HI-TOPK-032 is a novel, potent and selective TOPK kinase inhibitor which strongly suppressed TOPK kinase activity in vitro but had little effect on extracellular signal–regulated kinase 1 (ERK1), c-jun—NH2—kinase 1, or p38 kinase activities. Moreover, HI-TOPK-032 increased colon cancer cell apoptosis by controlling the abundance of p53, cleaved caspase-7, and cleaved PARP, and inhibited both anchorage-dependent and -independent colon cancer cell growth by decreasing ERK-RSK phosphorylation. A colon cancer xenograft model's tumor growth was inhibited in vivo by the administration of HI-TOPK-032. The development of HI-TOPK-032 as a possible treatment for colorectal cancer is under consideration. Apoptosis, inflammation, tumor development, and cancer growth are all significantly impacted by the serine-threonine mitogen-activated protein kinase kinase family member T-LAK cell-originated protein kinase (TOPK/PBK) (Cancer Res; 72(12); 3060–8).
Physicochemical Properties
| Molecular Formula | C20H11N5OS |
| Molecular Weight | 369.399241685867 |
| Exact Mass | 369.068 |
| Elemental Analysis | C, 65.03; H, 3.00; N, 18.96; O, 4.33; S, 8.68 |
| CAS # | 487020-03-1 |
| Related CAS # | 487020-03-1 |
| PubChem CID | 1936439 |
| Appearance | Pink to red solid powder |
| Density | 1.5±0.1 g/cm3 |
| Boiling Point | 415.3±45.0 °C at 760 mmHg |
| Flash Point | 204.9±28.7 °C |
| Vapour Pressure | 0.0±1.0 mmHg at 25°C |
| Index of Refraction | 1.808 |
| LogP | 2.71 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 2 |
| Heavy Atom Count | 27 |
| Complexity | 635 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C(C1=CC=CS1)NC2=CC3=C(C#N)C4=NC5=CC=CC=C5N=C4N3C=C2 |
| InChi Key | BCSBXWKRZUPFHW-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C20H11N5OS/c21-11-13-16-10-12(22-20(26)17-6-3-9-27-17)7-8-25(16)19-18(13)23-14-4-1-2-5-15(14)24-19/h1-10H,(H,22,26) |
| Chemical Name | N-(12-cyanoindolizino[2,3-b]quinoxalin-2-yl)thiophene-2-carboxamide |
| Synonyms | HI-TOPK-032; HI-TOPK032; HI-TOPK 032; HI TOPK-032; HI TOPK032; HI TOPK 032 |
| 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 | TOPK; ERK-RSK; p53; Caspase-7 |
| ln Vitro | HI-TOPK-032 has a substantial inhibitory effect on TOPK dormancy activity, but has no effect on p38, c-jun-NH2-dormancy1, or extracellular signal-regulated dormancy 1 (ERK1). HI-TOPK-032 modifies TOPK's ATP binding. This chemical reacts correspondingly with LYS30 and generates hydrogen bonds with GLY83 and ASP151. However, 40% of MEK1 activity in chromatin was reduced by the maximum dose (5 μM) of HI-TOPK-032. By controlling p53, localized caspase-7, and localized PARP, HI-TOPK-032 also prevented ERK-RSK phosphorylation. Wall-dependent and non-navigational circular growth is inhibited by an increase in the number of circular navigators [1]. |
| ln Vivo | Mice treated with 1 or 10 mg/kg of HI-TOPK-032 showed a significant growth inhibition of more than 60% of HCT-116 tumor growth when compared to the tumor treatment group. The mice exhibited good tolerance to the treatment group of HI-TOPK-032. The phosphorylation of ERK and ERK's immediate downstream protein RSK was markedly suppressed in the HI-TOPK-032 treatment group, while p53 expression was considerably elevated [1]. |
| Enzyme Assay | Through an in vitro kinase assay with [γ-32P]ATP and ERK1 (active, 500 ng), inactive RSK2 (ERK1 substrate, 1 μg), JNK1 (active, 50 ng), c-Jun (JNK1 substrate, 1 μg), p38 (active, 200 ng), and ATF2 (p38 substrate, 500 ng), the impact of HI-TOPK-032 on ERK1, JNK1, and p38 activity is evaluated. In a nutshell, 40 μL of reaction buffer is used along with 10 μCi of [γ-32P]ATP and HI-TOPK-032 (0.5, 1, 2, 5 μM). SDS-PAGE is used to separate the mixture after 30 minutes of room temperature incubation, during which time 10 μL of protein loading buffer is added to stop the reaction[1]. |
| Cell Assay | HI-TOPK-032 is administered at varying concentrations to HCT-116 colon cancer cells (1, 2, 5 μM). Following a period of 1, 2, or 3 days of incubation, 20 μL of CellTiter96 AQueous One Solution is introduced, and the cells are subsequently incubated for 1 hour at 37°C in an incubator with 5% CO2. At 492 nm, absorbance is measured[1]. |
| Animal Protocol | Mice: The four groups of mice are as follows: (i) the untreated vehicle group; (ii) the 1 mg and 10 mg HI-TOPK-032/kg body weight; (iii) the 10 mg and 1 mg body weight group; and (iv) the no cells and 10 mg HI-TOPK-032/kg body weight group. To inoculate each mouse's right flank, a suspension of HCT-116 cells is placed in serum-free McCoy 5A medium s.c. For twenty-five days, the vehicle or HI-TOPK-032 is injected three times a week. It calculates tumor volume[1]. |
| References |
[1]. Novel TOPK inhibitor HI-TOPK-032 effectively suppresses colon cancer growth. Cancer Res. 2012 Jun 15;72(12):3060-8. |
Solubility Data
| Solubility (In Vitro) | DMSO: 4~5 mg/mL (10.8~13.5 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: 10 mg/mL (27.07 mM) in 15% Cremophor EL + 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 0.25 mg/mL (0.68 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 2.5 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7071 mL | 13.5355 mL | 27.0709 mL | |
| 5 mM | 0.5414 mL | 2.7071 mL | 5.4142 mL | |
| 10 mM | 0.2707 mL | 1.3535 mL | 2.7071 mL |