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Purity: ≥98%
NSC-663284 (also known as SPS-8I1; DA3003-1) is a novel and potent inhibitor of the Cdc25 dual specificity phosphatases and SETD8 with anticancer activity. It inhibits Cdc25 phosphatases with an IC50 of 0.21 μM. NSC 663284 blocked cellular Erk dephosphorylation caused by ectopic Cdc25A expression. The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signaling processes and cell proliferation, but potent and selective inhibitors are lacking. DA3003-1 inhibited the growth of subcutaneous human colon HT29 xenografts in SCID mice. After a single i.v. dose of 5 mg/kg, DA3003-1 was not detectable in plasma or tissues beyond 5 min. In vitro studies showed that DA3003-1 was rapidly dechlorinated and conjugated to glutathione. Following DA3003-1 treatment of tumor-bearing SCID mice, reduced glutathione concentrations in HT29 tumor were decreased to a greater extent and remained decreased for longer than the reduced glutathione concentrations in liver and kidneys. These studies suggest that the minimal antitumor activity of DA3003-1 in mice may be due to its rapid metabolism.
| Targets |
PF-06700841 is a dual inhibitor of Tyrosine kinase 2 (TYK2) and Janus kinase 1 (JAK1).
For the enzyme: TYK2 IC50 = 23 nM, JAK1 IC50 = 17 nM, JAK2 IC50 = 77 nM, JAK3 IC50 = 6494 nM (ATP concentration = 1 mM). [1] |
|---|---|
| ln Vitro |
The NCI 60-cell human tumor panel showed a mean IC50 of 1.5 ± 0.6 μM for NSC 663284 (3-100 μM; 48 hours), while human breast cancer MDA-MB-435 and MDA-N cells had an IC50 of 0.2 μM. 1.7 μM is the IC50 value in human breast MCF-7 cells that have been grown [1]. NSC 663284's relative IC50 value for Cdc25B2 (IC50=0.21 μM) is greater than that of PTP1B (IC50>4.0 μM) or VHR (IC50=4.0 μM) [1].
In human whole blood assays, PF-06700841 inhibits cytokine-induced STAT phosphorylation. Against IFNα-induced pSTAT3 (lymphocytes, TYK2/JAK1), the IC50 (free) is 13 nM. It also inhibits IL-12-induced pSTAT4 (JAK2/TYK2) with an IC50 of 65 nM and IL-23-induced pSTAT3 (JAK2/TYK2) with an IC50 of 120 nM. For JAK1/JAK3-driven cytokines, it inhibits IL-15-induced pSTAT5 and IL-21-induced pSTAT3 with IC50 values of 238 nM and 204 nM, respectively. It inhibits IL-6-induced pSTAT1 (CD3+ cells, JAK1/JAK2) with an IC50 of 81 nM, but is less potent against IL-6-induced pSTAT3 (IC50 = 641 nM). It inhibits EPO-induced pSTAT5 (JAK2 homodimer) in HWB spiked with CD34+ progenitor cells with an IC50 of 577 nM. IL-10-induced pSTAT3 (TYK2/JAK1) and IL-27-induced pSTAT3 (JAK1/JAK2/TYK2) are inhibited with IC50s of 305 nM and 86 nM, respectively. [1] When profiled against a panel of 306 kinases at 1 µM under conditions with ATP at the apparent Km for each kinase, 21 kinases showed >50% inhibition. At a higher, more physiologically relevant ATP concentration of 1 mM, only 4 kinases (TYK2, JAK1, JAK2, and TNK1) showed >50% inhibition, demonstrating good kinase selectivity under pseudo-physiological conditions. [1] In a broad panel screen against receptors, ion channels, and transporters (CEREP) at 10 µM, significant inhibition (>50%) was only observed for kinase insert domain receptor (KDR/VEGFR2) with an enzymatic IC50 of 1600 nM. However, in a cell-based VEGFR2 signaling assay, the IC50 was >30 µM, indicating the in vitro kinase activity did not translate to functional cellular inhibition under higher ATP conditions. [1] |
| ln Vivo |
The growth of human floating HT29 xenografts obtained via SCID intravenous subcutaneous injection is inhibited by NSC 663284 (iv; 2, 3, and 5 mg/kg). After a single dosage of 5 mg/kg, NSC 663284 was not identifiable in tissues or veins for more than five minutes. Glutathione levels in HT29 tumors decreased more and persisted longer following NSC 663284 treatment in tumor-bearing SCID mice than in kidney and liver cancer [3].
PF-06700841 was evaluated in a therapeutic dosing paradigm in the rat adjuvant-induced arthritis model. Female Lewis rats with established disease were dosed orally once daily for 7 consecutive days with 3, 10, or 30 mg/kg/day of the tosylate salt or vehicle. Treatment with PF-06700841 significantly and dose-dependently reduced the increase in hind paw volume compared to the vehicle group. [1] |
| Enzyme Assay |
The enzymatic activity of PF-06700841 and related compounds against human JAK family kinases (JAK1, JAK2, JAK3, TYK2) was determined using a microfluidic mobility shift assay (Caliper assay). The assay monitors the phosphorylation of a fluorescently labeled synthetic peptide by the recombinant human kinase domain of each JAK member. Reaction mixtures contained 1 µM of the peptide substrate and ATP. For routine structure-activity relationship (SAR) and selectivity assessment, the ATP concentration was set at a pseudo-physiological level of 1 mM. Assay conditions were optimized to achieve a conversion rate of 20–30% phosphorylated product. Reactions were terminated with a stop buffer containing EDTA, and the level of phosphorylation was determined via mobility shift technology. Compounds were assayed at least twice, and IC50 values are reported as geometric means. [1]
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| Cell Assay |
Cellular potency and selectivity within the JAK/STAT signaling pathways were evaluated in human whole blood assays. Blood from healthy donors was collected into heparinized tubes. Specific cytokines were used to stimulate STAT phosphorylation in relevant cell populations at approximately EC90-EC95 concentrations. Following cytokine challenge and compound incubation, cells were fixed, permeabilized, and stained intracellularly for phosphorylated STAT proteins. The level of pSTAT inhibition by the test compound was quantified using flow cytometry. For the EPO assay, CD34+ progenitor cells were spiked into human whole blood before stimulation. Potencies (IC50) were determined from dose-response curves. An estimation of the unbound cellular potency was made using the total HWB IC50, the measured human plasma free fraction, and the red blood cell to plasma partitioning ratio. [1]
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| Animal Protocol |
Rat Adjuvant-Induced Arthritis (AIA) Therapeutic Model: Arthritis was induced in female Lewis rats (8-10 weeks old) via intradermal injection at the base of the tail with complete Freund's adjuvant containing Mycobacterium tuberculosis. Seven days post-immunization, baseline hind paw volume was measured. Rats showing an increase of ≥0.2 mL in a single hind paw were enrolled into the study. PF-06700841 p-toluenesulfonate (tosylate) salt was formulated in a vehicle containing 2% Tween 80 and 0.5% methylcellulose in deionized water. Animals were dosed orally via gavage once daily (q.d.) for 7 consecutive days with vehicle or compound at doses of 3, 10, or 30 mg/kg/day (based on salt). Hind paw volume was monitored. Animals were euthanized after 7 days of dosing. Blood was collected at 15 min (peak) and 24 h (trough) post-final dose for pharmacokinetic analysis and pharmacodynamic assessment of STAT phosphorylation. [1]
Rat Pharmacokinetic Study: The pharmacokinetics of PF-06700841 were studied in Sprague-Dawley rats following intravenous (1 mg/kg) and oral (3 mg/kg) administration of the tosylate salt. Specific formulation details for IV administration are not provided. [1] |
| ADME/Pharmacokinetics |
In Sprague-Dawley rats, plasma clearance was 31 mL/min/kg and volume of distribution was 2.0 L/kg after intravenous (1 mg/kg) and oral (3 mg/kg, tosylate) PF-06700841, with an oral bioavailability of 83%. After oral administration of 3 mg/kg, Cmax was 774 ng/mL and AUC∞ was 1340 ng·h/mL. [1]
In vitro metabolism in human liver microsomes showed low intrinsic clearance (Clint < 10 µL/min/mg protein). In human hepatocytes, Clint < 0.6 µL/min/million cells, indicating low metabolic turnover. [1] Plasma protein binding was low in both rats (free fraction, fu = 0.69) and humans (fu = 0.61). Partition between blood and plasma was not significant (human blood to plasma ratio was 1.2). [1] In in vitro rat, monkey and human models, the main biotransformation pathway is oxidative metabolism, primarily mediated by CYP450 enzymes (of which CYP3A4 is considered the major contributor). Metabolites include products of N-methylpyrazole oxidation, N-demethylation and N-dealkylation. Limited renal and bile clearance is expected. [1] Human pharmacokinetic parameters were predicted based on single-species allometric scaling of in vitro models (human liver microsomes, hepatocytes, recombinant CYP3A4) and rat data. The predicted human blood clearance (Clb) ranged from <0.8 to 5.8 mL/min/kg, with one method predicting a steady-state volume of distribution (Vss) of 1.7 L/kg. [1] The compound has good physical properties: pKa of the conjugate acid = 6.33; the tosylate salt is well soluble in PBS buffer (4.84 mg/mL at pH 7.64) and simulated gastric/intestinal fluid (>7 mg/mL). It exhibits high passive permeability (RRCK average Papp = 18.8 × 10⁻⁶ cm/s). [1] |
| Toxicity/Toxicokinetics |
The provided text focuses on pharmacological properties and does not include specific toxicity data for PF-06700841, such as LD50, hepatotoxicity, or nephrotoxicity. [1]
The compound was designed to selectively inhibit JAK2 to minimize potential hematopoietic side effects (e.g., decreased hemoglobin) associated with inhibition of the JAK2 homodimer/erythropoietin signaling pathway. Based on model predictions, the compound is not expected to inhibit the JAK2-driven EPO signaling pathway (predicted IC18) at the expected effective exposure level (covering IFNα IC80). [1] In rat AIA studies, no significant signs of toxicity were reported at the test doses (3, 10, 30 mg/kg/day for 7 days). [1] |
| References |
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| Additional Infomation |
6-Chloro-7-[2-(4-morpholino)ethylamino]quinoline-5,8-dione is a quinolone compound. NSC-663284 is a potent quinoline dione Cdc25 phosphatase inhibitor. PF-06700841 (compound 23) is a dual TYK2/JAK1 inhibitor originally designed to treat autoimmune diseases. It was designed to combine the efficacy of JAK1 inhibition (affecting γ-common chain cytokines, IL-6 and type I interferon) with the additional benefits of TYK2 inhibition (blocking IL-12 and IL-23 signaling and contributing to type I interferon blockade) while maintaining selectivity for JAK2 to control hematopoietic risks. [1] This compound is derived from a series of 2,4-diaminopyrimidine compounds. The lead compound optimization process focused on the 3,8-diazabicyclo[3.2.1]octane core structure with (S)-2,2-difluorocyclopropylamide group and N-methylpyrazole hinge-binding motif. X-ray crystallography confirmed its binding mode with TYK2 and JAK1. [1]
This compound has completed a Phase I clinical study in healthy volunteers and patients with psoriasis, and is currently undergoing a Phase II clinical trial for multiple indications (ClinicalTrials.gov registration numbers: NCT02969018, NCT02958865, NCT03395184, NCT02974868). [1] |
| Molecular Formula |
C15H16N3O3CL
|
|---|---|
| Molecular Weight |
321.75884
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| Exact Mass |
321.088
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| CAS # |
383907-43-5
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| PubChem CID |
379077
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| Appearance |
Pink to red solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
478.8±45.0 °C at 760 mmHg
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| Flash Point |
243.4±28.7 °C
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| Vapour Pressure |
0.0±1.2 mmHg at 25°C
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| Index of Refraction |
1.626
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| LogP |
0.33
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
22
|
| Complexity |
488
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
BMKPVDQDJQWBPD-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C15H16ClN3O3/c16-11-13(18-4-5-19-6-8-22-9-7-19)15(21)12-10(14(11)20)2-1-3-17-12/h1-3,18H,4-9H2
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| Chemical Name |
6-Chloro-7-(2-morpholin-4-yl-ethylamino)quinoline-5,8-dione
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| Synonyms |
SPS8I1; DA30031; NSC-663284; SPS-8I1; DA3003 1;NSC 663284; SPS 8I1; DA3003-1; NSC663284;
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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 |
| 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) |
DMSO : ≥ 100 mg/mL (~310.79 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.77 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 (7.77 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (7.77 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1079 mL | 15.5395 mL | 31.0791 mL | |
| 5 mM | 0.6216 mL | 3.1079 mL | 6.2158 mL | |
| 10 mM | 0.3108 mL | 1.5540 mL | 3.1079 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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