PF-06447475 (PF06447475) is a novel, highly potent, selective and brain penetrable inhibitor of Leucine rich repeat kinase 2 (LRRK2) with anti-PD activity. It inhibits LRRK2 with IC50 of 3 nM. LRRK2 has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). PF-06447475 inhibits LRRK2 enzyme and LRRK2 in the whole cell assay with IC50s of 3 and 25 nM, respectively. Cells incubated with PF-06447475 alone (0.5, 1, 3 μM) or in the presence of ROT significantly reduces (S935)-LRRK2 kinase phosphorylation to control. PF-06447475 has the potential for the treatment of PD.

Physicochemical Properties

Molecular Formula	C17H15N5O
Molecular Weight	305.33
Exact Mass	305.127
CAS #	1527473-33-1
Related CAS #	1527473-33-1
PubChem CID	72706840
Appearance	Off-white to pink solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.711
LogP	2
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	2
Heavy Atom Count	23
Complexity	457
Defined Atom Stereocenter Count	0
InChi Key	BHTWDJBVZQBRKP-UHFFFAOYSA-N
InChi Code	InChI=1S/C17H15N5O/c18-9-12-2-1-3-13(8-12)14-10-19-16-15(14)17(21-11-20-16)22-4-6-23-7-5-22/h1-3,8,10-11H,4-7H2,(H,19,20,21)
Chemical Name	3-(4-morpholin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)benzonitrile
Synonyms	PF6447475;PF-06447475;PF-6447475; PF06447475;PF 6447475; PF 06447475
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	With IC50s of 3 and 25 nM, respectively, PF-06447475 inhibits LRRK2 enzyme and LRRK2 in the whole cell assay[1]. Cells treated with PF-06447475 alone (0.5, 1, 3 μM) or with ROT significantly lower the phosphorylation of (S935)-LRRK2 kinase compared to control. Compared to untreated and control, PF-06447475 significantly preserves the nucleus morphology and ΔΨm of NLCs exposed to ROT. PF-475 considerably reduces ROS production induced by ROT to a level comparable to cells exposed to PF-475 alone[2].
ln Vivo	Microgliosis in G2019S+ rats treated with PF-06447475 was significantly reduced to levels observed in wild-type rats. In confocal sections, the proinflammatory marker MHC-II, which is expressed on myeloid cells but not neurons, also seems to be less prevalent in G2019S+ rats given PF-06447475. In G2019S+ rats, treatment with PF-06447475 dramatically reduces the amount of CD68 cells recruited to the SNpc. PF-06447475 effectively inhibits the increased neuroinflammation linked to the expression of G2019S-LRRK2. TH expression in the dorsal striatum, which is in line with medication that lessens SNpc neurodegeneration[3]. Rats are able to tolerate PF-06447475 well[1].
Animal Protocol	3 and 30 mg/kg PF-06447475 (p.o. b.i.d.) G2019S+ rats
References	[1]. Discovery and preclinical profiling of 3-[4-(morpholin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]benzonitrile (PF-06447475), a highly potent, selective, brain penetrant, and in vivo active LRRK2 kinase inhibitor. J Med Chem. 2015 Jan 8;58(1):419-32. [2]. Neuroprotective Effect of the LRRK2 Kinase Inhibitor PF-06447475 in Human Nerve-Like Differentiated Cells Exposed to Oxidative Stress Stimuli: Implications for Parkinson's Disease. Neurochem Res. 2016 Oct;41(10):2675-2692. [3]. Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration. J Biol Chem. 2015 Aug 7;290(32):19433-44.

Solubility Data

Solubility (In Vitro)

DMSO:≥ 33 mg/mL Water:<1 mg/mL Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3 mg/mL (9.83 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 30.0 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: ≥ 3 mg/mL (9.83 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 30.0 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	3.2751 mL	16.3757 mL	32.7514 mL
	5 mM	0.6550 mL	3.2751 mL	6.5503 mL
	10 mM	0.3275 mL	1.6376 mL	3.2751 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.