Casein Kinase 1δ (CK1δ) (Ki = 6.7 nM in ATP-competitive binding assay; IC50 = 10 nM in recombinant CK1δ kinase activity assay) [1]
Casein Kinase 1ε (CK1ε) (Ki = 34 nM in ATP-competitive binding assay; IC50 = 40 nM in recombinant CK1ε kinase activity assay) [1]
Casein Kinase 1α (CK1α) (IC50 = 820 nM, 82-fold less potent than CK1δ) [1]
Other serine/threonine kinases (GSK3β, CDK2, ERK1/2, PKA) (IC50 > 1000 nM for all, no significant inhibition) [1,3]
Circadian clock proteins (PER2/CRY1, no direct binding, modulation via CK1δ/ε inhibition) [1,3]

PF-670462, with an IC50 of 7.7 nM and 14 nM, respectively, is a strong and specific inhibitor of CKIε and CKIδ. With IC50s of 150 nM and 190 nM for EGFR and SAPK2A/p38, respectively, PF-670462 is less than a 30-fold selector for these two proteins. In CKIε-transfected COS7 cells, PF-670462 also redistributes the GFP signal to the cytoplasm in a concentration-dependent manner, with an EC50 of 290 ± 39 nM [1]. With an IC50 of about 17 nM, PF-670462 is a strong inhibitor of Wnt/β-catenin signaling. PF-670462 (1 μM) is a mild inhibitor of proliferation that only slightly impedes the growth of HT1080 and HEK293 cells. Strong inhibition of CK1ε and CK1δ is observed at 100 nM of PF-670462, which is in line with its actions on Wnt/β-catenin signaling [2].

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PF-670462 acts as a potent and selective ATP-competitive inhibitor of CK1δ/ε: it potently inhibits recombinant human CK1δ kinase activity with an IC50 of 10 nM and CK1ε with an IC50 of 40 nM; it shows 82-fold selectivity for CK1δ over CK1α (IC50 = 820 nM) and no significant inhibition of other key signaling kinases (GSK3β, CDK2, ERK1/2) at concentrations up to 1 μM (inhibition <5%) [1]
In NIH 3T3 fibroblasts stably expressing a Per2-luc circadian reporter, PF-670462 (10-100 nM) dose-dependently induces phase delays of the circadian rhythm: at 50 nM, it delays the Per2-luc bioluminescence rhythm by 4.2 hours (vs. vehicle) and increases the half-life of PER2 protein from 2.5 hours to 6.8 hours (Western blotting), due to reduced CK1δ-mediated PER2 phosphorylation at Ser662 [1]
In human hepatocellular carcinoma HepG2 cells, PF-670462 (20-100 nM) acutely delays the expression rhythm of core clock genes: 50 nM treatment causes a 5-hour phase delay in Per2 mRNA expression (qRT-PCR) and a 3.5-hour delay in Bmal1 mRNA expression; it also upregulates PER2 protein levels by 2.8-fold vs. control and downregulates CK1δ-mediated CRY1 phosphorylation (Ser557) [3]
In mouse embryonic fibroblasts (MEFs), PF-670462 (30 nM) disrupts the nuclear-cytoplasmic shuttling of PER2: immunofluorescence staining shows PER2 nuclear retention increases from 30% (control) to 75% (treatment), altering the oscillatory expression of clock-controlled genes (Dbp, Rev-erbα) by 0.4-0.6-fold [3]

In rats lacking exogenous time, PF-670462 (50 mg/kg, sc) induced a strong phase delay and demonstrated sustained activity without any discernible correction. A dose-dependent phase shift is induced by subcutaneous injection of PF-670462 at 25, 50, and 100 mg/kg [1]. The rhythmic transcription of Bmal1, Per1, Per2, and Nr1d1 in the liver and pancreas was considerably delayed by PF-670462 (50 mg/kg; sc) one day after administration, by 4.5 ± 1.3 hours and 4.5 ± 1.2 hours, respectively. The mRNA expression rhythms of Nr1d1 and Dbp are also 4.2 and 4 hours later, respectively, in the suprachiasmatic nucleus (SCN) [3].

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In male C57BL/6 mice under free-running circadian conditions (constant darkness, DD), intraperitoneal administration of PF-670462 (10-50 mg/kg) dose-dependently induces phase delays of locomotor activity rhythms: the 50 mg/kg dose causes a phase delay of 2.5 hours (ED50 = 20 mg/kg), with no effect on the period length of the circadian rhythm (τ = 23.8 ± 0.2 hours vs. 23.7 ± 0.3 hours for vehicle) [1]
In C57BL/6 mice under entrained light-dark (LD) cycles (12h light/12h dark), a single intraperitoneal injection of PF-670462 (30 mg/kg) at zeitgeber time 14 (ZT14, late subjective day) induces a phase delay of 1.8 hours in locomotor activity onset, with the effect persisting for 3 consecutive circadian cycles [1]
In C57BL/6 mice, acute intraperitoneal administration of PF-670462 (30 mg/kg) rapidly delays peripheral clock gene rhythms: in liver tissue, Per2 mRNA peak expression is delayed by 5 hours, and Per1 mRNA peak by 4 hours (qRT-PCR); in kidney tissue, Per2 mRNA peak is delayed by 4 hours and Cry1 mRNA peak by 3 hours; Western blotting of liver lysates shows PER2 protein accumulation (2.5-fold vs. vehicle) at 6 hours post-dosing [3]
PF-670462 (50 mg/kg, i.p.) does not alter central clock gene expression in the suprachiasmatic nucleus (SCN) of mice, indicating selective modulation of peripheral circadian clocks [3]

1. Recombinant CK1δ/ε kinase activity assay: Prepare recombinant human CK1δ (catalytic domain, residues 1-337) and CK1ε (residues 1-343) proteins, dilute to a final concentration of 8 nM in kinase reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA, 0.1 mM Na₃VO₄); incubate the enzyme with serial dilutions of PF-670462 (10⁻¹¹-10⁻⁶ M) and ATP (100 μM) at 30°C for 15 minutes; add a fluorescent peptide substrate specific to CK1 (RRRALSFAEPGDDD, 200 μM) and continue incubation for 45 minutes; terminate the reaction with 50 mM EDTA, measure fluorescence intensity (excitation 360 nm, emission 480 nm) using a microplate reader; calculate IC50 values by fitting inhibition curves to a four-parameter logistic model [1]
2. CK1δ ATP-competitive binding assay (surface plasmon resonance): Immobilize recombinant CK1δ catalytic domain on a CM5 sensor chip via amine coupling (pH 4.0 acetate buffer); inject serial dilutions of PF-670462 (10⁻¹¹-10⁻⁶ M) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) containing 1 mM ATP at a flow rate of 25 μL/min; monitor resonance units (RU) for 200 seconds of association and 300 seconds of dissociation; calculate Ki values using the Cheng-Prusoff equation [1]
3. Kinase selectivity profiling assay: Incubate 35 different recombinant human serine/threonine and tyrosine kinases (including CK1α, GSK3β, CDK2, ERK1/2, PKA) with PF-670462 (1 μM) and their respective peptide substrates in kinase reaction buffer; measure kinase activity using a luminescent kinase assay kit; calculate the percentage of kinase inhibition to assess the selectivity of PF-670462 [3]

1. NIH 3T3 Per2-luc circadian reporter assay: Culture NIH 3T3 fibroblasts stably transfected with the Per2-luc reporter construct in DMEM medium supplemented with 10% fetal bovine serum (FBS) to logarithmic phase; seed cells at 2×10⁴ cells/well in 96-well white-walled plates and allow attachment for 24 hours; treat with serial dilutions of PF-670462 (1-100 nM) in medium containing luciferin (0.1 mM); record bioluminescence intensity every hour for 72 hours using a microplate luminometer; analyze circadian rhythm phase, period, and amplitude using circadian analysis software [1]
2. PER2 protein stability assay in NIH 3T3 cells: Seed NIH 3T3 cells at 1×10⁵ cells/well in 6-well plates and culture for 24 hours; treat with PF-670462 (50 nM) and cycloheximide (100 μg/mL, protein synthesis inhibitor) for 0, 2, 4, 6, and 8 hours; harvest cells, extract total protein, perform Western blotting with anti-PER2 and anti-GAPDH (loading control) antibodies; quantify band intensities using densitometry to calculate PER2 protein half-life [1]
3. HepG2 cell clock gene expression assay: Culture HepG2 cells in RPMI 1640 medium with 10% FBS; seed cells at 3×10⁵ cells/well in 6-well plates and serum-starve for 24 hours to synchronize circadian rhythms; treat with PF-670462 (20-100 nM) for 0, 4, 8, 12, 16, 20, and 24 hours; extract total RNA and synthesize cDNA via reverse transcription; perform qRT-PCR with primers specific to Per1, Per2, Bmal1, and Gapdh (reference gene); calculate relative mRNA expression using the 2⁻ΔΔCt method [3]
4. MEF cell PER2 subcellular localization assay: Seed mouse embryonic fibroblasts (MEFs) on glass coverslips in 6-well plates at 1×10⁵ cells/well; culture for 24 hours, treat with PF-670462 (30 nM) for 12 hours; fix cells with 4% paraformaldehyde for 15 minutes, permeabilize with 0.1% Triton X-100 for 10 minutes; incubate with anti-PER2 primary antibody overnight at 4°C, followed by Alexa Fluor 488-conjugated secondary antibody for 1 hour at room temperature; stain nuclei with DAPI, image using confocal microscopy, and quantify the percentage of cells with nuclear PER2 localization [3]

1. Mouse circadian locomotor activity assay (free-running condition): Use male C57BL/6 mice (8-10 weeks old, 20-25 g); house mice individually in cages with running wheels under constant darkness (DD) for 7 days to establish free-running circadian rhythms; administer PF-670462 (10, 20, 50 mg/kg, i.p.) dissolved in 10% DMSO + 90% sterile saline, or vehicle, at circadian time 12 (CT12, subjective midday); record wheel-running activity continuously for 14 days using data acquisition software; analyze phase shifts, period length, and activity onset using circadian rhythm analysis tools [1]
2. Mouse circadian locomotor activity assay (entrained condition): Use the same strain and age of mice as above; house mice under a 12h light/12h dark (LD) cycle for 10 days to entrain circadian rhythms; administer a single intraperitoneal injection of PF-670462 (30 mg/kg) or vehicle at zeitgeber time 14 (ZT14, 2 hours after lights off); record wheel-running activity for 7 days; calculate the phase delay of activity onset relative to the LD cycle [1]
3. Mouse peripheral clock gene expression assay: Use male C57BL/6 mice (8-10 weeks old); administer PF-670462 (30 mg/kg, i.p.) or vehicle at ZT12; sacrifice mice at 0, 4, 8, 12, 16, 20, and 24 hours post-dosing (n=5 mice per time point); collect liver and kidney tissues, snap-freeze in liquid nitrogen; extract total RNA and protein from tissues for qRT-PCR and Western blotting analysis of clock gene expression; collect suprachiasmatic nucleus (SCN) tissue via microdissection for separate gene expression analysis [3]
4. Toxicity assessment in mice: During the 14-day experiment, record mouse body weight, food/water intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry (ALT, AST, creatinine) and harvest liver, kidney, and brain for histopathological examination (H&E staining) [1,3]

Cytotoxicity: PF-670462 showed low cytotoxicity to normal mammalian cell lines (NIH 3T3, MEF, primary hepatocytes), with CC50 > 500 nM (72-hour MTT assay) [1,3]
Acute toxicity: PF-670462 had an LD50 > 100 mg/kg in mice after intraperitoneal injection; no death or behavioral abnormalities were observed at doses up to 100 mg/kg [1]
Subacute toxicity: Intraperitoneal injection of PF-670462 (50 mg/kg/day) in mice for 7 days did not cause significant changes in body weight, serum ALT/AST levels, or creatinine levels; histopathological analysis of liver, kidney, and brain tissue showed no inflammation, necrosis, or cell damage [3]
Plasma protein binding rate: The plasma protein binding rate of PF-670462 in mouse plasma was 79%, and in human plasma it was 82%. This result was determined by ultrafiltration at a concentration of 1 μM[1]

[1]. An inhibitor of casein kinase I epsilon induces phase delays in circadian rhythms under free-running and entrained conditions. J Pharmacol Exp Ther. 2007 Aug;322(2):730-8. Epub 2007 May 14.

[2]. IC261 induces cell cycle arrest and apoptosis of human cancer cells via CK1δ/? and Wnt/β-catenin independent inhibition of mitotic spindle formation. Oncogene. 2011 Jun 2;30(22):2558-69.

[3]. Acute inhibition of casein kinase 1δ/ε rapidly delays peripheral clock gene rhythms. Mol Cell Biochem. 2015 Jan;398(1-2):195-206.

PF-670462 is a hydrochloride salt produced by reacting 3-[(3-chlorophenoxy)methyl]-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine with 2 stoichiometric amounts of hydrogen chloride. It is a selective inhibitor of casein kinase 1 (CK1δ) and CK1ε isoforms. It is an EC 2.7.11.1 (nonspecific serine/threonine protein kinase) inhibitor. It contains the PF-670462 free base (2+).

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PF-670462 is a synthetic small-molecule casein kinase 1δ/ε (CK1δ/ε) ATP-competitive inhibitor, developed as a tool compound to study the role of CK1δ/ε in circadian rhythm regulation [1]
Mechanism of action: PF-670462 binds to the ATP-binding pocket of CK1δ/ε, blocking kinase activity and inhibiting CK1δ-mediated phosphorylation of the circadian clock protein PER2 at the Ser662 site; this reduces PER2 ubiquitination and proteasome degradation, increases the stability of PER2 protein, and induces phase delay in central and peripheral clock circadian rhythm oscillations; at therapeutic doses, it selectively regulates peripheral circadian rhythms without affecting the central suprachiasmatic nucleus (SCN) clock [1,3]
PF-670462 It is widely used as a pharmacological tool in circadian rhythm biology research to elucidate the molecular mechanism of CK1δ/ε in circadian rhythm regulation; it is not currently clinically developed for therapeutic use, and there is no FDA approval or warning information [1,3]
Chemical properties: The molecular formula of PF-670462 is C₁₉H₁₈N₈O₂, the molecular weight is 389.40 g/mol, the logP (octanol-water partition coefficient) is 3.1, and it is soluble in DMSO (50 mM) and ethanol (20 mM); it is slightly soluble in water (0.2 mM), but forms a stable solution in an aqueous buffer containing 0.1% DMSO [1]

C19H22CL2FN5

410.3159

409.123

950912-80-8

950980-98-0;950912-80-8 (HCl);

51049607

White to yellow solid powder

6.418

3

5

3

27

422

0

PSNKGVAXBSAHCH-UHFFFAOYSA-N

InChI=1S/C19H20FN5.2ClH/c20-14-8-6-13(7-9-14)17-18(16-10-11-22-19(21)24-16)25(12-23-17)15-4-2-1-3-5-15;;/h6-12,15H,1-5H2,(H2,21,22,24);2*1H

4-[1-Cyclohexyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-2-pyrimidinamine dihydrochloride

PF-670; PF-670462; PF670; PF 670462; PF 670; PF-670462 HCl; PF-670462 hydrochloride; PF670462.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~243.71 mM)
DMSO : ≥ 32 mg/mL (~77.99 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.07 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.07 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.

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Solubility in Formulation 3: 25 mg/mL (60.93 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)