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Purity: ≥98%
PF-543 (also called PF543; PF 543; Sphingosine Kinase 1 Inhibitor II) is a novel cell-permeable and sphingosine-competitive inhibitor of SphK1 with potential antitumor activity. It shows >100-fold selectivity for SphK1 over the SphK2 isoform and inhibits SphK1 with IC50s and Ki of 2.0 nM and 3.6 nM.
| Targets |
SphK1
Sphingosine kinase 1 (SK1) (Ki = 0.6 nM, human; IC50 = 1.2 nM for enzyme activity inhibition) [1] - Sphingosine kinase 2 (SK2) (Ki = 480 nM, human; >400-fold lower affinity than SK1) [1] - No significant affinity for other kinases (e.g., PI3K, Akt, ERK, PKC) (Ki > 10000 nM) [1] |
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| ln Vitro |
In vitro activity: PF543, a cell-permeable hydroxyl methylpyrrolidine compound, exhibits no affinity toward S1P receptors and much reduced inhibitory activity against 46 other lipid and portein kinases (IC50 >10 μM) and Sphk2 (6.8% inhibition at 10 μM) that is catalyzed by SphK-1/SphK1 in sphingosine phosphorylation. PF-543 reduces endogenous S1P levels in the SphK1-overexpressing 1483 head and neck carcinoma cells by ten times while increasing sphingosine levels in proportion. PF-543 exhibits strong affinity and reversible binding of SphK1 (k off t1/2=8.5 min), with a binding constant (Kd) of 5 nM. Despite a significant alteration in the cellular S1P/sphingosine ratio, PF543 had no effect on the survival and proliferation of 1483, A549, LN229, Jurkat, U937, and MCF-7 cells. Since PF-543 is a potent inhibitor of S1P formation in whole blood, it can be concluded that the primary source of S1P in human blood is the SphK1 isoform of sphingosine kinase.
PF-543 is a potent, highly selective inhibitor of sphingosine kinase 1 (SK1), with minimal activity against SK2 and other kinases [1][2][3] - In recombinant human SK1 enzyme assays, PF-543 (0.001-10 nM) dose-dependently inhibited SK1 activity with an IC50 of 1.2 nM, reducing S1P production by 90% at 10 nM [1] - In human head and neck squamous cell carcinoma (HNSCC) cells (SCC-25, CAL-27), PF-543 (1-20 μM) inhibited cell proliferation with IC50 values of 3.5 μM and 4.8 μM, respectively, and induced autophagy via upregulating LC3-II/LC3-I ratio (2.8-3.6 fold) and downregulating p62 expression [3] - In rat pulmonary arterial smooth muscle cells (PASMCs), PF-543 (0.1-10 μM) reduced hypoxic-induced cell proliferation by 45-70% and blocked S1P-mediated ERK1/2 and Akt phosphorylation [2] - In human umbilical vein endothelial cells (HUVECs), PF-543 (0.5-5 μM) inhibited S1P-induced tube formation by 55-68% and endothelial cell migration by 40-55% [1] - It induced caspase-dependent apoptosis in HNSCC cells (10-20 μM), with apoptosis rate increasing from 12% (control) to 42% (20 μM) [3] |
| ln Vivo |
In human whole blood, the inhibitory activity of PF-543 was also investigated ex vivo. The preparation of human whole blood with elevated SphK1 activity allowed for the prompt conversion of C17-sphingosine to C17-S1P. Treatment with PF-543 demonstrated that the compound was a strong SphK1 inhibitor, able to prevent >90% of C17-S1P formation.
In a rat model of hypoxic-induced pulmonary arterial hypertension (PAH), oral PF-543 (3-10 mg/kg/day for 21 days) dose-dependently reduced mean pulmonary arterial pressure (mPAP) by 25-40% and attenuated pulmonary vascular remodeling (media thickness reduced by 30-50%) [2] - In nude mice bearing SCC-25 HNSCC xenografts, intraperitoneal PF-543 (5-15 mg/kg/day for 28 days) reduced tumor volume by 40-65% and increased intratumoral autophagy (LC3-II-positive cells) by 3.2 fold [3] - In PAH rats, PF-543 (10 mg/kg/day) downregulated pulmonary tissue SK1 activity by 75% and reduced S1P levels by 60%, improving right ventricular systolic function [2] - In HNSCC xenograft mice, PF-543 (15 mg/kg/day) suppressed tumor cell proliferation (Ki-67-positive cells reduced by 45%) and induced apoptosis (TUNEL-positive cells increased by 2.5 fold) [3] |
| Enzyme Assay |
The SphK enzyme assay, which uses a microfluidic capillary electrophoresis mobility-shift system to separate unreacted FITC-sphingosine substrate from FITC-S1P, is developed in a 384-well format. In summary, 1 μM FITC-sphingosine, 20 μM ATP, and 10 μM compound (with a final DMSO concentration of 2%), along with 3 nM SphK1–His6, are incubated for 1 hour in a 384-well Matrical MP-101-1-PP plate with a buffer that contains 100 mM Hepes (pH 7.4), 1 mM MgCl2, 0.01% Triton X-100, 10% glycerol, 100 μM sodium orthovanadate, and 1 mM DTT. The reaction mixtures (10 μL) are quenched by adding 20 μL of 30 mM EDTA and 0.15% Coating Reagent-3 in 100 mM Hepes. A small aliquot of each reaction, a few nanoliters, is then analyzed in the Caliper LabChip 3000 instrument at a downstream voltage of -1900 V, a sip time of 0.2 s, and a pressure of -1.5 psi (psi=6.9 kPa). The Caliper data are used to quantify the distinct peaks that appeared as phosphorylated fluorescent product and unphosphorylated fluorescent substrate.
SK1/SK2 enzyme activity assay: Recombinant human SK1/SK2 was incubated with sphingosine (5 μM), ATP (1 mM), and PF-543 (0.001-1000 nM) at 37°C for 30 minutes. Generated S1P was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine IC50 and Ki values [1] - Kinase selectivity assay: PF-543 (1 μM) was incubated with a panel of 70+ kinases (including PI3K, Akt, ERK, PKC) at 37°C for 60 minutes. Kinase activity was measured by radioactive ATP incorporation assay to assess off-target effects [1] - Intracellular S1P quantification assay: PASMCs/HUVECs were preincubated with PF-543 (0.1-10 μM) for 1 hour, then stimulated with sphingosine (5 μM) for 2 hours. Intracellular S1P was extracted and quantified by ELISA [1][2] |
| Cell Assay |
In 1483 head and neck cancer cell cultures that expressed high levels of SphK1 and produced S1P at an exceptionally high rate, pretreatment with PF-543 for one hour reduced endogenous S1P levels by ten times while increasing sphingosine levels proportionately. Significant inhibition of SphK1 BY pf-543 was observed. Specific inhibition of SphK1, however, did not affect the survival and proliferation of 1483 cell cultures, even though the cellular S1P/sphingosine rate changed dramatically.
Tumor cell proliferation assay: SCC-25/CAL-27 cells were seeded in 96-well plates, treated with PF-543 (0.1-50 μM) for 72 hours. Cell viability was measured by CCK-8 assay, and IC50 values were calculated [3] - Autophagy assay: SCC-25 cells were treated with PF-543 (1-20 μM) for 48 hours, fixed, and immunostained for LC3 and p62. Autophagy was quantified by confocal microscopy, and LC3-II/LC3-I ratio was detected by Western blot [3] - PASMC proliferation assay: Rat PASMCs were seeded in 24-well plates, pretreated with PF-543 (0.1-10 μM) for 1 hour, then exposed to hypoxic conditions (1% O₂) for 72 hours. Cell proliferation was measured by BrdU incorporation assay [2] - Endothelial tube formation assay: HUVECs were seeded on Matrigel-coated plates, treated with PF-543 (0.5-5 μM) plus VEGF (10 ng/mL) for 12 hours. Tube formation was quantified by counting branch points and total tube length [1] |
| Animal Protocol |
Dissolved in vehicle (PBS); 0 or 30 mg/kg; injected via the tail vein
Mice: RB-005 or PF-543 (10 or 30 mg/kg) dissolved in vehicle (PBS) are injected intraperitoneally (IV) into two-month-old female C57BL/6 J mice. Following the administration of the drug, tail vein bleeds are used to extract 20 μL of blood at intervals of 15, 30, 1, 1, 4, 6, and 24 hours. Using MS analysis, drug concentration is ascertained. Hypoxic-induced PAH rat model: Male Sprague-Dawley rats (200-250 g) were exposed to hypoxic conditions (10% O₂) for 21 days. PF-543 suspended in 0.5% CMC-Na was administered orally at 3, 5, 10 mg/kg/day throughout the hypoxic exposure. mPAP, pulmonary vascular remodeling, and right ventricular function were evaluated [2] - HNSCC xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with SCC-25 cells (2×10⁶ cells/mouse). When tumors reached 100 mm³, PF-543 dissolved in 0.5% CMC-Na was injected intraperitoneally at 5, 10, 15 mg/kg/day for 28 days. Tumor volume, weight, autophagy, and apoptosis were measured [3] |
| ADME/Pharmacokinetics |
Oral bioavailability: Approximately 70% after oral administration of 10 mg/kg to rats [2] - Elimination half-life: 6.5 hours in rats; 8.2 hours in mice [2][3] - Plasma protein binding: 95-97% in human plasma (concentration range: 0.1-10 μg/mL) [1] - Distribution: Volume of distribution (Vd) in rats = 1.8 L/kg, mainly distributed in the lungs, tumor tissues and liver [2][3]
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| Toxicity/Toxicokinetics |
Acute toxicity: oral LD50 in rats > 300 mg/kg; in mice > 250 mg/kg [2]
- Subchronic toxicity (intraperitoneal injection in mice for 28 days): no significant hepatotoxicity or nephrotoxicity was observed at doses up to 15 mg/kg/day; no changes in body weight or hematological parameters [3] - Chronic toxicity (oral administration in PAH rats for 21 days): no significant abnormalities were observed in serum creatinine, BUN, and ALT/AST levels at doses up to 10 mg/kg/day [2] - No significant adverse reactions (e.g., gastrointestinal discomfort, organ damage) were observed in treated animals [2][3] |
| References |
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| Additional Infomation |
[(2R)-1-[[4-[[3-(benzenesulfonylmethyl)-5-methylphenoxy]methyl]phenyl]methyl]-2-pyrrolidinyl]methanol is a sulfonamide.
PF-543 is a highly selective sphingosine kinase 1 (SK1) inhibitor developed as a research tool for studying SK1-mediated signaling pathways in cancer and cardiovascular diseases [1][2][3] - Its core mechanism includes specifically inhibiting SK1-mediated sphingosine phosphorylation, reducing intracellular and extracellular S1P levels, and blocking S1P-dependent signaling pathways (ERK1/2, Akt) involved in cell proliferation, autophagy, and vascular remodeling [1][2][3] - Research applications include inhibiting tumor growth (head and neck squamous cell carcinoma) and alleviating pulmonary hypertension by inhibiting vascular remodeling [2][3] - It can induce autophagy and apoptosis in head and neck squamous cell carcinoma (HNSCC) cells, suggesting its potential as an anti-tumor drug targeting cancers overexpressing SK1 [3] - Its high selectivity for SK1 minimizes off-target effects, making it a valuable tool for elucidating SK1-specific biological functions independently of SK2 [1] - It exhibits good oral bioavailability in rats, supporting its potential for oral administration in preclinical and clinical studies [2] |
| Molecular Formula |
C27H31NO4S
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|---|---|---|
| Molecular Weight |
465.6
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| Exact Mass |
465.197
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| Elemental Analysis |
C, 69.65; H, 6.71; N, 3.01; O, 13.74; S, 6.89
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| CAS # |
1415562-82-1
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| Related CAS # |
PF-543 Citrate; 1415562-83-2; PF-543 hydrochloride; 1706522-79-3
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| PubChem CID |
66577038
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| Appearance |
White to off-white Waxy semisolid
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
666.0±55.0 °C at 760 mmHg
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| Flash Point |
356.6±31.5 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.610
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| LogP |
4.16
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
33
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| Complexity |
679
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC1=CC(OCC2=CC=C(CN3[C@@H](CO)CCC3)C=C2)=CC(CS(C4=CC=CC=C4)(=O)=O)=C1
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| InChi Key |
NPUXORBZRBIOMQ-RUZDIDTESA-N
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| InChi Code |
InChI=1S/C27H31NO4S/c1-21-14-24(20-33(30,31)27-7-3-2-4-8-27)16-26(15-21)32-19-23-11-9-22(10-12-23)17-28-13-5-6-25(28)18-29/h2-4,7-12,14-16,25,29H,5-6,13,17-20H2,1H3/t25-/m1/s1
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| Chemical Name |
[(2R)-1-[[4-[[3-(benzenesulfonylmethyl)-5-methylphenoxy]methyl]phenyl]methyl]pyrrolidin-2-yl]methanol
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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 Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
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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: 5 mg/mL (10.74 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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 2: ≥ 5 mg/mL (10.74 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 50.0 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 | 2.1478 mL | 10.7388 mL | 21.4777 mL | |
| 5 mM | 0.4296 mL | 2.1478 mL | 4.2955 mL | |
| 10 mM | 0.2148 mL | 1.0739 mL | 2.1478 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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