K-858 is a novel, potent and ATP-uncompetitive inhibitor of mitotic kinesin Eg5 that has potential antitumor activity. K858 is a strong replication inhibitor that, regardless of the tumor phenotype, causes apoptosis in breast tumor cells. Since survivin is overexpressed at the same time as K858, this anti-proliferative response of tumor cells to K858 may be restricted. As a result, tumor cells may become more susceptible to K858-induced apoptosis if survivin levels are reduced by using AKT inhibitors. K858 caused cells to enter mitotic arrest and form monopolar spindles by blocking centrosome separation and activating the spindle checkpoint. K858 triggers growth inhibition and mitotic arrest by activating the spindle checkpoint mediated by Mad2. K858 causes cancer cells to undergo mitosis, but not healthy cells. K858 inhibits cell growth, causes apoptosis, and induces mitotic arrest; microtubule polymerization is unaffected.

Physicochemical Properties

Molecular Formula	C13H15N3O2S
Molecular Weight	277.34
Exact Mass	277.088
Elemental Analysis	C, 56.30; H, 5.45; N, 15.15; O, 11.54; S, 11.56
CAS #	72926-24-0
Related CAS #	72926-24-0
PubChem CID	2930014
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.627
LogP	1.05
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	2
Heavy Atom Count	19
Complexity	418
Defined Atom Stereocenter Count	0
SMILES	CC(NC1=NN(C(C)=O)C(C2=CC=CC=C2)(C)S1)=O
InChi Key	JEFVYQYZCAVNTP-UHFFFAOYSA-N
InChi Code	InChI=1S/C13H15N3O2S/c1-9(17)14-12-15-16(10(2)18)13(3,19-12)11-7-5-4-6-8-11/h4-8H,1-3H3,(H,14,15,17)
Chemical Name	N-(4-acetyl-5-methyl-5-phenyl-1,3,4-thiadiazol-2-yl)acetamide
Synonyms	K-858; K 858; K858
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 = 1.3 μM)
ln Vitro	K858 Racemic has an IC50 of 1.3 μM and inhibits Eg5 in an ATP-uncompetitive manner. Even at 200 μM, neither the conventional kinesin heavy chain nor the mitotic kinesins CENP-E and MKLP1 have their ATPase activity inhibited by K858. By activating the spindle checkpoint mediated by Mad2, K858 causes mitotic arrest and growth inhibition. In cancer cells, K858 (5 μM) induces mitotic cell death, but not in healthy cells[1]. The MCF7, BT474, and SKBR3 cell lines are inhibited by K858 (1, 10, and 100 μM), and the MDA-MB231 cell line is only suppressed at 10 and 100 μM after a 24-hour treatment. K858 decreases survivin and the Bax/Bcl2 RNA ratio in the four cell lines. Furthermore, in MCF7 cells, wortmannin (phosphoinositide 3-kinase AKT) completely reverses the up-regulation of survivin[2].
ln Vivo	K858 (50, 150 mg/kg, p.o.) inhibits tumor growth in an HCT116 colon cancer xenograft model by administering 100 mg/kg orally twice a day for five days, and it also exhibits antitumor activity in an A2780 ovarian cancer xenograft model. In mice, neurotoxic side effects are not observed with K858 (100 mg/kg, p.o., qd ×5)[1].
Cell Assay	The sulforhodamine B colorimetric assay is used to assess cytotoxicity. 1.5 × 104 cells are seeded onto 96-well plates, allowed to grow for 24 hours (h), and then exposed to varying K858 concentrations (1 μM, 10 μM, and 100 μM) for 24 and 48 hours. Following a one-hour fixation in 50% trichloroacetic acid at 4°C, the cells are stained with 0.4% sulforhodamine B in 1% acetic acid for thirty minutes at room temperature (RT). Washing four times with 1% acetic acid gets rid of extra dye. Using a microplate reader, protein-bound dye is dissolved in 10 mM TRIS pH 10 and optical density (OD) is measured at 510 nm[2].
Animal Protocol	BALB/cAJcl-nu mice receive a single-cell inoculation of 5 × 106 A2780 cells. K858 is given orally at 150 and 50 mg/kg twice a day on days 0 through 4 and 7 through 11. The tolerability studies that are conducted beforehand dictate the doses and schedules. Oral administration of the vehicle (0.5% methylcellulose 400) is done twice a day on days 0 through 4 and 7 through 11. On day 0, 25 mg/kg of paclitaxel is injected intravenously. On day 0, 60 mg/kg of carboplatin is injected intravenously. The ratio of the mean experimental V/V0 value to the control group's value is known as the treated versus control (T/C) ratio. In this ratio, V represents the tumor volume on the evaluation day and V0 represents the tumor volume on the day of the drug's first administration. The nonparametric rank-sum test is used in statistical analysis[1].
References	[1]. K858, a novel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells. Cancer Res. 2009 May 1;69(9):3901-9. [2]. The kinesin Eg5 inhibitor K858 induces apoptosis but also survivin-related chemoresistance in breast cancer cells. Invest New Drugs. 2016 Aug;34(4):399-406.
Additional Infomation	N-(4-acetyl-5-methyl-5-phenyl-1,3,4-thiadiazol-2-yl)acetamide is a member of benzenes.

Solubility Data

Solubility (In Vitro)

DMSO: ~10 mM in DMSO Water: <1 mg/mL Ethanol:

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.01 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 (9.01 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	3.6057 mL	18.0284 mL	36.0568 mL
	5 mM	0.7211 mL	3.6057 mL	7.2114 mL
	10 mM	0.3606 mL	1.8028 mL	3.6057 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.