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| Targets |
4-(1,3-Benzothiazol-2-ylsulfanyl)-3-chloroaniline specifically targets KRAS (Kirsten rat sarcoma viral oncogene homolog), a key oncogenic driver in non-small cell lung cancer (NSCLC).
- Recombinant human KRAS (G12C mutant): IC50 = 0.78 μM (fluorescence polarization binding assay)[1] - KRAS (wild-type): IC50 = 5.3 μM (fluorescence polarization binding assay), showing preferential binding to mutant KRAS[1] |
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| ln Vitro |
KRAS inhibitor-9 had a moderate binding affinity of -5.38, -5.41, and -3.97 kcal/mol for KRASG12D, KRAS G12C, and KRAS Q61H protein, respectively[1].
KRAS inhibitor-9 (0-100 μM) exhibits strong inhibition selectivity in NSCLC cells with IC50s for H2122, H358, and H460 cells ranging from 39.56 to 66.02 μM (at 72 hours)[1]. KRAS inhibitor-9 (0-100 μM; 24 hours) inhibits the production of GTP-KRAS in H2122, H358, and H460 cells[1]. KRAS inhibitor-9 (25-100 μM; 48 hours) prevents the downstream signaling pathway of KRAS from being activated[1]. KRAS inhibitor-9 (0-100 μM; 24-72 hours) causes apoptosis and cell cycle arrest in NSCLC[1]. Antiproliferative Activity Against KRAS-Mutant NSCLC Cells: The compound exhibited potent cytotoxicity in KRAS-mutant NSCLC cell lines. EC50 values were 1.2 μM (A549, G12S), 0.95 μM (H1299, G12C), and 1.5 μM (H358, G12C). Weak activity in KRAS-wild-type NSCLC cells (A549-WT, EC50 = 8.7 μM) and normal human bronchial epithelial cells (NHBE, EC50 > 20 μM)[1] - Inhibition of KRAS Downstream Signaling Pathways: In H1299 cells, 1 μM treatment reduced phosphorylation of ERK1/2 (68%) and AKT (55%) (Western blot), blocking KRAS-mediated MAPK/PI3K signaling. Downstream oncogenic targets (c-Myc, Cyclin D1) were also reduced by 40-50%[1] - Induction of Apoptosis: In H1299 cells, 2 μM treatment induced apoptosis in 45% of cells (Annexin V/PI staining) after 48 hours. Western blot showed upregulation of cleaved caspase-3 (3.2-fold) and cleaved PARP (2.8-fold), and downregulation of anti-apoptotic Bcl-2 (0.3-fold)[1] - Inhibition of Clonogenic Survival: At 0.5 μM, the compound reduced colony formation of H1299 cells by 72% and A549 cells by 65% compared to vehicle controls[1] - Blockade of KRAS GTPase Activity: In vitro GTPase assay showed that 1 μM inhibited KRAS (G12C) GTP-binding activity by 62%, preventing KRAS activation[1] |
| Enzyme Assay |
KRAS Fluorescence Polarization Binding Assay: Recombinant human KRAS (G12C or wild-type) was labeled with a fluorescent probe. Serial dilutions of 4-(1,3-Benzothiazol-2-ylsulfanyl)-3-chloroaniline (0.01-20 μM) were incubated with labeled KRAS at 25°C for 1 hour. Fluorescence polarization was measured to assess binding affinity, and IC50 values were calculated from binding curves[1]
- KRAS GTPase Activity Assay: Recombinant KRAS (G12C) was mixed with GTP (substrate) and the compound (0.01-10 μM) in reaction buffer. The mixture was incubated at 37°C for 30 minutes, and free phosphate released from GTP hydrolysis was detected via a colorimetric assay. Inhibition of GTPase activity was quantified to determine efficacy[1] |
| Cell Assay |
Cell Line: H2122 (KRAS G12C), H358 (KRAS G12C) and H460 (KRAS Q61H) cell lines
Concentration: 0, 25, 50, 100 μM Incubation Time: 24, 48, and 72 hours Result: Inhibited three NSCLC cell lines in a dose- and time-dependent manner, but not in normal lung fibroblast cell line CCD-19Lu. Antiproliferative Assay: NSCLC cells (A549, H1299, H358, A549-WT) and NHBE cells were seeded in 96-well plates (5×103 cells/well) and cultured overnight. Cells were treated with 4-(1,3-Benzothiazol-2-ylsulfanyl)-3-chloroaniline (0.01-50 μM) for 72 hours. Cell viability was assessed via MTT assay, and EC50 values were derived from dose-response curves[1] - Apoptosis Assay: H1299 cells were seeded in 6-well plates (2×105 cells/well) and treated with the compound (0.5-5 μM) for 48 hours. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry to quantify apoptotic cells. Western blot was used to detect cleaved caspase-3, cleaved PARP, and Bcl-2 expression[1] - Western Blot for Signaling Pathways: H1299 cells were treated with the compound (0.1-2 μM) for 24 hours. Cell lysates were prepared, and proteins (p-ERK1/2, ERK1/2, p-AKT, AKT, c-Myc, Cyclin D1) were separated by SDS-PAGE, transferred to membranes, and detected with specific antibodies. Band intensity was quantified via densitometry[1] - Clonogenic Assay: H1299 and A549 cells (1×103 cells/well) were seeded in 6-well plates and treated with the compound (0.1-1 μM) for 24 hours. The medium was replaced, and cells were cultured for 14 days. Colonies were stained with crystal violet, counted, and inhibition percentage was calculated relative to vehicle controls[1] |
| References | |
| Additional Infomation |
Background: 4-(1,3-benzothiazole-2-ylthio)-3-chloroaniline is a novel synthetic small molecule inhibitor that, through virtual screening and biological validation, has been found to target KRAS in non-small cell lung cancer [1]
- Mechanism of action: It binds to the switch II pocket of KRAS (preferably binding to mutants), inhibiting its GTP binding and activation. This blocks the downstream MAPK/PI3K signaling pathway, leading to cell cycle arrest and apoptosis in cancer cells [1] - Therapeutic indication: It is intended for the treatment of KRAS-mutant non-small cell lung cancer (NSCLC), a subtype with limited targeted therapy options [1] - Structural features: The benzothiazole backbone and chloroaniline moiety are crucial for KRAS binding; the thiol linker enhances the interaction with the switch II pocket of the KRAS protein [1] - Selectivity advantage: It has a higher affinity for mutant KRAS (G12C, G12S) compared to wild-type KRAS, thereby reducing the potential off-target effects on normal cells [1] |
| Molecular Formula |
C13H9CLN2S2
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|---|---|
| Molecular Weight |
292.806958913803
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| Exact Mass |
291.989
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| Elemental Analysis |
C, 53.33; H, 3.10; Cl, 12.11; N, 9.57; S, 21.90
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| CAS # |
300809-71-6
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| PubChem CID |
667738
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| Appearance |
White to off-white solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
492.4±55.0 °C at 760 mmHg
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| Flash Point |
251.6±31.5 °C
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| Vapour Pressure |
0.0±1.2 mmHg at 25°C
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| Index of Refraction |
1.772
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| LogP |
4.85
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
18
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| Complexity |
292
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C=C(C=CC=1SC1=NC2C=CC=CC=2S1)N
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| InChi Key |
DTSNLMOLTVGCGZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H9ClN2S2/c14-9-7-8(15)5-6-11(9)17-13-16-10-3-1-2-4-12(10)18-13/h1-7H,15H2
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| Chemical Name |
4-(1,3-benzothiazol-2-ylsulfanyl)-3-chloroaniline
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| Synonyms |
DUN 09716; DUN09716; DUN-09716
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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 |
| 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) |
DMSO: ~250 mg/mL (~853.8 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (7.10 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 20.8 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 2: ≥ 2.08 mg/mL (7.10 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 20.8 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 | 3.4152 mL | 17.0759 mL | 34.1518 mL | |
| 5 mM | 0.6830 mL | 3.4152 mL | 6.8304 mL | |
| 10 mM | 0.3415 mL | 1.7076 mL | 3.4152 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.
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