OTS964 HCl, the hydrochloride salt of OTS-964 and a dimethylated derivative of OTS 514, is a potent and selective inhibitor of TOPK (T-lymphokine-activated killer cell-originated protein kinase) with antitumor effects. With a high affinity and selectivity, it inhibits TOPK (IC50 = 28 nM). TOPK, a protein that is overexpressed in a number of cancers and tumors, is thought to function as an oncogene that encourages the growth of tumors. OTS964 may therefore have anticancer properties because it is a TOPK inhibitor. Both in vitro and in xenograft models of human lung cancer, it results in a cytokinesis defect and subsequent apoptosis of cancer cells. OTS964 administered as a liposomal formulation successfully caused total regression of transplanted tumors without causing any adverse reactions in mice, but it also caused hematopoietic adverse reactions (leukocytopenia associated with thrombocytosis).

Physicochemical Properties

Molecular Formula	C23H24N2O2S.HCL
Molecular Weight	428.98
Exact Mass	428.132
Elemental Analysis	C, 64.40; H, 5.87; Cl, 8.26; N, 6.53; O, 7.46; S, 7.47
CAS #	1338545-07-5
Related CAS #	OTS964;1338542-14-5
PubChem CID	89675898
Appearance	Off-white to light yellow solid powder
LogP	5.89
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	29
Complexity	563
Defined Atom Stereocenter Count	1
SMILES	Cl[H].Cl[H].S1C([H])=C([H])C2=C1C(N([H])C1C(C([H])([H])[H])=C([H])C(=C(C3C([H])=C([H])C(=C([H])C=3[H])[C@@]([H])(C([H])([H])[H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])C=12)O[H])=O
InChi Key	YHPWOYBWUWSJDW-UQKRIMTDSA-N
InChi Code	InChI=1S/C23H24N2O2S.ClH/c1-13-11-18(26)19(16-7-5-15(6-8-16)14(2)12-25(3)4)20-17-9-10-28-22(17)23(27)24-21(13)20;/h5-11,14,26H,12H2,1-4H3,(H,24,27);1H/t14-;/m0./s1
Chemical Name	9-[4-[(2R)-1-(dimethylamino)propan-2-yl]phenyl]-8-hydroxy-6-methyl-5H-thieno[2,3-c]quinolin-4-one;hydrochloride
Synonyms	OTS964 HCl; OTS-964 HCl; OTS 964 HCl
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: Please store this product in a sealed and protected environment, avoid exposure to moisture.
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	TOPK (IC50 = 28 nM); CDK11B (IC50 = 40 nM)
ln Vitro	OTS964 hydrochloride (10 nM; 48 hours) inhibits the growth of cancer cells[1]. OTS964 hydrochloride (10 nM; 48 hours) increases cancer cell death[1]. OTS964 (0.1-2 μM; 24 and 48 hours) exhibits a dose-dependent increase in LC3-II expression and a decrease in P62 expression[3].
ln Vivo	OTS964 hydrochloride causes tumors to continue to shrink even after treatment, eventually showing full regression[1]. OTS964 hydrochloride completely suppresses tumor growth in the end.
Cell Assay	At a specific density, 100 μl of cells are plated in 96-well plates. Prior to being exposed to compounds for 72 hours at 37°C, the cells are given an overnight period to adhere. At 450 nm in wavelength, plates are read using a spectrophotometer. Three duplicates of each assay are run. The z scores are computed to yield P values once the IC50 values have been measured. The log values of the IC50 for each of the 13 TOPK-positive cell lines are transformed to base ten (nM) and the mean and standard deviation are then computed.
Animal Protocol	Nude mice bearing LU-99 lung cancer cells[1] 40 mg/kg Intravenously; on days 1, 4, 8, 11, 15, and 18
References	[1]. TOPK inhibitor induces complete tumor regression in xenograft models of human cancer through inhibition ofcytokinesis. Sci Transl Med. 2014 Oct 22;6(259):259ra145. [2]. Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials. Sci Transl Med. 2019 Sep 11;11(509). [3]. TOPK inhibits autophagy by phosphorylating ULK1 and promotes glioma resistance to TMZ. Cell Death Dis. 2019 Aug 5;10(8):583.

Solubility Data

Solubility (In Vitro)

DMSO: >100 mg/mL Water: <1mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.83 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 (5.83 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 (5.83 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	2.3311 mL	11.6556 mL	23.3111 mL
	5 mM	0.4662 mL	2.3311 mL	4.6622 mL
	10 mM	0.2331 mL	1.1656 mL	2.3311 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.