Akt1 (Ki = 160 pM) [1]
Akt2, Akt3 (equipotent to Akt1 within cells) [1]
PKA (40-fold selective vs Akt1; Ki = 6.3 nM) [1]
PKCγ (150-fold selective vs Akt1; Ki = 24 nM) [1]
PKCδ (200-fold selective vs Akt1; Ki = 33 nM) [1]
GSK3β (260-fold selective vs Akt1; Ki = 41 nM) [1]
RSK2 (68-fold selective vs Akt1; Ki = 11 nM) [1]
CDK2 (150-fold selective vs Akt1; Ki = 24 nM) [1]
ERK2 (2,100-fold selective vs Akt1; Ki = 340 nM) [1]

A-443654 is 30,000 times more potent than the original lead molecule, with a Ki value of 160 pM. Compared to PKA, A-443654 has 40 times more selectivity for Akt. In cells, A-443654 inhibits Akt1, Akt2, or Akt3 in an equal manner. In all three cell lines, A-443654 lowered P-GSK3 in a dose-responsive way. At an EC50 of 0.1 μM, A-443654 suppresses the growth of tumor cells[1]. A-443654 caused morphological alterations in 10A and 10CA1a cells relatively quickly (in 2 to 4 hours), with 10CA1a cells being more sensitive to A-443654 than 10A cells. After 12 hours, A-443654 at 2 μM alone induced 10CA1a cells to separate from the plate, but not 10A cells. At 1 μM, on the other hand, 10CA1a cells separated from the plate. Rapamycin and A-443654's effects on the DNA content of 10A and 10CA1a cells were examined using FACScan. Conversely, after 8 hours, A-443654 at 2 and 5 μM decreased Bcl-2 levels in 10CA1a cells by 30% to 40%. Rapamycin and 2 or 5 μM A-443654 together, however, dramatically decreased Bcl-2 protein levels by around 40% to 50% in 10A cells and about 70% in 10CA1a cells [2]. A-443654 exhibits a relative growth inhibition of more than 3.5 times on mutant cells, which is the largest selective effect on mutant cells when compared to WT cells [3].

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In murine FL5.12 cells stably transfected with constitutively active myristoylated human Akt1, Akt2, or Akt3, A-443654 reduced the phosphorylation of the downstream target GSK3 in a dose-responsive manner, with equal potency against all three Akt isoforms. [1]
In MiaPaCa-2 pancreatic tumor cells, A-443654 (0-30 μM) reduced the phosphorylation of Akt downstream targets GSK3α/β, FOXO3, TSC2, and mTOR in a dose-dependent manner, as measured by Western blot. A concomitant increase in Akt phosphorylation at Thr³⁰⁸ and Ser⁴⁷³ was also observed. [1]
In HeLa cells transiently transfected with a FOXO3A-hrGFP construct, treatment with A-443654 induced the translocation of FOXO3A from the cytoplasm to the nucleus. [1]
In a cell proliferation assay (likely MTT or similar), A-443654 slowed the proliferation of tumor cells with an EC₅₀ of 0.1 μM. [1]

In the 3T3-Akt1 flank tumor model, subcutaneous administration of A-443654 (7.5 mg/kg/d) suppresses tumor growth. A-443654 (50 mg/kg, subcutaneously) causes the tumor cells around 3T3-Akt1 to undergo apoptosis. In MiaPaCa-2 tumors, A-443654 (30 mg/kg, subcutaneous injection) raises phosphorylated Akt1 levels [1].

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In a 3T3-Akt1 murine fibroblast flank tumor model in scid mice, subcutaneous administration of A-443654 (7.5 mg/kg/d, bid for 14 days) significantly inhibited the growth of established tumors (P < 0.02 from day 26 onward). An inactive analogue (2-methyl A-443654) showed no effect. Tumors regrew rapidly after dosing was stopped. [1]
In a MiaPaCa-2 human pancreatic cancer xenograft model, treatment with A-443654 (7.5 mg/kg/d, s.c., bid for 14 days), starting one day after tumor inoculation, significantly inhibited tumor growth (P < 0.03). The effect was comparable to that of gemcitabine (120 mg/kg/d i.p. on days 3, 6, 9, 12). [1]
In a MiaPaCa-2 xenograft model with established tumors, an intensified dosing regimen of A-443654 (50 mg/kg/d, s.c., tid on days 16, 20, and 24) proved more efficacious than the bid/daily schedule. The combination of A-443654 (same tid regimen) with rapamycin (20 mg/kg/d i.p. for 15 days) was statistically superior to either monotherapy from day 23 onward (P < 0.01). [1]
In MiaPaCa-2 tumor-bearing mice, a single dose of A-443654 (30 mg/kg, s.c.) increased phospho-Akt levels in tumors within 1 hour, as shown by immunohistochemistry. A single 50 mg/kg dose increased apoptosis (active caspase-3 staining) in 3T3-Akt1 tumors 8 hours after treatment. [1]
In an oral glucose tolerance test, fasted animals treated with A-443654 (20 mg/kg) showed elevated plasma insulin levels compared to vehicle-treated controls, without significant changes in blood glucose. [1]

Kinase inhibition assays were performed using a radioactive FlashPlate-based platform. Recombinant kinases (Akt1, PKA, PKCγ, PKCδ, GSK3β, RSK2, CDK2, ERK2, etc.) were incubated with biotinylated peptide substrates, [γ-³³P]ATP, and varying concentrations of A-443654. The reaction mixture was captured on streptavidin-coated FlashPlates, and radioactivity was measured. Ki values were calculated from the inhibition data. [1]
The binding mode of A-443654 was determined by X-ray crystallography of the compound complexed with PKA (a close homologue of Akt). The structure was solved to 2.7 Å resolution and refined to Rwork = 24.41% and RFree = 30.18%. It revealed three key hydrogen bond interactions: one with the hinge region backbone, one with a conserved lysine (Lys⁷² in PKA), and a set with Asn¹⁷¹ and Asp¹⁸⁴. [1]

Western Blot Analysis: Cells (e.g., MiaPaCa-2) were treated with 0-30 μM A-443654 for 2 hours. Lysates were prepared, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phospho-GSK3α/β, phospho-FOXO1A/3A, phospho-TSC2, phospho-mTOR, and phospho-Akt. Bands were visualized by chemiluminescence and quantified. [1]
FOXO3A Translocation Assay: HeLa cells were transiently transfected with a pFOXO3A-hrGFP plasmid. At 40 hours post-transfection, cells were treated with A-443654 or vehicle for 6 hours. Fluorescent live-cell images were captured by microscopy to visualize the subcellular localization of FOXO3A-GFP. [1]
Cell Proliferation Assay: The effect of A-443654 on cell viability/proliferation was assessed using the Alamar Blue assay. Cells were washed with PBS, and diluted Alamar Blue reagent was added. After incubation, fluorescence was measured (excitation 544 nm, emission 595 nm). EC₅₀ values were calculated. [1]

Tumor Efficacy Studies:** Immunocompromised male scid mice were inoculated subcutaneously with tumor cells (3T3-Akt1, MiaPaCa-2, or PC-3) in 50% Matrigel. For early treatment studies, therapy began the day after inoculation. For established tumor studies, treatment began when tumors reached a designated size (~250-270 mm³). A-443654 was administered subcutaneously in a vehicle of 0.2% HPMC. Dosing regimens varied: bid at 7.5 mg/kg/d for 14 days, or tid at 50 mg/kg/d on days 16, 20, and 24. Tumor size was measured twice weekly with calipers. [1]
* **Pharmacokinetic/Pharmacodynamic Studies:** Tumor-bearing mice were treated with A-443654 at various doses (3.8, 7.5, 15 mg/kg bid for 3 days). At time points after the final dose, plasma and tumor tissues were collected. Drug concentrations were determined by LC-MS or UV-HPLC. Tumor lysates were analyzed by Western blot for phospho-Akt, phospho-S6, and phospho-GSK3. [1]
* **Immunohistochemistry:** Tumors from treated mice were fixed, paraffin-embedded, and sectioned. Sections were stained with antibodies against active caspase-3 (apoptosis marker) or phospho-Akt, using standard immunohistochemical techniques with DAB color development. [1]
* **Glucose Tolerance Test:** Fasted mice were administered vehicle or A-443654 (20 mg/kg) 30 minutes before an oral glucose challenge (1 g/kg). Blood samples were collected, and plasma insulin levels were measured. [1]

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Tumor Efficacy Studies: Immunocompromised male scid mice were inoculated subcutaneously with tumor cells (3T3-Akt1, MiaPaCa-2, or PC-3) in 50% Matrigel. For early treatment studies, therapy began the day after inoculation. For established tumor studies, treatment began when tumors reached a designated size (~250-270 mm³). A-443654 was administered subcutaneously in a vehicle of 0.2% HPMC. Dosing regimens varied: bid at 7.5 mg/kg/d for 14 days, or tid at 50 mg/kg/d on days 16, 20, and 24. Tumor size was measured twice weekly with calipers. [1]
Pharmacokinetic/Pharmacodynamic Studies: Tumor-bearing mice were treated with A-443654 at various doses (3.8, 7.5, 15 mg/kg bid for 3 days). At time points after the final dose, plasma and tumor tissues were collected. Drug concentrations were determined by LC-MS or UV-HPLC. Tumor lysates were analyzed by Western blot for phospho-Akt, phospho-S6, and phospho-GSK3. [1]
Immunohistochemistry: Tumors from treated mice were fixed, paraffin-embedded, and sectioned. Sections were stained with antibodies against active caspase-3 (apoptosis marker) or phospho-Akt, using standard immunohistochemical techniques with DAB color development. [1]
Glucose Tolerance Test: Fasted mice were administered vehicle or A-443654 (20 mg/kg) 30 minutes before an oral glucose challenge (1 g/kg). Blood samples were collected, and plasma insulin levels were measured. [1]

A-443654 is not orally bioavailable. It was administered subcutaneously in all in vivo studies. [1]
Following subcutaneous administration in mice at 3.8, 7.5, and 15 mg/kg bid, plasma concentrations of A-443654 remained above the cellular EC₅₀ (0.1 μM) for approximately 5 hours. [1]
Concentrations of A-443654 in tumor tissue were significantly higher than in plasma and remained above the cellular EC₅₀ for the entire 12-hour dosing interval. [1]

The therapeutic window for A-443654 is narrow. Efficacy was achieved at doses approximately 2-fold lower than the maximally tolerated dose (MTD). [1]
The dosing period for A-443654 was limited to 14 days due to severe injection site irritation (a local toxicity). [1]
Systemic toxicities observed in treated animals included malaise and weight loss (typically >10% at supratherapeutic doses), consistent with abnormalities in glucose metabolism. [1]
A-443654 induced a significant increase in plasma insulin levels in glucose challenge tests, consistent with a homeostatic response to maintain blood glucose and mirroring the phenotype of Akt2 knockout animals. [1]

[1]. Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo. Mol Cancer Ther. 2005 Jun;4(6):977-86.

[2]. Rapamycin sensitizes Akt inhibition in malignant human breast epithelial cells. Cancer Lett. 2010 Oct 1;296(1):74-87.

[3]. Inhibition of Akt inhibits growth of glioblastoma and glioblastoma stem-like cells. Mol Cancer Ther. 2009 Feb;8(2):386-93.

[4]. Estrogen receptor alpha and beta regulate actin polymerization and spatial memory through an SRC-1/mTORC2-dependent pathway in the hippocampus of female mice. J Steroid Biochem Mol Biol. 2017 Nov;174:96-113.

(2S)-1-(1H-indol-3-yl)-3-[[5-(3-methyl-2H-indazol-5-yl)-3-pyridyl]oxy]-2-propane is a member of the indole class of compounds.

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A-443654 is a potent, ATP-competitive, and reversible inhibitor of Akt kinases, belonging to the indazole-pyridine series. It was discovered through a high-throughput screen followed by structure-guided optimization. [1]
The compound binds to the ATP-binding site of Akt. X-ray crystallography with the related kinase PKA showed that it forms three critical hydrogen bonds: one with the hinge region, one with a conserved lysine, and one with residues that normally coordinate a magnesium ion (Asn and Asp). [1]
In cells, A-443654 inhibits the phosphorylation of multiple downstream effectors of Akt (GSK3, FOXO3, TSC2, mTOR) and induces a compensatory increase in Akt phosphorylation. This increase is dependent on PI3K activity. [1]
A-443654 showed antitumor activity in multiple xenograft models (3T3-Akt1, MiaPaCa-2, PC-3). Its effects were cytostatic, with tumors regrowing after cessation of treatment. It showed enhanced efficacy when combined with paclitaxel or rapamycin. [1]
The observed toxicities (weight loss, malaise, hyperinsulinemia) are consistent with mechanism-based inhibition of Akt, particularly Akt2, which is involved in insulin signaling. [1]

397.19

552325-16-3

10172943

White to off-white solid powder

1.3±0.1 g/cm3

722.0±60.0 °C at 760 mmHg

390.4±32.9 °C

0.0±2.3 mmHg at 25°C

1.722

3.65

3

4

6

30

562

1

N[C@H](COC1=CN=CC(C2=CC3=C(NN=C3C)C=C2)=C1)CC4=CNC5=CC=CC=C54

YWTBGJGMTBHQTM-IBGZPJMESA-N

InChI=1S/C24H23N5O/c1-15-22-10-16(6-7-24(22)29-28-15)17-9-20(13-26-11-17)30-14-19(25)8-18-12-27-23-5-3-2-4-21(18)23/h2-7,9-13,19,27H,8,14,25H2,1H3,(H,28,29)/t19-/m0/s1

(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine

A443564 A 443564 A-443564

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 : ≥ 100 mg/mL (~251.59 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.29 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.29 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.29 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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