GLS1 Inhibitor-4 (compound 41e) is a potent GLS1 inhibitor (antagonist) with IC50 of 11.86 nM. GLS1 Inhibitor-4 displays antiproliferation activity, good metabolic stability, and strong GLS1 binding affinity. GLS1 Inhibitor-4 blocks glutamine metabolism and induces ROS production. GLS1 Inhibitor-4 can cause apoptosis and exhibit anti-tumor activity.

Physicochemical Properties

Molecular Formula	C29H27F3N10O2S2
Molecular Weight	668.72
CAS #	2768599-97-7
Appearance	Typically exists as solid at room temperature
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	IC50: 11.86 nM (GLS1)[1]
ln Vitro	Compound 41e, GLS1 Inhibitor-4, exhibits antiproliferative activity against HCT116 and MDA-MB-436, CT26, and H22 cells, with IC50s of 0.051, 0.37, 0.32, and 1.34 µM, respectively[1]. With 96% stability in human plasma, GLS1 Inhibitor-4 exhibits good liver microsomal stability and plasma stability[1]. Strong binding affinity between GLS1 protein and GLS1 Inhibitor-4 is demonstrated by a dissociation constant (Kd) of 52 nM[1]. In a dose-dependent manner, GLS1 Inhibitor-4 (0.1, 0.5, and 1 µM) prevents HCT116 cells from forming colonies[1]. Several important metabolites downstream of glutamate are found to have their concentrations lowered within 12 hours by GLS1 Inhibitor-4 (100, 300 nM, 12 h)[1]. In HCT116 cells, inhibitor-4 (30, 50, 200 nM; 6 h) raises ROS levels in a dose-dependent manner[1]. In HCT116 cancer cells, GLS1 Inhibitor-4 (1 mmol/L; 12 h) suppresses aerobic glycolysis and dramatically reduces basal and maximal OCRs (oxygen consumption rates) after 12 h[1]. In a dose-dependent manner, GLS1 Inhibitor-4 (30, 50, 200 nM; 24 h) induces apoptosis[1].
ln Vivo	In a dose-dependent manner, GLS1 Inhibitor-4 (50, 100 mg/kg; ip; twice daily for 21 consecutive days) exhibits antitumor activity[1].
Cell Assay	Apoptosis Analysis[1] Cell Types: HCT116 cells Tested Concentrations: 30, 50, 200 nM Incubation Duration: 24 h Experimental Results: Induced approximately 28% and 95% more apoptotic cells at concentrations of 50 and 200 nM, respectively. And upregulated the expression of apoptotic protein cleaved PARP in a dose-dependent manner.
Animal Protocol	Animal/Disease Models: Sixweeks old BALB/c SPF nude mice ( HCT116 tumor nude mouse xenograft model)[1] Doses: 50, 100 mg/kg Route of Administration: Ip; twice a day for 21 days Experimental Results: Inhibited the tumor growth at a dose-dependent manne with the tumor growth inhibition (TGI) values of 35.5% at 50 mg/kg and 47.5% at 100 mg/kg, respectively.
References	[1]. Discovery of novel glutaminase 1 allosteric inhibitor with 4-piperidinamine linker and aromatic heterocycles. Eur J Med Chem. 2022 Jun 5;236:114337.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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 (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *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). 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)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL 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). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.4954 mL	7.4770 mL	14.9539 mL
	5 mM	0.2991 mL	1.4954 mL	2.9908 mL
	10 mM	0.1495 mL	0.7477 mL	1.4954 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.