PF-06409577 (PF06409577) is a potent, selective, orally bioavailable, allosteric activator of AMPK α1β1γ1 (5′ adenosine monophosphate-activated protein kinase) with the potential to be used for diabetic nephropathy. With an EC50 value of 7 nM in the TR-FRET assay, it stimulates AMPK α1β1γ1 . The major human cytochrome P450 isoforms' microsomal activities were not inhibited by it (IC50 > 100 M), and it did not exhibit any discernible inhibition of hERG in a patch-clamp assay (100 M). Treatment for diabetic nephropathy may be possible with PF-06409577. An early model of diabetic nephropathy using PF-06409577 demonstrated effectiveness.

Physicochemical Properties

Molecular Formula	C19H16CLNO3
Molecular Weight	341.79
Exact Mass	341.082
Elemental Analysis	C, 66.77; H, 4.72; Cl, 10.37; N, 4.10; O, 14.04
CAS #	1467057-23-3
Related CAS #	1467057-23-3
PubChem CID	71748255
Appearance	White to off-white solid powder
LogP	4.558
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	3
Heavy Atom Count	24
Complexity	487
Defined Atom Stereocenter Count	0
SMILES	ClC1=CC2=C(C(C(=O)O)=CN2)C=C1C1C=CC(=CC=1)C1(CCC1)O
InChi Key	FHQXLWCFSUSXBF-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H16ClNO3/c20-16-9-17-14(15(10-21-17)18(22)23)8-13(16)11-2-4-12(5-3-11)19(24)6-1-7-19/h2-5,8-10,21,24H,1,6-7H2,(H,22,23)
Chemical Name	6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic acid
Synonyms	PF-06409577; PF-6409577; PF 06409577; PF6409577; PF06409577; PF 6409577
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	AMPK α2β1γ1 (EC50 = 6.8 nM); AMPK α1β1γ1 (EC50 = 7 nM)
ln Vitro	PF-06409577 activates AMPK isoforms α1β1γ1 and α2β1γ1 with EC50 values of 7.0 nM and 6.8 nM, respectively, but was significantly less active against α1β2γ1/α2β2γ1/α2β2γ3 isoforms α1β1γ1 and α2β1γ1 with EC50 values greater than 4000 nM[1].100 µM) of the microsomal activities of major human cytochrome P450 isoforms.
ln Vivo	PF-06409577 exhibits efficacy in a preclinical model of diabetic nephropathy[1]. It exhibits high plasma protein binding in rat (plasma unbound fraction, fu,p = 0.0044), dog (fu,p = 0.028), monkey (fu,p = 0.032), and human (fu,p = 0.017). Following iv administration, PF-06409577 demonstrates moderate plasma clearance (CLp) in rats (22.6 mL/min/kg), dogs (12.9 mL/min/kg), and monkeys (8.57 mL/min/kg) and was well distributed with steady state distribution volumes (Vdss) ranging from 0.846-3.15 L/kg. When given orally to rats, dogs, and monkeys, crystalline PF-06409577 in 0.5% methylcellulose suspension is quickly absorbed (Tmax = 0.25-1.20 h). Rats, dogs, and monkeys had oral bioavailability (F) values that were 15%, 100%, and 59%, respectively. Compared to other preclinical species and humans, PF-06409577 is subject to a greater degree of first pass intestinal glucuronidation in rats[2].
Enzyme Assay	PF-06409577 is prepared in DMSO. PF-06409577 is incubated with fully phosphorylated AMPK in assay buffer at room temperature for 15 min followed by addition of PP2a and another incubation for 60 min at room temperature. Okadaic acid (50 nM final), 50 nM Cy-5 SAMS peptide, and ATP equal to Km for each isoform are added before the phosphatase treatment is stopped and the kinase assay is started. The kinase reaction is quenched by adding 10 mM EDTA and 2 nM Eu-pACC antibody to the detection buffer after the reactions have been incubated for an additional 60 min. Excitation at 320 nM and emission measurements at 665 and 615 nM, respectively, are used to measure kinase activity.
Cell Assay	PF-06409577 possesses similar potency toward the human and rat α1β1γ1 isoforms. In broad panel screening against other receptors, channels, PDEs and kinases, PF-06409577 exhibits minimal off-target pharmacology. PF-06409577 shows no detectable inhibition of hERG in a patch-clamp assay (100 µM) and is not an inhibitor (IC50>100 µM) of the microsomal activities of major human cytochrome P450 isoforms.
Animal Protocol	Rats: For 68 days, daily administration of ramipril in drinking water (1 mg/kg/day), PF-06409577 at 10, 30, or 100 mg/kg (p.o.), PF-249 at 3, 10, or 30 mg/kg (p.o.), or 0.5% methylcellulose (p.o.) is started and continued. After 14, 28, 42, and 60 days of dosing, all lean and obese rats have their 24-hour urine collected, and the volume is recorded. All rats receive a final dose on day 63 following a 16-hour overnight fast. One hour after the last dose, a 100 L tail vein blood sample is taken and processed to determine the total protein and insulin levels. Blood glucose is also measured using a glucometer. Isoflurane is then used to put each rat to sleep. The right kidney is collected and immediately freeze-clamped and transferred to liquid nitrogen storage; the left kidney is fixed in 10% formalin. Rats are then euthanized by exsanguination from the vena cava[1]
References	[1]. Int J Mol Sci . 2017 Jun 30;18(7):1408. [1]. J Med Chem . 2016 Sep 8;59(17):8068-81.

Solubility Data

Solubility (In Vitro)

DMSO: ~68 mg/mL (~199.9 mM) Water: <1 mg/mL Ethanol: ~68 mg/mL (~199.9 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.09 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 20.8 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: ≥ 2.08 mg/mL (6.09 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 20.8 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: ≥ 2.08 mg/mL (6.09 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 20.8 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	2.9258 mL	14.6289 mL	29.2577 mL
	5 mM	0.5852 mL	2.9258 mL	5.8515 mL
	10 mM	0.2926 mL	1.4629 mL	2.9258 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.