GNE-495 is a novel, potent and selective MAP4K4(mitogen-activated protein kinase kinase kinase kinase 4) inhibitor with anIC50of 3.7 nM. In order to study the function of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4 in short) in various disease contexts, potent inhibitors had to be found. For illuminating the potential for therapeutic intervention, compounds that can be used to conduct such studies in vivo would be especially important. An advanced compound 13 (GNE-495) that showed excellent potency and good PK was created through a structure-based design effort combined with property-guided optimization aimed at reducing the ability of the inhibitors to cross into the CNS. It was then used to demonstrate in vivo efficacy in a retinal angiogenesis model, recapitulating effects that were seen in the inducible Map4k4 knockout mice.

Physicochemical Properties

Molecular Formula	C22H20FN5O2
Molecular Weight	405.4249
Exact Mass	405.16
Elemental Analysis	C, 65.18; H, 4.97; F, 4.69; N, 17.27; O, 7.89
CAS #	1449277-10-4
Related CAS #	1449277-10-4
PubChem CID	89730041
Appearance	Yellow solid powder
Density	1.5±0.1 g/cm3
Boiling Point	734.5±60.0 °C at 760 mmHg
Flash Point	398.0±32.9 °C
Vapour Pressure	0.0±2.4 mmHg at 25°C
Index of Refraction	1.712
LogP	2.54
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	4
Heavy Atom Count	30
Complexity	668
Defined Atom Stereocenter Count	0
SMILES	FC1=C([H])C([H])=C([H])C(=C1[H])C1C([H])=C([H])C2=C(C(N([H])[H])=NC([H])=C2C(N([H])C2([H])C([H])([H])N(C2([H])[H])C(C2([H])C([H])([H])C2([H])[H])=O)=O)N=1
InChi Key	FYXCIBJXJYBWPX-UHFFFAOYSA-N
InChi Code	InChI=1S/C22H20FN5O2/c23-14-3-1-2-13(8-14)18-7-6-16-17(9-25-20(24)19(16)27-18)21(29)26-15-10-28(11-15)22(30)12-4-5-12/h1-3,6-9,12,15H,4-5,10-11H2,(H2,24,25)(H,26,29)
Chemical Name	8-amino-N-[1-(cyclopropanecarbonyl)azetidin-3-yl]-2-(3-fluorophenyl)-1,7-naphthyridine-5-carboxamide
Synonyms	GNE 495; GNE495; GNE-495
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	MAP4K4 (IC50 = 3.7 nM)
ln Vitro	GNE-495 is a strong MAP4K4 inhibitor that works well to promote angiogenesis. GNE-495 exhibits minimum body stability, permeability, efficient ideal cellular equilibrium, and MAP4K4 adsorption [1].
ln Vivo	High doses of GNE-495—25 and 50 mg/kg—were given intraperitoneally to newborn mouse pups. In every species examined, GNE-495 demonstrates favorable internal characteristics, including low clearance, moderate terminal half-phase decay, and appropriate leverage levels (F=37–47%) [1].
Animal Protocol	Rats, Mice and Pups For the brain cassette study, three male Sprague-Dawley (SD) rats receive an intravenous (IV) bolus dose of six test substances (for example, GNE-495; 0.5 mg/kg). GNE-495 is injected intravenously in bolus doses of 1 mg/kg in female CD-1 mice for the mouse PK study. GNE-495 (5 mg/kg) is additionally injected intravenously (PO) into female CD-1 mice. The rat brain cassette PK is dosed at 2 mL/kg, and all other doses are administered at 5 mL/kg. Water and food are available to animals at all times, and they are not fasted before receiving a dose. Three blood samples (~60 μL) are taken from each mouse up to either 9 or 24 hours after the compound of interest is given. This is done using a serial sampling method, and it involves taking three blood samples from each mouse. After being mixed with K2EDTA and placed on ice or in a cold Kryorack immediately after collection, the blood is then centrifuged to separate the plasma. Blood samples are centrifuged for 10–15 minutes at 4°C while being spun at a speed of 1000-2000× g within an hour of being drawn. This process yields the plasma. The plasma samples are stored at -70 to -80°C until analysis. For neonate PK, 3-day-old CD1 pups are injected with 25 mg/kg and 50 mg/kg GNE-495. Blood samples are taken intraperitoneally at the times specified, retinas are taken one hour after the dose, snap frozen in liquid nitrogen, and kept at -80°C until analysis. The concentrations of plasma and retinal lysate are assessed using LC/MS/MS.
References	[1]. Structure-Based Design of GNE-495, a Potent and Selective MAP4K4 Inhibitor with Efficacy in Retinal Angiogenesis. ACS Med Chem Lett. 2015 Jun 29;6(8):913-8.

Solubility Data

Solubility (In Vitro)

DMSO: ~2.2 mg/mL (~5.4 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 0.22 mg/mL (0.54 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 2.2 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: ≥ 0.22 mg/mL (0.54 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 2.2 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: ≥ 0.22 mg/mL (0.54 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 2.2 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	2.4666 mL	12.3329 mL	24.6658 mL
	5 mM	0.4933 mL	2.4666 mL	4.9332 mL
	10 mM	0.2467 mL	1.2333 mL	2.4666 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.