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Purity: ≥98%
GSK2194069 is a novel, potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS with an IC50 of 7.7 ± 4.1 nM in an assay detecting released CoA. Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer.
| Targets |
β-ketoyl reductase (KR) of fatty acid synthase (FASN) (IC50 = 7.7 nM)
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| ln Vitro |
Fatty acid synthase (FAS) in cancer cell lines (KATO-III, MKN45, A549, SNU-1) is inhibited by GSK2194069 (100 nM; 24 hours) without lowering the amounts of FAS-producing proteins [1]. With a half-maximal effective concentration (EC50) value of 15.5 ± 9 nM (n = 78), GSK2194069 lowers phosphatidylcholine levels in A549 cells, which is linked to decreased palmitate production [1]. FASN expression levels were higher in LNCaP cells, while GSK2194069 (5 μM and 20 μM) demonstrated greater effectiveness in FASN-positive LNCaP cells compared to FASN-negative PC3 cells [2]. The proliferation of LNCaP-LN3 human prostate cancer cells is inhibited by GSK2194069 (50 μM; 24 hours) [3]. The metabolomic features of GSK2194069 (60.4 nM; 24 h) in LNCaP-LN3 cells include reduced L-acetylcarnitine, stearoylcarnitine, empty carnitine, and palmitoyl-L-carnitine [3].
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| ln Vivo |
Our aim was to synthesize and evaluate a series of small molecule triazolones based on GSK2194069, an FASN inhibitor with IC50 = 7.7 ± 4.1 nM, for PET imaging of FASN expression. These triazolones were labeled with carbon-11 in good yield and excellent radiochemical purity, and binding to FASN-positive LNCaP cells was significantly higher than FASN-negative PC3 cells. Despite these promising characteristics, however, these molecules exhibited poor in vivo pharmacokinetics and were predominantly retained in lymph nodes and the hepatobiliary system. Future studies will seek to identify structural modifications that improve tumor targeting while maintaining the excretion profile of these first-generation 11C-methyltriazolones [2].
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| Enzyme Assay |
The activity of the probes was determined against LNCaP cells, which highly express FASN, and PC3 prostate cancer cells, which express FASN at much lower levels and served as our negative controls. Each of the compounds exhibited higher binding to LNCaP cells than to PC3 cells after the harvested counts were normalized to protein content. Binding was characterized by a rapid initial rate of uptake, followed by a period of equilibrium. Peak cell binding was reached after 10–15 min and was two-fold higher in LNCaP cells. Efflux of radioactivity was negligible up to 50 min after addition of activity. Peak binding of [11C]5 and [11C]6 to LNCaP cells was comparable (4.23 ± 0.14 vs. 5.06 ± 0.18% added activity), while peak binding of [11C]4 was slightly lower (2.37 ± 0.21% added activity). Binding of [11C]5 to LNCaP cells was significantly higher than PC3 cells (p = 0.03) [2].
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: A549 Tested Concentrations: 0, 10, 100, 1000 nM Incubation Duration: 48 hrs (hours) or 120 hrs (hours) Experimental Results: No reduction in FAS protein levels. Western Blot Analysis [2] Cell Types: FASN positive LNCaP cells, FASN negative PC3 cells Tested Concentrations: 1 nM-0.1 mM Incubation Duration: 48 hrs (hours) Experimental Results: Dramatically inhibited the growth of tumor cells, and had a better effect on reducing LNCaP cells. |
| Animal Protocol |
MicroPET/CT Imaging [2]
Method A: Tumor-bearing mice were injected intravenously with 100 µL of the final product solution, containing 8–10 MBq [11C]4, 9–11 MBq [11C]5, or 9–11 MBq [11C]6. The mice were then placed on the imaging bed and a 60 min dynamic acquisition was performed by microPET/CT. The acquisition typically began 15 min after injection of the radiotracer. A CT scan was performed immediately upon conclusion of the microPET scan for attenuation correction and anatomical co-registration. The images were processed using open-source image processing software (AMIDE). Method B: A 500 µL aliquot containing approximately 111 MBq (38 ng) [11C]5 in 10% EtOH/saline was added to a vial containing 50 mg Captisol® in 0.5 mL saline, prepared 4 h previously. The mixture was shaken vigorously for 10 min. A control solution was prepared by diluting a 500 µL aliquot containing approximately 111 MBq (38 ng) [11C]5 in 10% EtOH/saline with 0.5 mL saline. Two male athymic nu/nu mice bearing LNCaP xenograft tumors (150–500 mm3) were injected intravenously with 100 µL of the solution, containing 9–11 MBq [11C]5 and 5 mg Captisol®. In parallel, two mice were injected intravenously with 100 µL of the control solution, containing 9–11 MBq. The mice were then placed on the imaging bed and a 60 min dynamic acquisition was performed by microPET/CT. The acquisition typically began 15 min after injection of [11C]5. A CT scan was performed immediately upon conclusion of the microPET scan for attenuation correction and anatomical co-registration. The images were processed using AMIDE. |
| References |
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| Molecular Formula |
C25H24N4O3
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|---|---|
| Molecular Weight |
428.4831
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| Exact Mass |
428.185
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| CAS # |
1332331-08-4
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| PubChem CID |
67376285
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| Appearance |
Light yellow solid
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| LogP |
3.712
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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 |
5
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| Heavy Atom Count |
32
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| Complexity |
773
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O=C(C1([H])C([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])C2=NN([H])C(N2C2C([H])=C([H])C(C3C([H])=C([H])C4=C(C([H])=C([H])O4)C=3[H])=C([H])C=2[H])=O)C1([H])[H]
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| InChi Key |
AQTPWCUIYUOEMG-INIZCTEOSA-N
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| InChi Code |
InChI=1S/C25H24N4O3/c30-24(18-1-2-18)28-11-9-16(15-28)13-23-26-27-25(31)29(23)21-6-3-17(4-7-21)19-5-8-22-20(14-19)10-12-32-22/h3-8,10,12,14,16,18H,1-2,9,11,13,15H2,(H,27,31)/t16-/m0/s1
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| Chemical Name |
4-[4-(5-Benzofuranyl)phenyl]-5-[[(3S)-1-(cyclopropylcarbonyl)-3-pyrrolidinyl]methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one
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| Synonyms |
GSK-2194069; GSK 2194069; GSK2194069.
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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 : ≥ 100 mg/mL (~233.38 mM)
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| 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. View More
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. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3338 mL | 11.6692 mL | 23.3383 mL | |
| 5 mM | 0.4668 mL | 2.3338 mL | 4.6677 mL | |
| 10 mM | 0.2334 mL | 1.1669 mL | 2.3338 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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