Litronesib (formerly also known as LY2523355; LY-2523355; KF-8961) is a novel, potent and selective inhibitor of the kinesin-related motor protein Eg5 with potential anticancer activity. Litronesib specifically inhibits the activity of Eg5, which in actively dividing tumor cells may cause disruption of mitosis, apoptosis, and ultimately cell death. Additionally referred to as KIF11 or kinesin spindle protein-5, the ATP-dependent Eg5 kinesin-related motor protein is a plus-end directed kinesin motor protein that is crucial for the regulation of spindle dynamics, including assembly and maintenance, during mitosis. Look for open or closed clinical trials that are utilizing this agent. (NCI Lexicon).

Physicochemical Properties

Molecular Formula	C23H37N5O4S2
Molecular Weight	511.7010
Exact Mass	511.229
Elemental Analysis	C, 53.99; H, 7.29; N, 13.69; O, 12.51; S, 12.53
CAS #	910634-41-2
Related CAS #	Litronesib Racemate;546111-97-1
PubChem CID	25167017
Appearance	White to off-white solid powder
Density	1.23 g/cm3
LogP	4.499
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	11
Heavy Atom Count	34
Complexity	865
Defined Atom Stereocenter Count	1
SMILES	C([C@]1(SC(NC(=O)C(C)(C)C)=NN1C(=O)C(C)(C)C)C1C=CC=CC=1)NS(=O)(=O)CCNCC
InChi Key	YVAFBXLHPINSIK-QHCPKHFHSA-N
InChi Code	InChI=1S/C23H37N5O4S2/c1-8-24-14-15-34(31,32)25-16-23(17-12-10-9-11-13-17)28(19(30)22(5,6)7)27-20(33-23)26-18(29)21(2,3)4/h9-13,24-25H,8,14-16H2,1-7H3,(H,26,27,29)/t23-/m0/s1
Chemical Name	N-[(5R)-4-(2,2-dimethylpropanoyl)-5-[[2-(ethylamino)ethylsulfonylamino]methyl]-5-phenyl-1,3,4-thiadiazol-2-yl]-2,2-dimethylpropanamide
Synonyms	LY2523355; KF 89617; LY-2523355; KF89617; LY 2523355; KF-89617; Eg5 KinesinRelated Motor Protein Inhibitor
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
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	Eg5 (IC50 = 14 μM)
ln Vitro	Litronesib (LY-2523355; KF-89617) is a selective Eg5 inhibitor. Litronesib (25 nM) induces cancer cells to death during mitotic arrest, and this needs sustained activation of spindle-assembly checkpoint (SAC)[1].
ln Vivo	Litronesib (LY2523355; KF89617; 1.1, 3.3, 10, and 30 mg/kg, i.v.) shows antitumor activity in a dose-dependently, and causes a dramatic increase in cancer cells immuno-positive for histone H3 phosphorylation in Colo205 xenograft tumors[1].
Cell Assay	In 96-well plates coated with poly-d-lysine, cancer cells are plated and incubated for an entire night. Following that, cells are given different lengths of time to respond to the indicated concentrations of lidocaine. After that, the cells are permeabilized for 10 minutes with cold methanol, fixed for 45 minutes with 3.7% formaldehyde in PBS or 30 minutes with 1× Prefer fixative solution at room temperature, and then fixed for another 10 minutes with 0.2% Triton X-100 in PBS. PBS is used to wash the cells three times. Following an hour of incubation with 100 μg/mL DNase-free RNase and 10 μg/mL propidium iodide, the cells are scanned to determine the mitotic index (MI), which is calculated as the percentage of cells with condensed DNA. Cells are cultivated with anti-phospho-histone H3 antibody or anti-phospho-histone H2AX at 1:1,000 dilution with 5% bovine serum albumin (BSA) in PBS for an overnight period at 4°C in MI based on histone H3 phosphorylation or apoptosis analysis inspection. Alexa 488 secondary antibody (1:1,000 in PBS-2% BSA) is incubated on cells for 60 minutes at room temperature after they have been washed three times with 0.2% Triton-X 100 in PBS. The cells are staining for 15 minutes in PBS containing 10 μg/mL propidium iodide and 100 μg/mL RNaseA after being rinsed three times with purified water. In Acumen Explorer eX3 microplate cytometer, the stained cells are scanned. Phospho-histone H3-Ser10 or phospho-histone H2AX positivity as a percentage of cells is used to express the results[1].
Animal Protocol	In nude mice, primary human tumor xenograft models are created and kept alive. Tumor volume is measured serially using caliper measurements to determine the antitumor efficacy in subcutaneous xenograft tumor-bearing mice (10 mice per treatment group) derived from either established cancer cell lines or fragments of human tumor explants. Female BDF1 mice weighing 20–23 g are used to create the p388 syngeneic tumor model for high-content imaging analysis. The mice are house-acclimated for a week prior to use in experiments. The STR assay is used to validate the p388 murine lymphocytic leukemia cells, which are then cultured in RPMI1640 medium with 10% FBS. Three rounds of serum-free medium washing precede the intraperitoneal injection of 1.25 million cells into mice for inoculation. Litronesib is administered to mice on the fifth post-implantation day, either as an intravenous bolus or as an intravenous infusion, at the prescribed doses and times. Following the death of the mice, the ascitic (intraperitoneal) fluid containing the p388 tumor cells is extracted and subjected to acumen, flow cytometry, and TUNEL assays for apoptosis, G2-M, and phospho-histone H3. Blood samples are obtained via cardiac puncture for pharmacokinetic analysis. EDTA is used to create plasma, and the amount of lidotrexeb in the plasma is measured[1].
References	[1]. A Novel Eg5 Inhibitor (LY2523355) Causes Mitotic Arrest and Apoptosis in Cancer Cells and Shows Potent Antitumor Activity in Xenograft Tumor Models. Mol Cancer Ther. 2015 Nov;14(11):2463-72.
Additional Infomation	Litronesib has been used in trials studying the treatment of Solid Tumors, Ovarian Cancer, Gastric Cancer, Prostate Cancer, and Acute Leukaemia, among others. Litronesib is an inhibitor of the kinesin-related motor protein Eg5 with potential antineoplastic activity. Litronesib selectively inhibits the activity of Eg5, which may result in mitotic disruption, apoptosis and consequently cell death in tumor cells that are actively dividing. The ATP-dependent Eg5 kinesin-related motor protein (also known as KIF11 or kinesin spindle protein-5) is a plus-end directed kinesin motor protein that plays an essential role during mitosis, particularly in the regulation of spindle dynamics, including assembly and maintenance.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 50 mg/mL (~97.7 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.89 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 (4.89 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (4.89 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 25.0 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	1.9543 mL	9.7714 mL	19.5427 mL
	5 mM	0.3909 mL	1.9543 mL	3.9085 mL
	10 mM	0.1954 mL	0.9771 mL	1.9543 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.