GSK656 (GSK-3036656 and GSK-070) is a novel and potent antitubercular agent which acts as an inhibitor of Mycobacterium tuberculosis (Mtb) leucyl-tRNA synthetase (LeuRS), with an IC50 of 0.2 μM. There is an urgent need to develop new and safer antitubercular agents that possess a novel mode of action. GSK3036656 (abbreviated as GSK656) shows potent inhibition of Mtb LeuRS (IC50 = 0.20 μM) and in vitro antitubercular activity (Mtb H37Rv MIC = 0.08 μM). Additionally, it is highly selective for the Mtb LeuRS enzyme with IC50 of >300 μM and 132 μM for human mitochondrial LeuRS and human cytoplasmic LeuRS, respectively. In addition, it exhibits remarkable PK profiles and efficacy against Mtb in mouse TB infection models with superior tolerability over initial leads. This compound has been progressed to clinical development for the treatment of tuberculosis.

Physicochemical Properties

Molecular Formula	C10H14BCL2NO4
Molecular Weight	293.939461231232
Exact Mass	293.039
Elemental Analysis	C, 40.86; H, 4.80; B, 3.68; Cl, 24.12; N, 4.77; O, 21.77
CAS #	2131798-13-3
Related CAS #	Ganfeborole;2131798-12-2
PubChem CID	134691736
Appearance	Solid powder
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	4
Heavy Atom Count	18
Complexity	261
Defined Atom Stereocenter Count	1
SMILES	B1(C2=C(C=CC(=C2[C@H](O1)CN)Cl)OCCO)O.Cl
InChi Key	FUOKBESTQMGROA-DDWIOCJRSA-N
InChi Code	InChI=1S/C10H13BClNO4.ClH/c12-6-1-2-7(16-4-3-14)10-9(6)8(5-13)17-11(10)15;/h1-2,8,14-15H,3-5,13H2;1H/t8-;/m1./s1
Chemical Name	(S)‑3-(Aminomethyl)-4-chloro-7-(2-hydroxyethoxy)benzo[c][1,2]oxaborol-1(3H)‑ol hydrochloride
Synonyms	GSK3036656; GSK-3036656; GSK 3036656; GSK070; GSK-070; GSK 070; GSK656; GSK-656; GSK 656; GSK656 HCl. GSK656 hydrochloride
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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
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	Mtb LeuRS(IC50= 0.2 μM)
ln Vitro	The highly selective inhibitor ganfeborole hydrochloride has an IC50 of 0.2 μM for Mycobacterium tuberculosis (Mtb) leucyl-tRNA synthetase (LeuRS), and it exhibits IC50s of >300 μM and 132 μM for human mitochondrial and cytoplasmic LeuRS, respectively. The antitubercular activity of ganfeborole hydrochloride against Mtb H37Rv is demonstrated by a minimal inhibitory concentration (MIC) of 80 nM. Additionally, ganfeborole hydrochloride shows EC50 values of 381 μM and 137 μM, respectively, against HepG2 cells and HepG2 protein synthesis[1].
ln Vivo	Mice infected with M. tuberculosis H37Rv exhibit strong antitubercular activity against ganfeborole hydrochloride, with an ED99 of 0.4 mg/kg[1].
Cell Assay	The day before subculturing the plates, HepG2 (HB-8065) cells are cultured in fresh medium (essential minimum Eagle medium, EMEM, supplemented with 5% fetal calf serum and 2 mM l-glutamine). On the day of the test, cells (10 000 cells/well) are seeded in a clear-bottomed, black, 96-well collagen-coated microplate, with the exception of column 11, where 100 mL of culture medium is dispensed. A 100% DMSO stock solution for GSK656 is made. To reach a final concentration of 0.5% of DMSO, ten consecutive 1:2 dilutions of GSK656 are prepared. A 1:200 dilution is then made in medium. Phosphate buffer saline is used to dissolve resazurin tablets at a 0.0042% concentration. The cells are incubated for 24 hours at 37°C, 5% CO2, and 95% relative humidity. Two replicates of the cells are then added, each with 150 μL of culture medium that contains the appropriate test concentrations of GSK656 dilutions. For the blank control, only 150 μL of 0.5% DMSO are added. GSK656 is then exposed to the cells for 48 hours. The medium is then taken out, and each well is filled with resazurin solution before being incubated for an additional 1.5 hours. In a Microplate reader 1420 Multilabel HTS counter, Victor 2, fluorescence is measured at an excitation wavelength of 515 nm and an emission wavelength of 590 nm[1].
Animal Protocol	Mice Eight to ten-week-old female C57BL/6 mice free of specific pathogens are given a week to adjust. In summary, mice receive an intratracheal infection of M. tuberculosis H37Rv at a rate of 100,000 CFU/mouse. GSK656 is given for eight days in a row, beginning the day after infection. In order to conduct the chronic assay, mice are intraperitoneally infected with 100 CFU/mouse, and GSK656 is given seven days a week for eight weeks, beginning six weeks after the initial infection. 24 hours after the last dose, the lungs are harvested, and each lung lobe is aseptically removed, homogenized, and frozen. After plating homogenates onto 10% OADC-Middlebrook 7H11 medium, they are kept at 37°C for 21 days. GSK656 is given intravenously as a single dose of 5 mg/kg in saline or orally as a single dose of 30 mg/kg in 1% methylcellulose (1% MC).Regarding the IV route, 15 μL of blood is extracted via puncture from the lateral tail vein in each of the three mice (n = 3) at 5, 15, and 30 minutes as well as 1, 2, 4, 8, and 24 hours after the dose; concerning the oral route, 15 μL of blood is extracted from the lateral tail vein in each of the three mice (n = 3) at 15, 30, and 45 minutes as well as 1, 2, 4, 8, and 24 hours after the dose[1].
References	J Med Chem.2017 Oct 12;60(19):8011-8026.

Solubility Data

Solubility (In Vitro)

DMSO : ~67.5 mg/mL (~229.64 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.51 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 (8.51 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (8.51 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (8.51 mM)  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.4021 mL	17.0103 mL	34.0205 mL
	5 mM	0.6804 mL	3.4021 mL	6.8041 mL
	10 mM	0.3402 mL	1.7010 mL	3.4021 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.