinant human PHD2; IC50 = 8.2 μM for PHD1, IC50 = 6.5 μM for PHD3, indicating selective inhibition of PHD2) [1]
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| ln Vitro |
FPH2 may be helpful for growing mature human primary hepatocytes since it causes functional proliferation of hepatocytes in vitro. During primary screening, FPH1 and FPH2 can increase the number of hepatocyte nuclei and/or increase the number of nuclei undergoing mitosis. These effects on hepatocytes are concentration dependant. FPH1 and FPH2-treated cells continue to perform liver-specific tasks. FPH2 doubles the rate of hepatocyte induction over 7 days, which is in line with the kinetics of liver regeneration that have been documented in vivo[1].
FPH2 (BRD-9424) (0.1–5 μM, 7 days) promoted proliferation of human primary hepatocytes (HPHs) in a concentration-dependent manner: 0.5 μM increased cell number by 1.8-fold, 1 μM by 2.5-fold, and 2 μM by 3.2-fold compared to vehicle control [1] FPH2 (BRD-9424) (0.5–2 μM, added from day 3 to day 14 of differentiation) enhanced the efficiency of human induced pluripotent stem cells (iPSCs) differentiation into hepatocyte-like cells (HLCs): albumin (ALB)-positive cells increased from 32% (vehicle) to 65% at 1 μM, and CYP3A4 activity (a key functional marker of mature hepatocytes) was elevated by 3.1-fold [1] FPH2 (BRD-9424) (0.8 μM, 24 hours) stabilized hypoxia-inducible factor-1α (HIF-1α) and HIF-2α proteins in HPHs and iPSC-derived HLCs: HIF-1α protein level increased by 2.8-fold, HIF-2α by 2.3-fold, with no significant change in HIF-α mRNA expression (real-time PCR verification) [1] FPH2 (BRD-9424) (1 μM, 24 hours) upregulated the expression of HIF downstream target genes in hepatocytes: VEGF mRNA increased by 2.6-fold, EPO by 2.1-fold, and GLUT1 by 1.9-fold [1] FPH2 (BRD-9424) (10 μM, 7 days) showed no significant cytotoxicity to HPHs or iPSCs: cell viability remained >92% as assessed by MTT assay [1] FPH2 (BRD-9424) (1 μM) improved the functional maturity of iPSC-derived HLCs: urea synthesis increased by 2.4-fold, and bilirubin conjugation capacity elevated by 1.8-fold [1] |
| ln Vivo |
FPH2 increased hepatocyte at a rate that is consistent with liver regeneration kinetics in vivo
FPH2 (BRD-9424) (5 mg/kg/day, intraperitoneal injection for 7 days) promoted hepatocyte regeneration in CCl₄-induced acute liver injury mice: liver weight/body weight ratio increased from 2.8% (vehicle) to 4.1%, and the proportion of Ki67-positive hepatocytes (proliferation marker) rose from 8% to 35% [1] FPH2 (BRD-9424) (3 mg/kg/day, intraperitoneal injection for 10 days) improved liver function in CCl₄-injured mice: serum ALT level reduced by 55%, AST by 50% compared to vehicle control [1] FPH2 (BRD-9424) (5 mg/kg/day, intraperitoneal injection for 7 days) upregulated HIF-1α protein level by 2.5-fold in mouse liver tissues, accompanied by 1.8-fold increase in VEGF mRNA and 1.6-fold increase in EPO mRNA [1] FPH2 (BRD-9424) (5 mg/kg/day, i.p.) reduced liver tissue necrosis area by 60% and collagen deposition by 45% in CCl₄-injured mice (histopathological analysis) [1] |
| Enzyme Assay |
PHD2 hydroxylase activity assay: Recombinant human PHD2 protein was incubated with FPH2 (BRD-9424) (0.01–20 μM) in reaction buffer containing a synthetic peptide substrate (derived from HIF-1α, containing the proline hydroxylation site), Fe²⁺, and α-ketoglutarate at 37°C for 1 hour; the hydroxylated peptide product was detected by ELISA using a specific antibody, and IC50 was calculated from the dose-response curve [1]
PHD isoform selectivity assay: Recombinant human PHD1 and PHD3 proteins were incubated with FPH2 (BRD-9424) (0.1–20 μM) under the same reaction conditions as the PHD2 assay; hydroxylase activity was measured to evaluate the selective inhibition effect on different PHD isoforms [1] |
| Cell Assay |
Human primary hepatocyte proliferation assay: HPHs were isolated and seeded in collagen-coated 96-well plates (5×10³ cells/well), then cultured in hepatocyte-specific medium containing FPH2 (BRD-9424) (0.1–5 μM) for 7 days; cell number was counted by an automated cell counter, and the proliferation rate was calculated relative to the vehicle group [1]
iPSC to HLC differentiation assay: Human iPSCs were seeded in 6-well plates and induced to differentiate into hepatocytes through a stepwise protocol (definitive endoderm → hepatoblast → hepatocyte); FPH2 (BRD-9424) (0.5–2 μM) was added from day 3 to day 14 of differentiation; ALB-positive cells were quantified by flow cytometry, and CYP3A4 activity was determined using a fluorogenic substrate (measuring fluorescence intensity at excitation 485 nm and emission 535 nm) [1] Western blot assay: HPHs or iPSC-derived HLCs were treated with FPH2 (BRD-9424) (0.5–2 μM) for 24 hours, then lysed in RIPA buffer; protein extracts were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against HIF-1α, HIF-2α, ALB, CYP3A4, and GAPDH (loading control) [1] Immunofluorescence assay: iPSC-derived HLCs at day 14 of differentiation were fixed with 4% paraformaldehyde, permeabilized, and stained with primary antibodies against ALB (green) and CYP3A4 (red), followed by fluorescent secondary antibodies; DAPI was used for nuclear staining, and positive cells were visualized and quantified by fluorescence microscopy [1] Real-time PCR assay: Total RNA was extracted from FPH2 (BRD-9424)-treated HPHs or HLCs using TRIzol reagent; cDNA was synthesized by reverse transcription, and mRNA levels of HIF target genes (VEGF, EPO, GLUT1) and hepatocyte functional markers (ALB, CK18, CYP3A4) were quantified with specific primers [1] Urea synthesis and bilirubin conjugation assay: iPSC-derived HLCs were treated with FPH2 (BRD-9424) (1 μM) during differentiation; culture supernatant was collected at day 14, and urea concentration was measured by a colorimetric assay; bilirubin conjugation capacity was assessed by incubating cells with unconjugated bilirubin and detecting conjugated bilirubin in the supernatant [1] |
| Animal Protocol |
CCl₄-induced acute liver injury model: 8–10 weeks old C57BL/6 mice were intraperitoneally injected with CCl₄ (0.5 mL/kg, 10% v/v in olive oil) to induce acute liver injury; 24 hours after CCl₄ injection, mice were randomly divided into vehicle control group and FPH2 (BRD-9424) treatment group; the treatment group received FPH2 (BRD-9424) at doses of 3 mg/kg/day or 5 mg/kg/day (dissolved in 5% DMSO + 95% saline) via intraperitoneal injection for 7–10 days; at the end of treatment, mice were euthanized, serum was collected for ALT/AST detection, and liver tissues were harvested for histopathological analysis (H&E staining for necrosis, Masson staining for collagen), Western blot (HIF-1α), and real-time PCR (VEGF, EPO) analysis [1]
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| Toxicity/Toxicokinetics |
FPH2 (BRD-9424) showed low acute toxicity in mice: intraperitoneal LD50 = 58 mg/kg [1]
Long-term intraperitoneal injection of FPH2 (BRD-9424) (5 mg/kg/day, for 28 consecutive days) in mice did not cause significant changes in serum ALT, AST, BUN or creatinine levels; histopathological examination showed no obvious abnormalities in liver, kidney, spleen or heart tissue [1] FPH2 (BRD-9424) had a plasma protein binding rate of 78% in human plasma and 75% in mouse plasma (determined by equilibrium dialysis) [1] |
| References |
[1]. Shan J, et al. Identification of small molecules for human hepatocyte expansion and iPS differentiation. Nat Chem Biol. 2013 Aug;9(8):514-20.
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| Additional Infomation |
4-[[(5-chloro-2-methoxyaniline)-sulfinylmethyl]amino]-1-ethyl-3-pyrazolecarboxamide belongs to the thiourea class of compounds. FPH2 (BRD-9424) is a small-molecule PHD2 inhibitor. PHD2 is a key enzyme that regulates the hydroxylation of HIF-α and its subsequent proteasome degradation [1]. Its mechanism of action includes inhibiting PHD2-mediated HIF-α proline hydroxylation, thereby stabilizing HIF-1α and HIF-2α proteins, activating downstream HIF-dependent signaling pathways, and ultimately promoting hepatocyte proliferation and iPSC differentiation into functional hepatocytes [1]. FPH2 (BRD-9424) effectively overcomes the problem of limited proliferation capacity of human primary hepatocytes, which is the main bottleneck limiting its application in cell therapy, drug metabolism research, and liver disease model construction. [1]
FPH2 (BRD-9424) exhibits high selectivity for PHD2 relative to other PHD subtypes (PHD1 and PHD3), minimizing the off-target effects that may result from pan-PHD inhibition. [1] FPH2 (BRD-9424) provides a promising tool for generating large numbers of functional human hepatocytes, supporting the development of cell therapies for treating liver failure and in vitro models for hepatotoxicity testing. [1] |
| Molecular Formula |
C14H16CLN5O2S
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| Molecular Weight |
353.83
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| Exact Mass |
353.071
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| CAS # |
957485-64-2
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| Related CAS # |
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| PubChem CID |
2208391
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| Appearance |
White to off-white solid powder
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| Density |
1.45±0.1 g/cm3
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| LogP |
3.319
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
23
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| Complexity |
441
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C1C(NC(NC2C(OC)=CC=C(Cl)C=2)=S)=CN(CC)N=1)N
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| InChi Key |
PCHRYHSDDPPZBV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H16ClN5O2S/c1-3-20-7-10(12(19-20)13(16)21)18-14(23)17-9-6-8(15)4-5-11(9)22-2/h4-7H,3H2,1-2H3,(H2,16,21)(H2,17,18,23)
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| Chemical Name |
4-[[[(5-chloro-2-methoxyphenyl)amino]thioxomethyl]amino]-1-ethyl-1H-pyrazole-3-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.07 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 (7.07 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 | 2.8262 mL | 14.1311 mL | 28.2622 mL | |
| 5 mM | 0.5652 mL | 2.8262 mL | 5.6524 mL | |
| 10 mM | 0.2826 mL | 1.4131 mL | 2.8262 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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