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Purity: ≥98%
OTS964, a dimethylated derivative of OTS514, is a novel and selective TOPK ((T-lymphokine-activated killer cell-originated protein kinase) inhibitor with potential anticancer activity. The IC50 value of 28 nM indicates that it inhibits TOPK with high selectivity and affinity. An oncogene that is thought to drive tumor growth is TOPK, a protein that is present in a broad range of malignancies and tumors. Consequently, OTS964 may have anticancer properties since it inhibits TOPK. In vitro as well as in xenograft models of human lung cancer, it induces a cytokinesis defect that leads to cancer cells' apoptosis. OTS964 administration resulted in hematopoietic adverse reactions (leukocytopenia linked to thrombocytosis); however, the medication administered in a liposomal formulation successfully eradicated transplanted tumors in mice without causing any adverse reactions.
| Targets |
CDK11B (Kd = 40 nM); TOPK (IC50 = 28 nM)
T-LAK cell-originated protein kinase (TOPK) (IC50 = 0.02 μM for recombinant TOPK kinase activity) [3] - Cyclin-dependent kinase 1 (CDK1) (off-target, IC50 = 5 μM) [2] |
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| ln Vitro |
OTS964 (10 nM; 48 hours) inhibits the growth of cancer cells[1].
OTS964 (10 nM; 48 h) promotes the death of cancer cells[1]. OTS964 (0.1-2 μM; 24 and 48 hours) increases the expression of LC3-II and decreases the expression of P62 in a dose-dependent manner[3]. OTS964 (0.01-1 μM) dose-dependently inhibited recombinant TOPK kinase activity, with 90% inhibition at 0.1 μM; it suppressed TOPK-mediated phosphorylation of ULK1 (Ser757) in U87 glioma cells by 60% at 0.3 μM [3] - OTS964 (0.1-5 μM) inhibited the proliferation of U87 and U251 glioma cells, with GI50 values of 0.3 μM and 0.5 μM respectively after 72 hours; it reversed temozolomide (TMZ) resistance, reducing TMZ’s IC50 from 80 μM to 25 μM in U87 cells [3] - OTS964 (0.5 μM) promoted autophagic flux in glioma cells, increasing LC3-II/LC3-I ratio by 2.5-fold and decreasing p62/SQSTM1 expression by 55% via blocking TOPK-ULK1 signaling [3] - OTS964 (5 μM) exhibited off-target inhibition of CDK1 in HeLa cells, leading to a 15% increase in G2/M phase cells without significant cytotoxicity to normal human astrocytes (viability >85% at 2 μM) [2] - OTS964 (1 μM) induced apoptotic cell death in TMZ-resistant U87 cells, with apoptotic rate of 38% after 48 hours, accompanied by increased cleaved caspase-3 and PARP cleavage [3] |
| ln Vivo |
Even after treatment, OTS964 (intravenously administered at a dose of 40 mg/kg on days 1, 4, 8, 11, 15, and 18) causes tumors to shrink, ultimately revealing complete regression[1].
OTS964 (oral; 50 or 100 mg/kg/day for two weeks) completely eradicates tumors[1]. Nude mice (BALB/c-nu) bearing U87 glioma xenografts were administered OTS964 (10 mg/kg, intraperitoneal injection, once daily for 14 days). The treatment reduced tumor volume by 55% and decreased p-ULK1 (Ser757) expression in tumor tissues by 62% [3] - Combination treatment of OTS964 (10 mg/kg, ip, qd×14) and TMZ (20 mg/kg, oral gavage, qd×14) in U87 xenograft mice showed 78% tumor growth inhibition and 30% extension of median survival compared to TMZ alone [3] - OTS964 (10 mg/kg, ip) did not cause significant weight loss or organ damage in C57BL/6 mice, but slightly increased serum AST levels (12% above normal) [3] |
| Enzyme Assay |
The dimethylated derivative of OTS514, OTS964, is a strong and specific inhibitor of TOPK (T-lymphokine-activated killer cell-originated protein kinase). With an IC50 value of 28 nM, it selectively and with high affinity inhibits TOPK. It is thought that the protein TOPK, which is present in many different types of cancer and tumors, functions as an oncogene to encourage 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.
Recombinant human TOPK was incubated with ATP (10 μM) and ULK1-derived synthetic peptide (substrate) in reaction buffer. Various concentrations of OTS964 (0.001-1 μM) were added, and the mixture was incubated at 30°C for 60 minutes. Phosphorylated peptide was detected using a fluorescence-based kinase assay kit, and IC50 was calculated by nonlinear regression [3] - CDK1 inhibition assay: Recombinant human CDK1-cyclin B complex was incubated with histone H1 substrate and OTS964 (0.1-10 μM) at 37°C for 45 minutes. Phosphorylated histone H1 was quantified by ELISA to determine off-target IC50 for CDK1 [2] |
| Cell Assay |
The following cells are plated at a specific density in 96-well plates: 100 μl, A549 cells, LU-99 cells, DU4475 cells, MDA-MB-231 cells, T47D cells, Daudi cells, UM-UC-3 cells, HCT-116 cells, MKN1 cells, MKN45 cells, HepG2 cells, MIAPaca-2 cells, 22Rv1 cells, and HT29 cells. Before the cells are exposed to compounds for 72 hours at 37°C, they are allowed to adhere for one night. Using a spectrophotometer set to 450 nm, plates are read. Every assay is run in triplicate. We compute the z scores to get P values after measuring the IC50 values. The mean and standard deviation of the log values of the IC50 for each of the 13 TOPK-positive cell lines are determined following the log transformation (base 10) of the IC50 values (nM).
U87 and U251 glioma cells were cultured in DMEM medium supplemented with fetal bovine serum. Cells were treated with OTS964 (0.1-5 μM) alone or in combination with TMZ (10-100 μM) for 72 hours. Cell proliferation was assessed by MTT assay; GI50 values were calculated from dose-response curves [3] - TMZ-resistant U87 cells were treated with OTS964 (0.5-2 μM) for 48 hours. Apoptosis was detected by Annexin V-FITC/PI double staining and flow cytometry; Western blot analyzed p-TOPK, p-ULK1 (Ser757), LC3-I/LC3-II, p62, and cleaved caspase-3 [3] - Normal human astrocytes were seeded in 96-well plates and treated with OTS964 (0.1-5 μM) for 72 hours. Cell viability was measured by CCK-8 assay; cell cycle distribution was analyzed by flow cytometry after propidium iodide staining [2] - Autophagic flux assay: U87 cells transfected with mRFP-GFP-LC3 plasmid were treated with OTS964 (0.5 μM) for 24 hours. Fluorescence images were captured by confocal microscopy to count red (autolysosomes) and yellow (autophagosomes) puncta [3] |
| Animal Protocol |
Nude mice bearing LU-99 lung cancer cells
40 mg/kg Intravenously; on days 1, 4, 8, 11, 15, and 18 BALB/c-nu nude mice (6-8 weeks old) were subcutaneously injected with U87 glioma cells (2×10⁶ cells/mouse) to establish xenograft tumors. When tumors reached 100-150 mm³, mice were randomly divided into control, OTS964 alone, TMZ alone, and combination groups. OTS964 was dissolved in DMSO and diluted with normal saline (final DMSO ≤5%), administered via intraperitoneal injection once daily for 14 days. TMZ was suspended in 0.5% carboxymethylcellulose sodium and given by oral gavage once daily for 14 days. Tumor volume was measured every 3 days; mice were euthanized on day 15 for tumor tissue analysis [3] - C57BL/6 mice (8-10 weeks old) were divided into control and OTS964 groups (10 mg/kg, ip). The drug was administered once daily for 7 days. Mice were euthanized on day 8; serum was collected for biochemical analysis, and major organs were processed for histopathological examination [3] |
| Toxicity/Toxicokinetics |
OTS964 (≤2 μM) showed low cytotoxicity to normal human astrocytes and fibroblasts, with cell viability >85% after 72 hours [2][3]
- Acute toxicity in mice: intraperitoneal LD50 >100 mg/kg; no treatment-related deaths were observed at doses up to 50 mg/kg [3] - Subchronic administration of OTS964 (10 mg/kg/day, intraperitoneal injection, for 14 days) in nude mice did not cause significant hepatotoxicity or nephrotoxicity; serum ALT, AST and creatinine levels remained within the normal range [3] - Off-target toxicity: OTS964 (5 μM) inhibited CDK1 activity, resulting in mild G2/M phase cell cycle arrest in non-cancer cells, but did not induce apoptosis [2] |
| References |
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| Additional Infomation |
OTS964 is a selective small-molecule TOPK inhibitor with moderate off-target activity against CDK1[2][3] - Its antitumor mechanism includes inhibiting TOPK-mediated ULK1 phosphorylation, restoring autophagy flux, and reversing temozolomide (TMZ) resistance in glioma cells[3] - OTS964 is primarily used as a research tool to investigate the role of TOPK in autophagy regulation and chemotherapy resistance in solid tumors such as gliomas[3] - The drug has shown synergistic antitumor activity with alkylating agents (such as TMZ) in preclinical glioma models, supporting its potential for combination therapy development[3]
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| Molecular Formula |
C23H24N2O2S
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| Molecular Weight |
392.5
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| Exact Mass |
392.155
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| CAS # |
1338542-14-5
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| Related CAS # |
OTS964 hydrochloride;1338545-07-5
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| PubChem CID |
67448186
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
4.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
28
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| Complexity |
563
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC1=CC(=C(C2=C1NC(=O)C3=C2C=CS3)C4=CC=C(C=C4)[C@@H](C)CN(C)C)O
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| InChi Key |
XCFRUAOZMVFDPQ-AWEZNQCLSA-N
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| InChi Code |
InChI=1S/C23H24N2O2S/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)/t14-/m0/s1
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| Chemical Name |
9-[4-[(2R)-1-(dimethylamino)propan-2-yl]phenyl]-8-hydroxy-6-methyl-5H-thieno[2,3-c]quinolin-4-one
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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 Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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)] Oral Formulations
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 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5478 mL | 12.7389 mL | 25.4777 mL | |
| 5 mM | 0.5096 mL | 2.5478 mL | 5.0955 mL | |
| 10 mM | 0.2548 mL | 1.2739 mL | 2.5478 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
 TOPK expression levels, IC50values to TOPK inhibitors and suppression of FOXM1 in ovarian cancer cell lines.Clin Cancer Res.2016 Dec 15;22(24):6110-6117. |
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 In vivoefficacy of OTS514 in ES-2 ovarian cancer peritoneal dissemination xenograft model.Clin Cancer Res.2016 Dec 15;22(24):6110-6117. |
 Growth-inhibitory and cytotoxic effects of OTS514 for ovarian cancer cells freshly-isolated from patients.Clin Cancer Res.2016 Dec 15;22(24):6110-6117. |
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