Dual sphingosine kinase 1 and 2 (Sphk1 and Sphk2) [1].
SKI-II HCl targets sphingosine kinase, the enzyme responsible for the conversion of sphingosine to sphingosine-1-phosphate (S1P), a bioactive lipid that promotes cell proliferation, survival, and migration. By acting as a substrate-competitive and reversible inhibitor, SKI-II prevents the production of S1P, thereby inhibiting SphK-mediated signaling pathways. It is a highly specific inhibitor of sphingosine kinase.

SKI-II reduced viability of HepG2 cells in a dose-dependent manner with IC50 of 8.02 μM after 72 h treatment, compared to 55.22 μM for 5-FU [1].
Combination of SKI-II (5 μM) with 5-FU (20 μM) synergistically inhibited cell proliferation (CI < 1) [1].
SKI-II (5 μM) augmented 5-FU-induced apoptosis: co-treatment increased early apoptotic cells by 23.81% and secondary necrotic cells by 29.44%, while viable cells decreased by 54.04% compared to untreated cells [1].
SKI-II combined with 5-FU markedly reduced HepG2 cell migration after 48 h compared to 5-FU alone or control (p < 0.05) [1].
SKI-II combined with 5-FU effectively inhibited clonogenic survival: significant reduction in colony formation after 6 h treatment compared to 5-FU alone [1].
Western blot showed that SKI-II plus 5-FU completely inhibited phosphorylation of IGF-1R β-subunit, reduced p-FAK, p-c-Raf, p-ERK1/2, p-NF-κB-p65, osteopontin, SIRT1, and p-p38 compared to 5-FU alone; no effect on p-Akt [1].
In vitro, SKI-II HCl is a selective non-lipid inhibitor of sphingosine kinase with an IC₅₀ of 0.5 μM. It inhibits the activity of sphingosine kinase in a dose-dependent manner. The compound inhibits acute myelogenous leukemia cell growth in vitro. Sphingosine kinases 1 and 2 play tumor-promoting roles in different cancer types including hepatocellular carcinoma (HCC) and thus represent promising pharmacological targets.

In vivo, SKI-II HCl has shown antitumor activity in preclinical models, including inhibition of acute myelogenous leukemia cell growth. As a selective sphingosine kinase inhibitor, it has the potential to treat various cancers that are driven by dysregulation of the sphingolipid signaling pathway. However, specific in vivo efficacy data in animal models are not extensively detailed in the available literature.

In vitro sphingosine kinase assays for SKI-II HCl involve measuring the conversion of sphingosine to S1P by purified sphingosine kinase enzyme. The enzyme is incubated with sphingosine and ATP in the presence of varying concentrations of SKI-II, and the production of S1P is measured using radiometric or mass spectrometry-based methods. IC₅₀ values are calculated from dose-response curves. The mechanism of inhibition (substrate-competitive, reversible) is confirmed by kinetic analysis.

Human hepatocellular carcinoma HepG2 cells were cultured in DMEM supplemented with 10% foetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO2 humidified atmosphere [1].
Cell viability assay (MTT): Cells were seeded into 96-well plates at 3×10^3 cells/well and treated for 72 h. Then MTT was added at final concentration 5 mg/mL for 3 h, formazan dissolved in DMSO, absorbance measured at 570 nm. Combination index calculated by CompuSyn Software [1].
Apoptosis detection by Annexin V: Cells were seeded in 6-well plates at 1×10^5 cells/well, treated for 48 h, then stained with Annexin V and PI, visualized by fluorescence microscope at 40x magnification. Percentages of early apoptotic (PI-/AnnV+), late apoptotic/primary necrotic (PI+/AnnV+), secondary necrotic (PI+), and viable (PI-/AnnV-) cells were calculated [1].
Wound healing migration assay: Cells were seeded in 6-well plates at 1×10^5 cells/well, a scratch made with a pipette tip, washed with PBS, incubated with test agents. Wound images taken at 0, 24, 48 h, wound area measured by Image J software [1].
Clonogenic assay: Cells were seeded in 6-well plates at 300 cells/well, treated for 2, 4, 6 h, then washed and cultured for 7 days under agent-free conditions. Colonies fixed with acetic acid/methanol (1:7), stained with 0.5% crystal violet, colonies with >50 cells counted [1].
Western blot: After treatment, 1.5×10^5 cells lysed with RIPA buffer with protease and phosphatase inhibitors. 50 μg proteins resolved by SDS-PAGE, transferred to PVDF membranes, blocked with BSA or non-fat milk, probed with primary antibodies against: phospho-FAK, SirT1, osteopontin, phospho-Erk1/2, phospho-p38; phospho-IGF1 receptor β, phospho-c-Raf, phospho-NF-κB; phospho-Akt and α-tubulin. Secondary antibodies used, signal visualized by chemiluminescence, densitometric analysis by Quantity One software [1].
In vitro cell-based assays for SKI-II HCl are performed using cancer cell lines, such as acute myelogenous leukemia cells. Cells are cultured in appropriate media and treated with SKI-II at various concentrations. Sphingosine kinase activity is measured by assessing S1P levels in cell lysates. Cell viability is measured by MTT or CellTiter-Glo assays to determine IC₅₀ values. Apoptosis is evaluated by caspase-3/7 activation and Annexin V/PI staining. The effects on cell proliferation and migration can also be assessed.

In vivo animal studies for SKI-II HCl would typically involve mouse xenograft models of cancer, such as acute myelogenous leukemia. Immunodeficient mice are injected with cancer cells to establish tumors. When tumors reach a certain size, SKI-II is administered intraperitoneally or orally at doses determined from pharmacokinetic studies. Tumor growth is measured over time to assess efficacy. Pharmacodynamic studies would also be performed to confirm sphingosine kinase inhibition and S1P reduction in tumor tissue.

Specific pharmacokinetic properties of SKI-II HCl, such as half-life and oral bioavailability, are not extensively detailed in the available literature. As a small molecule with a molecular weight of 339.24 g/mol, it is expected to be absorbed and distributed to tissues following administration. It is soluble in DMSO and is typically formulated for in vivo administration. The compound's pharmacokinetic profile in animal models requires further elucidation.

Comprehensive toxicological data for SKI-II HCl are not widely available in public literature. As a research compound, it is intended for laboratory use only and is not for human therapeutic use. Standard safety precautions should be followed when handling this compound. The compound is supplied with a purity of ≥98%. SKI-II HCl is not approved for clinical use.

Biochem Biophys Res Commun.2017Jun 10;487(4):782-788.

SKI-II enhances sensitivity to 5-fluorouracil in hepatocellular carcinoma cells via suppression of osteopontin and FAK/IGF-1R signalling, leading to inhibition of NF-κB activity and downstream SIRT1/p38 MAPK signalling. The combination treatment at sub-toxic concentrations (5 μM SKI-II and 20 μM 5-FU) synergistically inhibits cell proliferation, migration, clonogenic survival and induces apoptosis [1]. SKI-II is a dual Sphk1/Sphk2 inhibitor with potential as an adjunct to 5-FU treatment in HCC [1].
SKI-II HCl is a substrate-competitive, reversible, and highly specific inhibitor of sphingosine kinase. It has an IC₅₀ of 0.5 μM and inhibits acute myelogenous leukemia cell growth in vitro and in vivo. Sphingosine kinases 1 and 2 are promising pharmacological targets for cancer therapy. SKI-II HCl is a valuable research tool for studying the role of sphingosine kinase in cancer and other diseases. This product is for research use only.

C15H12CL2N2OS

339.239580154419

338.005

1177741-83-1

312636-16-1

16760659

Typically exists as solid at room temperature

5.787

3

4

3

21

304

0

ClC1C=CC(=CC=1)C1=CSC(=N1)NC1C=CC(=CC=1)O.Cl

ZDRVLAOYDGQLFI-UHFFFAOYSA-N

InChI=1S/C15H11ClN2OS.ClH/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);1H

2-(4-Hydroxyanilino)-4-(4-chlorophenyl)thiazole hydrochloride

SKI-II HCl; SKI II HCl; SKI-I; SKI-II Hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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