GSK-3α (pKi = 8.2 nM); GSK-3β (pKi = 7.5 nM); cdk5 (pKi = 6.4 nM); cdk2 (pKi = 5.9 nM); cdk1 (pKi = 5.7 nM)
GSK3α (Ki = 6.9 nM [pKi=8.2])
GSK3β (Ki = 31 nM [pKi=7.5]) [1]

AZD1080 is a selective, orally active, brain permeable GSK3 inhibitor, inhibits human GSK3 with a Ki of 6.9 nM and 31 nM, respectively. It also exhibits >14-fold selectivity against cdk2, cdk5, cdk1, and Erk2. With an IC50 of 324 nM, AZD1080 prevents tau phosphorylation in cells that express human tau. [1]

---

AZD1080 inhibited tau phosphorylation at Ser396 in 3T3 fibroblasts stably expressing 4-repeat human tau with an IC50 of 324 nM, whereas the non-selective GSK3 inhibitor LiCl had an IC50 of 1.5 mM. At 1 µM, maximal inhibition of tau phosphorylation was observed at 1-2 h and remained for at least 8 h [1].
AZD1080 exhibited high permeability in Caco-2 cells (17 × 10⁻⁶ cm/s) and in a bovine endothelial blood-brain barrier cell assay (8 × 10⁻³ cm/min) [1].

AZD1080 inhibits tau phosphorylation in rat brain after oral dministration, with a brain/plasma exposure ratio of 0.5 to 0.8 at peak concentrations. In mice, AZD1080 restores cognitive deficits and heals damaged synapses. Acute oral administration of AZD1080 reduces the ratio of phosphorylated to total glycogen synthase (GS) in a dose-dependent manner, with a mean maximal inhibitory effect of 49% at the highest dose (10 μmol/kg) at 2 hours after dosing. [1]

---

AZD1080 (3 or 10 µmol/kg, oral gavage) inhibited tau phosphorylation at Thr231 in rat hippocampus, with maximal inhibition of 38±2% and 48±2% at 6 h post-dose, and the effect persisted up to 24 h (15±7% and 30±3% inhibition, respectively) [1].
In a mouse contextual fear conditioning (CFC) model, acute oral administration of AZD1080 (15 µmol/kg) did not reverse MK-801-induced memory deficits. However, subchronic oral treatment (twice daily for 3 days) at 4 or 15 µmol/kg significantly blocked MK-801-induced deficits (p<0.05 at 4 µmol/kg; p<0.01 at 15 µmol/kg) when training was performed 3 h after last dose. The effect was also observed at 1.5 h and 5 h after last dose (4 µmol/kg) [1].
In ex vivo LTP experiments using mouse hippocampal slices, subchronic treatment with AZD1080 (15 µmol/kg, twice daily for 3 days) prevented the disruption of LTP induction caused by 500 nM MK-801 (LTP amplitude: 121±5% vs. MK-801 alone 107±3%, p<0.05). AZD1080 alone had no significant effect on LTP induction compared to vehicle [1].
In adult rats, acute oral AZD1080 (1-10 µmol/kg) dose-dependently reduced the phosphorylated to total glycogen synthase (GS) ratio in peripheral blood mononuclear cells (PBMC), with maximal inhibition of 49±2% at 10 µmol/kg at 2 h post-dose, returning to baseline by 24 h [1].

The GSK3β, Cdk2, and Cdk5 Ki’s are determined using scintillation proximity assays and kinetic analyses. The GSK3α assay is conducted as part of the GSK3α assay. For the purpose of determining the Ki value for GSKα , the KM value of ATP is 10 M. Cdk1 is inhibited in this process. To determine the Ki value, 51 M of ATP is used as the KM value. An Erk2 SPA kit, p42 MAPK kinase (20 U/well), and biotinylated MBP are used to measure Erk2 activity. In order to determine the Ki value, the KM value of ATP is 71 μM[1].

---

The Ki values for GSK3β, Cdk2, and Cdk5 were determined using scintillation proximity assays and kinetic analyses. The KM value of ATP used to calculate the Ki was 10 µmol/L for GSK3α, 51 µmol/L for Cdk1, and 71 µmol/L for Erk2. Recombinant human GSK3α was used for the GSK3α assay. Cdk1 inhibition was performed according to a previously described method. Erk2 activity was determined using a Ser/Thr kinase SPA kit with p42 MAPK kinase and biotinylated MBP [1].
The selectivity of AZD1080 (at 10 µM) was tested against 24 protein kinases. Enzymatic activity was measured in the presence of 0.1 mM ATP. Off-target selectivity was also evaluated on 65 targets including adenosine receptors, adrenergic receptors, calcium channels, dopamine receptors, GABA receptors, glutamate receptors, histamine receptors, muscarinic receptors, serotonin receptors, and others. AZD1080 showed less than 50% effect at 10 µM on all tested targets [1].

The four-repeat tau protein is engineered to express consistently in 3T3 fibroblasts. These cells have high endogenous concentrations of GSK3, which can constitutively phosphorylate tau protein. LiCl prevents this phosphorylation from occurring. Cultures are twice washed with 5 mM MgCl2-PBS after treatment with various chemicals. Extracts for Western blot analysis are prepared by homogenizing cells in ice-cold extraction buffer consisting of 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM NaF, 1% Triton X-100, 1 mM sodium orthovanadate, 10 mM EDTA, and protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin). The samples are homogenized at 4 °C, and the Bradford method is used to calculate the protein content. On a 10% SDS-PAGE gel, the total protein (25 μg) is electrophoresed before being transferred to a nitrocellulose membrane. The primary antibodies tau Ser(P)-396, tau5, and anti-GSK3β are used in the experiments at a 1:1000 dilution each. At 4 °C overnight, the filters are incubated with the antibody in 5% nonfat dried milk. For immunodetection, a secondary antibody (1:5000) is used, then ECL detection tools. Densitometric scanning is used to measure immunoreactivity.

---

3T3 fibroblasts stably expressing 4-repeat human tau were treated with AZD1080 at various concentrations. Cells were lysed and total tau and phosphorylated tau at Ser396 were detected by Western blotting. The percent inhibition of tau phosphorylation was calculated compared to vehicle-treated cells [1].
Caco-2 cell permeability was measured using standard protocols. Bovine endothelial blood-brain barrier cell assay was used to determine permeability values [1].

Mice: A total of 161 male C57BL/6 mice between the ages of 8 and 12 weeks are used. The animals are housed in standard cages with three to five mice each, and they are given regular rodent chew and unlimited access to tap water. Each experimental group typically consists of 9–12 mice, with 2-4 mice in the satellite groups (see below for information on determining compound exposure in plasma and the brain). Acute or subchronic (twice daily for 3 days) oral gavage administration of AZD1080 (4.0 or 15 μmol/kg) or vehicle (water with 0.5% ascorbic acid, 0.01% EDTA, pH 2.0) is done. After the final AZD1080 administration, the training trial is conducted 1.5, 3, or 5 hours later.
Rats: The rats used are 71 adult male Sprague-Dawley rats (250-300 g). The rats are given an acute dose of AZD1080 (1, 3 or 10 mol/kg) or a vehicle (5 mL/kg) of water with 0.5% ascorbic acid, 0.01% EDTA, and pH 2.0. After administration, the rats are sedated and blood from the abdominal aorta is collected in heparin microtainer tubes at 1, 2, 3, 6, or 24 hours. Blood samples are used to isolate peripheral blood mononuclear cells (PBMC). To process the plasma and conduct the subsequent bioanalysis, separate blood samples are obtained.

---

For rat PK/PD studies, young (11-12 days old) Sprague-Dawley rats received AZD1080 (3 or 10 µmol/kg) or vehicle (water with 0.5% ascorbic acid, 0.01% EDTA, pH 2.0) via oral gavage (6 mL/kg). At 1, 2, 3, 6, or 24 h after administration, rats were decapitated, hippocampus was dissected, and tau phosphorylation was measured by sandwich immunoassay [1].
For mouse cognition studies, male C57BL/6 mice (8-12 weeks) received AZD1080 (4 or 15 µmol/kg) or vehicle (water with 0.5% ascorbic acid, 0.01% EDTA, pH 2.0) orally (10 mL/kg) either acutely or subchronically (twice daily for 3 days). MK-801 (0.1 or 0.15 mg/kg) or saline was given subcutaneously 30 min before training. Contextual fear conditioning (CFC) was performed: training included a 2 s foot shock (0.7 mA), and freezing behavior was measured 24 h later [1].
For ex vivo LTP studies, mice were dosed twice daily for 3 days with vehicle or AZD1080 (15 µmol/kg). Mice were killed 2 h after final dose. Transverse hippocampal slices (400 µm) were prepared and maintained in aCSF at 30-32°C. For slices from AZD1080-treated mice, 50 nM AZD1080 was included in the aCSF. MK-801 (300-500 nM) was added to perfusion for 10 min, followed by a minimal tetanus protocol (four trains of five pulses at 200 Hz delivered at 5 min intervals). LTP was induced by 100 Hz stimulation for 1 s. fEPSP amplitudes were recorded [1].
For PBMC studies in rats, adult male Sprague-Dawley rats received acute oral AZD1080 (1, 3, or 10 µmol/kg) or vehicle (same formulation) at 5 mL/kg. Blood was collected at 1, 2, 3, 6, or 24 h post-dose. PBMC were isolated using density gradient centrifugation, and phosphorylated GS (P-GS) and total GS (T-GS) were measured by ELISA [1].

AZD1080 had good oral bioavailability in rats (15-24%) with a half-life of 7.1 h [1].
The brain/plasma exposure ratio of AZD1080 at peak concentrations was 0.5-0.8 in rats, demonstrating good brain permeability [1].
In healthy volunteers, after multiple ascending doses (15 mg once daily for 14 days), steady-state exposure of AZD1080 was reached within 5 days, and the half-life was approximately 20 h. Limited accumulation in plasma was observed, with no apparent age effect on pharmacokinetics. Mean maximal plasma concentration was 86±16 nM at 1 h post-dose [1].
In rats, peak plasma concentrations after oral administration were 147±4 nM at 1 µmol/kg, 150±3 nM at 3 µmol/kg, and 464±21 nM at 10 µmol/kg, all at 1 h post-dose [1].
In mouse brain, concentrations of AZD1080 were 19±3 nM (4 µmol/kg) and 61±10 nM (15 µmol/kg) at 3 h after subchronic dosing [1].

In a Phase 1 multiple ascending dose study in healthy middle-aged volunteers, AZD1080 (15 mg once daily for 14 days) demonstrated an acceptable safety and tolerability profile [1].

[1]. AZD1080, a novel GSK3 inhibitor, rescues synaptic plasticity deficits in rodent brain and exhibits peripheral target engagement in humans. J Neurochem. 2013 May;125(3):446-56.

AZD1080 belongs to the hydroxyindole class of compounds with the structure 1H-indole, substituted at positions 2, 3, and 5 with hydroxyl, 5-(morpholino-4-ylmethyl)pyridin-2-yl, and cyano groups, respectively. It is a potent, blood-brain barrier-crossing inhibitor of human GSK3α and GSK3β, with Ki values of 6.9 nM and 31 nM, respectively. This drug was initially developed by AstraZeneca for the treatment of Alzheimer's disease (clinical trials have since been terminated). It has multiple functions, including as an EC 2.7.11.26 (tau protein kinase) inhibitor, a tau protein aggregation inhibitor, an antitumor drug, and an inducer of apoptosis. AZD1080 belongs to the morpholino, pyridine, hydroxyindole, nitrile, and tertiary amine classes of compounds.

---

AZD1080 is a clinical candidate for Alzheimer's disease and related tauopathies. It inhibits GSK3 by binding within the ATP pocket of the catalytic domain, forming three hydrogen bonds to Val-135. Subchronic but not acute treatment with AZD1080 reverses synaptic plasticity deficits in dysfunctional systems, likely via modification of proteins downstream of GSK3β signaling. AZD1080 also inhibits peripheral GSK3 activity in humans, measured as the ratio of phosphorylated to total glycogen synthase in lymphocytes, with a mean maximal reduction of 23±4% at 6 h after 15 mg dose on Day 1, returning to baseline by 24 h [1].

C19H18N4O2

334.3718

334.142

C, 68.25; H, 5.43; N, 16.76; O, 9.57

612487-72-6

612487-72-6

135564570

Orange solid powder

1.4±0.1 g/cm3

594.0±50.0 °C at 760 mmHg

313.0±30.1 °C

0.0±1.7 mmHg at 25°C

1.716

1.36

2

5

3

25

501

0

O1C([H])([H])C([H])([H])N(C([H])([H])C2=C([H])N=C(C([H])=C2[H])C2=C(N([H])C3C([H])=C([H])C(C#N)=C([H])C2=3)O[H])C([H])([H])C1([H])[H]

JULOXTBHCHEFBE-ZCXUNETKSA-N

InChI=1S/C19H18N4O2/c20-10-13-1-3-16-15(9-13)18(19(24)22-16)17-4-2-14(11-21-17)12-23-5-7-25-8-6-23/h1-4,9,11,21H,5-8,12H2,(H,22,24)/b18-17-

(Z)-3-(5-(morpholinomethyl)pyridin-2(1H)-ylidene)-2-oxoindoline-5-carbonitrile

AZD-1080;AZD-1080; AZD 1080

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~52 mg/mL (155.5 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.48 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 25.0 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: ≥ 2.5 mg/mL (7.48 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 25.0 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: 30% PEG400+0.5% Tween80+5% propylene glycol: 20 mg/mL

---

 (Please use freshly prepared in vivo formulations for optimal results.)