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Purity: ≥98%
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.
| Targets |
AMPK α2β1γ1 (EC50 = 6.8 nM); AMPK α1β1γ1 (EC50 = 7 nM)
The target of PF-06409577 is adenosine monophosphate-activated protein kinase (AMPK), specifically acting as a direct activator of AMPK. [1] PF-06409577 is a direct activator of AMPK, with a preference for the β1 subunit of AMPK (α1β1γ1 isoform), [2] |
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| 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.
1. In molecular docking studies, PF-06409577 was docked to the α1β1γ1 isoform of AMPK. The indole moiety of PF-06409577 showed a slight deflection compared with its original conformation in the X-ray crystal structure, and hydrogen bonds were formed between the compound and the active site of AMPK α1β1γ1 isoform, which is the basis for its direct activation of AMPK [2] |
| 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].
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| 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.
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| 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.
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| 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]
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| ADME/Pharmacokinetics |
The core and aryl side chain of the lead compound (indazole acid) were optimized to improve the oral absorption rate of PF-06409577, but the specific ADME parameters (such as absorption rate, distribution, metabolism, excretion, half-life, oral bioavailability, etc.) were not described in the literature [1].
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| References | |
| Additional Infomation |
1. PF-06409577 (chemical name: 6-chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic acid) is an indole acid compound and a direct activator of AMPK. It was developed based on human genetic association data and has the potential to treat diabetic nephropathy. The discovery of this compound originated from the identification of an indazole amide lead compound by high-throughput screening (HTS). After truncating to its minimum pharmacophore to obtain an indazole acid lead compound, the core and aryl side chain were optimized to improve oral absorption. Finally, PF-06409577 was identified. This compound has entered the first human trial for the treatment of diabetic nephropathy [1]
2. AMPK is a protein kinase involved in maintaining cellular energy homeostasis. Direct β1-selective AMPK activators (including PF-06409577) have regained attention due to their potential in treating patients with diabetic nephropathy. In structure-based virtual screening and molecular docking studies, PF-06409577 was used as a reference compound for screening AMPK activators, and its binding mode with the AMPK α1β1γ1 isoform was analyzed to guide the identification of novel β1-selective AMPK activators [2]. |
| Molecular Formula |
C19H16CLNO3
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| Molecular Weight |
341.79
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| Exact Mass |
341.082
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| Elemental Analysis |
C, 66.77; H, 4.72; Cl, 10.37; N, 4.10; O, 14.04
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| CAS # |
1467057-23-3
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| Related CAS # |
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| PubChem CID |
71748255
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| Appearance |
White to off-white solid powder
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| LogP |
4.558
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
24
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| Complexity |
487
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=CC2=C(C(C(=O)O)=CN2)C=C1C1C=CC(=CC=1)C1(CCC1)O
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| InChi Key |
FHQXLWCFSUSXBF-UHFFFAOYSA-N
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| 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)
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| Chemical Name |
6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic acid
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| 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. View More
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. |
| 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Status | Interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT02286882 | Terminated | Drug: PF-06409577 or Placebo |
Healthy | Pfizer | November 2014 | Phase 1 |
(A) In vitro AMPK activity for selected AMPK heterotrimers (αβγ) activated with PF-06409577. (B) In vitro AMPK activity for selected AMPK heterotrimers (αβγ) activated with PF-249. (C) Ribbon representation of crystal structure of AMPKα1β1γ1 bound to PF-249. (D) Close-up view of the ligand-protein interface. (E) AMPKβsubunit levels in kidney tissue were measured by quantitative ELISA and plotted as percentage of AMPK heterotrimer containing theβ1 subunit.J Pharmacol Exp Ther.2017 May;361(2):303-311. |
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(A) Western blot for AMPKβ1,β2, and pan-αin 293FT cells transfected with scrambled siRNA or siRNA targeting AMPKβ1. (B) ELISA quantification of ACC phosphorylation status in 293FT treated with siRNA and PF-06409577. (C) ELISA quantification of ACC phosphorylation status in 293FT treated with siRNA and PF-06409577.J Pharmacol Exp Ther.2017 May;361(2):303-311. |
(A) Cumulative urinary albumin excretion over a 24-hour period at multiple time points during a dosing study in obese ZSF1 rats treated with vehicle or PF-06409577; 10 or 11 animals per group. (B) Kidney AMPK phosphorylation status in terminal samples after 8 weeks of dosing, 1 hour after the last dose.J Pharmacol Exp Ther.2017 May;361(2):303-311. |
(A) Cumulative albumin excretion over a 24-hour period at multiple time points during a dosing study in ZSF1 rats treated with vehicle, PF-249, PF-06409577, or ramipril (1 mg/kg/d in drinking water); 12 animals per group. (B) Kidney AMPK phosphorylation status in terminal samples after 8 weeks of dosing, 1 hour after the last dose.J Pharmacol Exp Ther.2017 May;361(2):303-311. |
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(A–C) Representative histology images of phospo-S6 (p-S6) reactivity in kidneys collected after 8 weeks of dosing vehicle or PF-06409577 to ZSF1 rats; 12 animals per group. Glomeruli are indicated by the arrows and stain area was quantified. (D) Quantitative measure of p-S6 stain area in glomeruli of five lean ZSF1 treated with vehicle, seven obese ZSF1 treated with vehicle, and seven obese ZSF1 animals treated with PF-06409577.J Pharmacol Exp Ther.2017 May;361(2):303-311. |
Quantitative PCR measurements of mRNA for (A)Col1a1, (B)Col4a1, (C)Nox4, and (D)Ppargc1ain kidney samples from animals treated with 3, 10, and 30 mg/kg PF-249, 100 mg/kg PF-06409577 (PF-577), or ramipril for 8 weeks; 8–10 animals per group. Significance compared with the obese vehicle group.J Pharmacol Exp Ther.2017 May;361(2):303-311. |
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