C16 (GW-506033X; C-16; PKR Inhibitor) is a brain penetrant protein kinase (PKR) inhibitor with anti-inflammatory effects. The double-stranded RNA-dependent protein kinase (PKR), an apoptotic inducer, regulates much pro-inflammatory cytokine production. It binds the ATP-binding site of PKR and blocks autophosphorylation (IC50 =186-210 nM). It can decrease Aβ42-induced inflammatory cytokine release and apoptosis in neuronal cultures, and prevents neuroinflammation and neuronal loss in an acute excitotoxic rat model.

Physicochemical Properties

Molecular Formula	C13H8N4OS
Molecular Weight	268.294
Exact Mass	268.041
CAS #	608512-97-6
PubChem CID	6490494
Appearance	Light yellow to yellow solid powder
Density	1.6±0.1 g/cm3
Boiling Point	674.6±55.0 °C at 760 mmHg
Flash Point	361.8±31.5 °C
Vapour Pressure	0.0±2.1 mmHg at 25°C
Index of Refraction	1.851
LogP	0.8
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	1
Heavy Atom Count	19
Complexity	427
Defined Atom Stereocenter Count	0
SMILES	C1=CC2=C(C\3=C1NC(=O)/C3=C\C4=CN=CN4)SC=N2
InChi Key	VFBGXTUGODTSPK-BAQGIRSFSA-N
InChi Code	InChI=1S/C13H8N4OS/c18-13-8(3-7-4-14-5-15-7)11-9(17-13)1-2-10-12(11)19-6-16-10/h1-6H,(H,14,15)(H,17,18)/b8-3-
Chemical Name	6,8-dihydro-8-(1H-imidazol-5-ylmethylene)-7H-pyrrolo[2,3-g]benzothiazol-7-one
Synonyms	GW 506033XC16 GW-506033X PKR InhibitorGW506033X
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	In primary neuronal cultures, PKR-IN-C16 (compound C16) releases the PKR-induced translation block [1]. In SH-SY5Y cells, PKR-IN-C16 (0.1 or 0.3 μM; 24 hours) prevents endoplasmic reticulum stress-induced neuronal cell death [1]. In addition to inhibiting PKR phosphorylation, PKR-IN-C16 (1-1000 nM; 4 hours) also stops amyloid beta-induced caspase-3 activation in SH-SY5Y cells [2].
ln Vivo	PKR-IN-C16 (compound C16) (60 or 600 μg/kg; i.p.; 3 times) prevents both the inflammatory response in the model and PKR-induced neuronal loss in rats. It also prevents quinolinic acid (QA)-induced acute excitotoxicity.
Cell Assay	Western Blot Analysis[2] Cell Types: Human SH-SY5Y Cell Tested Concentrations: 1, 10, 20, 200, 1000 nM Incubation Duration: 4 hrs (hours) Experimental Results: Significant levels of phosphorylated PKR in cells exposed to 20 μM beta-amyloid reduce.
Animal Protocol	Animal/Disease Models: normotensive male Wistar rats, excitotoxic neuroinflammation model, unilateral striatal injection of quinolinic acid (QA) to induce inflammation [1] Doses: 60 or 600 μg/kg Mode of Route of Administration: intraperitoneal (ip) injection; 24 hrs (hrs (hours)) before, 2 hrs (hrs (hours)) after QA injection and 24 hrs (hrs (hours)) after QA injection Experimental Results: diminished expression of PKR active catalytic domain, preventing contralateral IL-1β levels from increasing at 600 μg/kg (97% inhibition ). Neuronal loss induced by 600 μg/kg QA injection was diminished by 47%, and the number of positively cleaved caspase-3 neurons was diminished by 37%.
References	[1]. The specific PKR inhibitor C16 prevents apoptosis and IL-1β production in an acute excitotoxic rat model with a neuroinflammatory component. Neurochem Int. 2014 Jan;64:73-83. [2]. Activated double-stranded RNA-dependent protein kinase and neuronal death in models of Alzheimer's disease. Neuroscience. 2006;139(4):1343-54.

Solubility Data

Solubility (In Vitro)

DMSO : ~10 mg/mL (~37.27 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1 mg/mL (3.73 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 + to the above solution and mix evenly; then add 450 μL of 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.7273 mL	18.6366 mL	37.2731 mL
	5 mM	0.7455 mL	3.7273 mL	7.4546 mL
	10 mM	0.3727 mL	1.8637 mL	3.7273 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.