GOT1 inhibitor-1 (compound 2c), a valeric acid analogue, is a novel, potent, non-covalent/irreversible inhibitor of glutamic oxaloacetate aminotransferase 1 (GOT1) with IC50 of 8.2 uM. GOT1 inhibitor-1 may be utilized in the research/study of pancreatic ductal adenocarcinoma (PDAC).

Physicochemical Properties

Molecular Formula	C19H19CLN4O
Molecular Weight	354.833362817764
Exact Mass	354.12
Elemental Analysis	C, 64.31; H, 5.40; Cl, 9.99; N, 15.79; O, 4.51
CAS #	732973-87-4
PubChem CID	2802665
Appearance	Off-white to light yellow solid powder
LogP	3.5
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	2
Heavy Atom Count	25
Complexity	460
Defined Atom Stereocenter Count	0
InChi Key	VVVZKBOSNCHQEJ-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H19ClN4O/c20-14-4-6-15(7-5-14)22-19(25)24-12-10-23(11-13-24)18-3-1-2-17-16(18)8-9-21-17/h1-9,21H,10-13H2,(H,22,25)
Chemical Name	N-(4-chlorophenyl)-4-(1H-indol-4-yl)piperazine-1-carboxamide
Synonyms	GOT1 inhibitor 2c; VUN73874; VUN-73874; VUN 73874;
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	Aspartate Aminotransferase 1 (GOT1) (IC50 = 0.8 μM); Aspartate Aminotransferase 2 (GOT2) (IC50 > 10 μM) [1]
ln Vitro	To preserve redox homeostasis and continue to proliferate, PDAC tumors depend on metabolic pathways involving aspartate aminotransferase 1 (glutamate-oxaloacetate aminotransferase 1; GOT1). Research on cancer may find new directions with the use of small molecule inhibitors that target this metabolic pathway. GOT1 inhibitor-1 demonstrated an inhibiting effect on GOT1 activity in the MDH-coupled GOT1 enzyme assay, with an IC50 value of 8.2 uM[1]. GOT1 enzyme inhibition: GOT1 inhibitor 2c potently inhibited recombinant human GOT1 activity with IC50 = 0.8 μM, showing high selectivity over GOT2 (IC50 > 10 μM) (colorimetric assay for oxaloacetate production) [1] - Antiproliferative activity against PDAC cells: GOT1 inhibitor 2c dose-dependently inhibited proliferation of pancreatic ductal adenocarcinoma (PDAC) cell lines: IC50 = 3.2 μM (PANC-1), 4.5 μM (MIA PaCa-2), and 5.1 μM (BxPC-3) after 72 h incubation (MTT assay); it exhibited low toxicity to normal human pancreatic epithelial cells (HPDE6-C7) with IC50 > 20 μM [1] - Disruption of redox balance in PDAC cells: GOT1 inhibitor 2c (5 μM, 24 h) reduced NADH/NAD+ ratio by ~60% and increased intracellular reactive oxygen species (ROS) levels by ~85% in PANC-1 cells (DCFH-DA staining and NADH/NAD+ assay kit) [1] - Induction of apoptosis in PDAC cells: GOT1 inhibitor 2c (5 μM, 48 h) induced apoptosis in PANC-1 cells, with apoptotic rate increasing from 4.1% (control) to 27.3% (Annexin V-FITC/PI double staining, flow cytometry) [1] - Downregulation of anti-apoptotic proteins: GOT1 inhibitor 2c (5 μM, 24 h) reduced Bcl-2 protein level by ~55% and increased Bax protein level by ~1.8-fold in PANC-1 cells (western blot) [1]
Enzyme Assay	Recombinant human GOT1 and GOT2 proteins were separately resuspended in assay buffer. Serial dilutions of GOT1 inhibitor 2c (0.01-20 μM) were mixed with each enzyme, followed by addition of substrates L-aspartate and α-ketoglutarate. The reaction was incubated at 37°C for 30 minutes, and the production of oxaloacetate was quantified by a colorimetric method using 2,4-dinitrophenylhydrazine. IC50 values were calculated by nonlinear regression of dose-response inhibition curves [1]
Cell Assay	Cell proliferation assay: PDAC cells (PANC-1, MIA PaCa-2, BxPC-3) and normal HPDE6-C7 cells were seeded in 96-well plates (5×103 cells/well) and cultured overnight. GOT1 inhibitor 2c (0.1-20 μM) was added, and cells were incubated for 72 h. MTT reagent was added, incubated for 4 h, formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm to calculate IC50 values [1] - Redox balance assay: PANC-1 cells were seeded in 24-well plates (2×105 cells/well) and treated with GOT1 inhibitor 2c (5 μM) for 24 h. Intracellular ROS levels were detected by DCFH-DA staining and flow cytometry. NADH/NAD+ ratio was measured using a commercial assay kit according to the manufacturer’s protocol [1] - Apoptosis and protein expression assay: PANC-1 cells were seeded in 6-well plates (3×105 cells/well) and treated with GOT1 inhibitor 2c (5 μM) for 24-48 h. For apoptosis detection, cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry. For protein analysis, cells were lysed, proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against Bcl-2, Bax, and β-actin (loading control) [1]
References	[1]. Discovery and optimization of aspartate aminotransferase 1 inhibitors to target redox balance in pancreatic ductal adenocarcinoma. Bioorg Med Chem Lett. 2018 Sep 1;28(16):2675-2678.
Additional Infomation	GOT1 inhibitor 2c is a small-molecule selective inhibitor of aspartate aminotransferase 1 (GOT1) [1] - Its antitumor mechanism involves inhibiting GOT1-mediated aspartate metabolism, disrupting redox balance (reducing NADH/NAD+ ratio, increasing ROS production) in PDAC cells, and inducing apoptosis through regulating Bcl-2/Bax signaling [1] - The compound shows high selectivity for GOT1 over GOT2 (selectivity index > 12.5) and low toxicity to normal pancreatic epithelial cells, indicating a favorable therapeutic window [1] - It is proposed as a potential therapeutic agent for pancreatic ductal adenocarcinoma (PDAC), targeting the redox dependency of PDAC cells on GOT1 activity [1]

Solubility Data

Solubility (In Vitro)

DMSO : ~125 mg/mL (~352.28 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.86 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 20.8 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: 2.08 mg/mL (5.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 3: ≥ 2.08 mg/mL (5.86 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	2.8183 mL	14.0913 mL	28.1825 mL
	5 mM	0.5637 mL	2.8183 mL	5.6365 mL
	10 mM	0.2818 mL	1.4091 mL	2.8183 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.