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.

Physicochemical Properties

Molecular Formula	C15H11CLN2OS
Molecular Weight	302.78
Exact Mass	302.028
Elemental Analysis	C, 59.50; H, 3.66; Cl, 11.71; N, 9.25; O, 5.28; S, 10.59
CAS #	312636-16-1
Related CAS #	312636-16-1
PubChem CID	753704
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	507.1±60.0 °C at 760 mmHg
Flash Point	260.5±32.9 °C
Vapour Pressure	0.0±1.4 mmHg at 25°C
Index of Refraction	1.709
LogP	3.91
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	20
Complexity	304
Defined Atom Stereocenter Count	0
SMILES	OC1=CC=C(NC2=NC(C3=CC=C(Cl)C=C3)=CS2)C=C1
InChi Key	ZFGXZJKLOFCECI-UHFFFAOYSA-N
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)
Chemical Name	4-[[4-(4-chlorophenyl)-1,3-thiazol-2-yl]amino]phenol
Synonyms	SphK-I2; SKI-II; SKI II; Sphingosine kinase inhibitor II; SKIII
HS Tariff Code	2934.99.9001
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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	SK1/2 ( IC50 = 78/45 μM )
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]
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]
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.
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.
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
References	[1]. Cancer Res . 2003 Sep 15;63(18):5962-9. [2]. J Pharmacol Exp Ther . 2006 Aug;318(2):596-603. [3]. Asian Pac J Cancer Prev . 2012;13(2):625-31. [4]. J Pharmacol Sci . 2010;114(3):304-10.
Additional Infomation	4-[[4-(4-chlorophenyl)-2-thiazolyl]amino]phenol is a substituted aniline.

Solubility Data

Solubility (In Vitro)

DMSO: ~61 mg/mL (~201.5 mM) Water: <1 mg/mL Ethanol: ~61 mg/mL (~201.5 mM)

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. Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% Propylene glycol : 20 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

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.