Gepotidacin (formerly GSK2140944) is a novel, potent, first-in-class, triazaacenaphthylene antibacterial that inhibits bacterial DNA gyrase and topoisomerase IV via a unique mechanism and has demonstrated in vitro activity against Neisseria gonorrhoeae, including drug-resistant strains, and also targets pathogens associated with other conventional and biothreat infections. Broth microdilution was used to evaluate the MIC and minimum bactericidal concentration (MBC) activity of gepotidacin and comparators against 25 N. gonorrhoeae strains (including five ciprofloxacin-nonsusceptible strains). Gepotidacin activity was also evaluated against three N. gonorrhoeae strains (including a ciprofloxacin-nonsusceptible strain) for resistance development, against three N. gonorrhoeae strains (including two tetracycline- and azithromycin-nonsusceptible strains) using time-kill kinetics and checkerboard methods, and against two N. gonorrhoeae strains for the investigation of postantibiotic (PAE) and subinhibitory (PAE-SME) effects. The MIC50 and MIC90 for gepotidacin against the 25 N. gonorrhoeae isolates tested were 0.12 and 0.25 μg/ml, respectively. The MBC50 and MBC90 for gepotidacin were 0.25 and 0.5 μg/ml, respectively. Gepotidacin was bactericidal, and single-step resistance selection studies did not recover any mutants, indicating a low rate of spontaneous single-step resistance. For combinations of gepotidacin and comparators tested using checkerboard methods, there were no instances where antagonism occurred and only one instance of synergy (with moxifloxacin; fractional inhibitory concentration, 0.375). This was not confirmed by in vitro time-kill studies. The PAE for gepotidacin against the wild-type strain ranged from 0.5 to >2.5 h, and the PAE-SME was >2.5 h. These in vitro data indicate that further study of gepotidacin is warranted for potential use in treating infections caused by N. gonorrhoeae.

Physicochemical Properties

Molecular Formula	C24H28N6O3
Molecular Weight	448.5175
Exact Mass	448.222
Elemental Analysis	C, 64.27; H, 6.29; N, 18.74; O, 10.70
CAS #	1075236-89-3
Related CAS #	Gepotidacin (S enantiomer);2319789-82-5
PubChem CID	25101874
Appearance	White to yellow solid powder
LogP	1.415
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	5
Heavy Atom Count	33
Complexity	893
Defined Atom Stereocenter Count	1
SMILES	O=C1C([H])=NC2C([H])=C([H])C(N3C=2N1[C@@]([H])(C3([H])[H])C([H])([H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N([H])C([H])([H])C1C([H])=C2C(=C([H])N=1)OC([H])([H])C([H])([H])C2([H])[H])=O
InChi Key	PZFAZQUREQIODZ-LJQANCHMSA-N
InChi Code	InChI=1S/C24H28N6O3/c31-22-4-3-20-24-29(22)15-19(30(24)23(32)13-27-20)14-28-7-5-17(6-8-28)25-11-18-10-16-2-1-9-33-21(16)12-26-18/h3-4,10,12-13,17,19,25H,1-2,5-9,11,14-15H2/t19-/m1/s1
Chemical Name	(2R)-2-[(4-{[(3,4-dihydro-2H-pyrano[2,3-c]pyridin- 6-yl)methyl]amino}piperidin-1-yl)methyl]-1,2-dihydro- 3H,8H-2a,5,8a-triazaacenaphthylene-3,8-dione
Synonyms	GSK-2140944; GSK2140944; GSK 2140944; GSK-2140944E; GSK2140944E; GSK 2140944E; Gepotidacin
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

ln Vitro	Gepotidacin is a novel, first-in-class triazaacenaphthylene antibacterial that targets pathogens linked to other common and biothreat infections. It also inhibits bacterial DNA gyrase and topoisomerase IV through a unique mechanism. It has demonstrated in vitro activity against both gram-positive and gram-negative bacteria, including drug-resistant strains. Against the 25 N. gonorrhoeae isolates tested, the MIC50 and MIC90 values for gepotidacin are 0.12 and 0.25 μg/mL, respectively[1]. The following bacteria have different gepotidacin MIC90s (in μg/mL): Escherichia coli, 2; Moraxella catarrhalis, ≤0.06; Haemophilus influenzae, 1; Clostridium perfringens, 0.5; and Shigella spp., 1[2]. Acute bacterial skin and skin structure infections (ABSSSIs) can be caused by pathogens that gepotidacin is reactive against in vitro[3].
ln Vivo	GSK2140944's minimum inhibitory concentrations (MICs) for the six MRSA isolates range from 0.125 to 0.5 mg/L. The range of ELF penetration ratios is 1.1 to 1.4. In neutropenic mice, maximal decreases of 1.1 to 3.1 log10 CFU have been observed. For stasis and 1-log-unit decreases, the mean fAUC/MIC ratios needed are 59.3 ± 34.6 and 148.4 ± 83.3, respectively.
Animal Protocol	Mice: GSK2140944 s.c. in single doses of 6.25, 50, or 200 mg/kg is given to groups of 48 infected mice at 3 h postinoculation (0 h) for neutropenic pharmacokinetic studies. Using a cardiac puncture, blood samples are taken from groups of six mice at five minutes and 0.25, 0.5, 1, 1.5, 2, 3, and 4 hours after the dose for doses of 6.25 or 50 mg/kg, and at five minutes and 0.25, 0.5, 1, 1.5, 2, 4, and 6 hours after the dose for 200 mg/kg[4].
References	[1]. In Vitro Activity of Gepotidacin (GSK2140944) against Neisseria gonorrhoeae. Antimicrob Agents Chemother. 2017 Feb 23;61(3). [2]. In Vitro Activity of Gepotidacin, a Novel Triazaacenaphthylene Bacterial Topoisomerase Inhibitor, against a Broad Spectrum of Bacterial Pathogens. Antimicrob Agents Chemother. 2016 Jan 4;60(3):1918-23. [3]. Efficacy, Safety, and Tolerability of Gepotidacin (GSK2140944) in the Treatment of Patients with Suspected or Confirmed Gram-Positive Acute Bacterial Skin and Skin Structure Infections. Antimicrob Agents Chemother. 2017 May 24;61(6). [4]. Pharmacodynamic Profile of GSK2140944 against Methicillin-Resistant Staphylococcus aureus in a Murine Lung Infection Model. Antimicrob Agents Chemother. 2015 Aug;59(8):4956-61.
Additional Infomation	Gepotidacin has been used in trials studying the treatment of Gonorrhea, Infections, Bacterial, and Infections, Respiratory Tract. Drug Indication Treatment of uncomplicated urinary tract infections Treatment of uncomplicated urogenital gonorrhea

Solubility Data

Solubility (In Vitro)

DMSO : ~7.14 mg/mL (~15.92 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 0.71 mg/mL (1.58 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 7.1 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: ≥ 0.71 mg/mL (1.58 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 7.1 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: ≥ 0.71 mg/mL (1.58 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 7.1 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: ≥ 0.71 mg/mL (1.58 mM)  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2296 mL	11.1478 mL	22.2955 mL
	5 mM	0.4459 mL	2.2296 mL	4.4591 mL
	10 mM	0.2230 mL	1.1148 mL	2.2296 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.