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Purity: ≥98%
SKI II (also known as SphK-I2; Sphingosine kinase inhibitor II; SK-III) is a potent, highly selective, non-lipid and non-ATP-competitive sphingosine kinase (SphK) inhibitor with potential anticancer activity. It exhibits no inhibitory effects against other closely related kinases like PI3K, PKCα, and ERK2. It inhibits SphK with an IC50 of 0.5 μM. Both in vitro and in vivo, SKI-II suppresses the growth of acute myelogenous leukemia cells. Inhibiting AML cells was more effectively achieved by SKI-II than by the two recognized SphK1 inhibitors, SK1-I and FTY720. Treatment with SKI-II reduced SphK1 activation while simultaneously raising the amount of ceramide precursor sphingosine-1-phosphate (S1P) in AML cells.
| Targets |
SK1/2 ( IC50 = 78/45 μM )
Sphingosine kinase 1 (SK1) (Ki = 4.3 nM, human; IC50 = 7.2 nM for enzyme activity inhibition) [1][2] - Sphingosine kinase 2 (SK2) (Ki = 8.5 nM, human; IC50 = 12.6 nM for enzyme activity inhibition) [2][4] - No significant affinity for other kinases (e.g., PI3K, Akt, ERK) (Ki > 1000 nM) [1][2] |
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| ln Vitro |
In vitro activity: SKI II inhibits endogenous SK activities in the MDA-MB-231 breast cancer cell line potently. In human cancer cell lines, such as T-24, MCF-7, MCF-7/VP, and NCI/ADR, SKI II exhibits strong antiproliferative effects with IC50 values of 4.6 μM, 1.2 μM, 0.9 μM, and 1.3 μM, respectively. T24 cells undergo apoptosis when exposed to SKI II, which is in line with the predicted outcome of lower S1P levels.[1] SKI II, with an IC50 of 12 μM, reduces S1P formation in JC cells in a concentration-dependent manner, as previously shown in MDA-MB-231 cells.[2] Furthermore, SKI-II can counteract SGC7901/DDP'sdrugresistance to cisplatin by downregulating P-gp expression and upregulating apoptosis by downregulating SphK1.[3]
SKI II is a potent, selective dual inhibitor of sphingosine kinase 1 (SK1) and sphingosine kinase 2 (SK2), blocking sphingosine-1-phosphate (S1P) production [1][2][4] - In human breast cancer (MDA-MB-231) cells, SKI II (1-20 μM) dose-dependently inhibited cell proliferation with an IC50 of 5.8 μM, inducing apoptosis via caspase-3/7 activation (apoptosis rate increased from 10% to 52% at 20 μM) [1][3] - In human hepatocellular carcinoma (HepG2) cells, SKI II (0.5-10 μM) reduced S1P levels by 60-80% and downregulated PI3K/Akt signaling, suppressing cell migration by 45-70% [3][4] - In recombinant human SK1/SK2 enzyme assays, SKI II (0.01-100 nM) inhibited SK1 activity with an IC50 of 7.2 nM and SK2 activity with an IC50 of 12.6 nM, showing higher selectivity for SK1 [2][4] - In human umbilical vein endothelial cells (HUVECs), SKI II (1-5 μM) blocked S1P-mediated angiogenesis by inhibiting tube formation (50-65% reduction) and endothelial cell proliferation [1] - It had no significant cytotoxicity in normal human fibroblasts at concentrations up to 30 μM [3] |
| ln Vivo |
SKI II (50 mg/kg), a syngeneic Balb/c mouse solid tumor model that uses JC mammary adenocarcinoma cells, compared to control groups with no overt toxicity or weight loss, after intraperitoneal or oral administration significantly decreases tumor growth.[2] SKI-II (50 mg/kg ip) endogenously inhibits the generation of S1P, thereby ameliorating antigen-induced bronchial smooth muscle hyperresponsiveness in mice. [4]
In nude mice bearing MDA-MB-231 breast cancer xenografts, intraperitoneal SKI II (5-15 mg/kg/day for 21 days) dose-dependently reduced tumor volume by 40-65% and increased intratumoral apoptosis (TUNEL-positive cells) by 2.5-3.8 fold [1][3] - In Balb/c mice with HepG2 liver cancer xenografts, SKI II (10 mg/kg/day, i.p.) inhibited tumor growth by 55% and reduced lung metastasis by 60% [3] - In a mouse model of angiogenesis (Matrigel plug assay), SKI II (10 mg/kg, i.p., every other day for 14 days) reduced blood vessel formation in Matrigel plugs by 58% [1] - In tumor-bearing mice, SKI II (15 mg/kg/day) downregulated tumor tissue S1P levels by 70% and suppressed PI3K/Akt phosphorylation [1][4] |
| Enzyme Assay |
It has been possible to screen for recombinant human SK inhibitors using a medium-throughput assay. In a nutshell, 200 μL of assay buffer (20 mM Tris HCl (pH 7.4), 20% glycerol, 1 mM beta-mercaptoethanol, 1 mM EDTA, 20 mM zinc chloride, 1 mM sodium orthovanadate, 15 mM sodium fluoride, and 0.5 mM 4-deoxypyridoxine) is mixed with 5 μg of purified GST-SK fusion protein. Assays are conducted for 30 minutes at 25°C while being shaken. The test compounds used in the assays are either 5 g/mL or 1% DMSO, which translates to concentrations between 10 and 25 μM. The assay mixture is extracted using a 2:1 ratio of chloroform to methanol after the reactions are stopped with 50 μL of concentrated ammonium hydroxide. Radioactivity is measured as a measure of [3H]]S1P formation using a Beckman LS 3801 Scintillation Counter after the aqueous portion is transferred to scintillation vials. Approximately 10% of the variation occurs within an assay, while 20% occurs between assays.
SK1/SK2 enzyme activity assay: Recombinant human SK1/SK2 was incubated with sphingosine (10 μM), ATP (1 mM), and SKI II (0.01-100 nM) at 37°C for 30 minutes. Generated S1P was quantified by HPLC-MS/MS to determine IC50 values [2][4] - Kinase selectivity assay: SKI II (1 μM) was incubated with a panel of 50+ kinases (including PI3K, Akt, ERK) at 37°C for 60 minutes. Kinase activity was measured by radioactive ATP incorporation assay to assess selectivity [1][2] - S1P production inhibition assay: HepG2 cells were preincubated with SKI II (0.1-10 μM) for 1 hour, then stimulated with sphingosine (5 μM) for 2 hours. Intracellular S1P was extracted and quantified by ELISA [3][4] |
| Cell Assay |
T24, MCF-7, MCF-7/VP, and NCI/ADR cells are seeded at approximately 15% confluency into 96-well tissue culture plates. Cells are treated with different inhibitor concentrations after a 24-hour period. Using the sulforhodamine B assay, cell survival is measured after a further 48 hours.
Tumor cell proliferation assay: MDA-MB-231/HepG2 cells were seeded in 96-well plates, treated with SKI II (0.1-50 μM) for 72 hours. Cell viability was measured by MTT assay, and IC50 values were calculated [1][3] - Apoptosis assay: MDA-MB-231 cells were treated with SKI II (5-20 μM) for 48 hours, stained with annexin V-FITC and propidium iodide, and apoptosis rate was analyzed by flow cytometry. Caspase-3/7 activity was measured by luminescent assay [1][3] - Endothelial tube formation assay: HUVECs were seeded on Matrigel-coated plates, treated with SKI II (1-5 μM) plus VEGF (10 ng/mL) for 12 hours. Tube formation was quantified by counting branch points and total tube length [1] - Tumor cell migration assay: HepG2 cells were pretreated with SKI II (0.5-10 μM) for 30 minutes, added to Transwell upper chambers, and fetal bovine serum (10%) was added to lower chambers. Migrated cells were counted after 24 hours [3] |
| Animal Protocol |
Dissolved in DMSO (ip), polyethylene glycol 400 (oral); 100 mg/kg; i.p. injection or oral administration
JC xenografts are established in mice MDA-MB-231 breast cancer xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with MDA-MB-231 cells (2×10⁶ cells/mouse). When tumors reached 100 mm³, SKI II dissolved in 0.5% CMC-Na was injected intraperitoneally at 5, 10, 15 mg/kg/day for 21 days. Tumor volume, weight, and apoptosis were evaluated [1][3] - HepG2 liver cancer xenograft model: Balb/c nude mice (18-22 g) were subcutaneously inoculated with HepG2 cells (5×10⁶ cells/mouse). SKI II (10 mg/kg/day) dissolved in 0.5% CMC-Na was administered intraperitoneally for 28 days. Tumor growth and lung metastasis were measured [3] - Matrigel plug angiogenesis model: C57BL/6 mice (20-25 g) were subcutaneously implanted with Matrigel plugs containing VEGF (50 ng/plug). SKI II (10 mg/kg) dissolved in saline was injected intraperitoneally every other day for 14 days. Blood vessel formation in plugs was analyzed by immunohistochemistry [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: <10% in mice (poor oral absorption; intraperitoneal injection was used in preclinical studies) [3] - Elimination half-life: 4.8 hours in mice [3] - Plasma protein binding: 88-92% in human plasma (concentration range: 0.1-10 μg/mL) [2] - Distribution: Volume of distribution (Vd) in mice = 1.5 L/kg, mainly distributed in tumor tissue and liver [3]
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| Toxicity/Toxicokinetics |
Acute toxicity: Intraperitoneal LD50 in mice = 120 mg/kg; Intraperitoneal LD50 in rats = 80 mg/kg [3]
- Subchronic toxicity (intraperitoneal injection in mice for 28 days): No significant hepatotoxicity or nephrotoxicity was observed at doses up to 15 mg/kg/day; mild transient neutropenia (≤15% reduction) occurred at 20 mg/kg/day [3] - No significant changes were observed in serum creatinine, blood urea nitrogen, alanine aminotransferase/aspartate aminotransferase or electrolyte levels at therapeutic doses [3][4] - Preclinical studies have shown that this product has no significant drug interaction with chemotherapy drugs (e.g., doxorubicin) [1][3] |
| References | |
| Additional Infomation |
4-[[4-(4-chlorophenyl)-2-thiazolyl]amino]phenol is a substituted aniline.
SKI II is a selective dual SK1/SK2 inhibitor developed as a research tool for studying sphingolipid signaling and anti-tumor therapy[1][2][4] - Its core mechanism includes inhibiting SK1/SK2-mediated sphingosine phosphorylation, reducing intracellular and extracellular S1P levels, and blocking S1P-dependent signaling pathways (PI3K/Akt, NF-κB) involved in cell proliferation, survival, and angiogenesis[1][4] - Research applications include inhibiting tumor growth and metastasis (breast cancer, liver cancer) and inhibiting pathological angiogenesis[1][3] - It can induce tumor cell apoptosis and inhibit endothelial cell function, making it a potential candidate drug for treating S1P-overexpressing tumors. [1][3] - This kinase exhibits high selectivity for SK1/SK2 compared to other kinases, minimizing off-target effects, and therefore can be used as a specific tool for elucidating sphingolipid-mediated biological processes. [2][4] |
| Molecular Formula |
C15H11CLN2OS
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| Molecular Weight |
302.78
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| Exact Mass |
302.028
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| Elemental Analysis |
C, 59.50; H, 3.66; Cl, 11.71; N, 9.25; O, 5.28; S, 10.59
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| CAS # |
312636-16-1
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| Related CAS # |
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| PubChem CID |
753704
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
507.1±60.0 °C at 760 mmHg
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| Flash Point |
260.5±32.9 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.709
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| LogP |
3.91
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
20
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| Complexity |
304
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| Defined Atom Stereocenter Count |
0
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| SMILES |
OC1=CC=C(NC2=NC(C3=CC=C(Cl)C=C3)=CS2)C=C1
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| InChi Key |
ZFGXZJKLOFCECI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C15H11ClN2OS/c16-11-3-1-10(2-4-11)14-9-20-15(18-14)17-12-5-7-13(19)8-6-12/h1-9,19H,(H,17,18)
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| Chemical Name |
4-[[4-(4-chlorophenyl)-1,3-thiazol-2-yl]amino]phenol
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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: This product is not stable in solution, please use freshly prepared working solution for optimal results. |
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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: ≥ 2.75 mg/mL (9.08 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 27.5 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: ≥ 2.75 mg/mL (9.08 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 27.5 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: 30% PEG400+0.5% Tween80+5% Propylene glycol : 20 mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3027 mL | 16.5136 mL | 33.0273 mL | |
| 5 mM | 0.6605 mL | 3.3027 mL | 6.6055 mL | |
| 10 mM | 0.3303 mL | 1.6514 mL | 3.3027 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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