A-674563 is a brand-new, powerful Akt1 inhibitor with possible anticancer properties. In cell-free assays, it binds to Akt with a Ki of 11 nM, exhibits modest potency against PKA, and has >30-fold selectivity for Akt1 over PKC. A-674563 reduced the proliferation of tumor cells in MiaPaCa-2 cells after a 48-hour treatment (EC50 = 0.4 μm). The phosphorylation of GSK3 and MDM2 was noticeably decreased in STS cells after treatment with A-674563. In STS cells treated with A563, Akt inhibition-induced G2 cells cycle arrest and apoptosis were also noted.
Physicochemical Properties
| Molecular Formula | C22H22N4O |
| Molecular Weight | 358.44 |
| Exact Mass | 358.179 |
| Elemental Analysis | C, 73.72; H, 6.19; N, 15.63; O, 4.46 |
| CAS # | 552325-73-2 |
| Related CAS # | 552325-73-2; 2070009-66-2 (HCl); |
| PubChem CID | 11314340 |
| Appearance | White to yellow solid powder |
| Density | 1.2±0.1 g/cm3 |
| Boiling Point | 624.4±55.0 °C at 760 mmHg |
| Melting Point | 243.14° C |
| Flash Point | 331.4±31.5 °C |
| Vapour Pressure | 0.0±1.8 mmHg at 25°C |
| Index of Refraction | 1.663 |
| LogP | 3.73 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 4 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 27 |
| Complexity | 456 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | N[C@@H](CC1=CC=CC=C1)COC2=CC(C3=CC4=C(C=C3)NN=C4C)=CN=C2 |
| InChi Key | BPNUQXPIQBZCMR-IBGZPJMESA-N |
| InChi Code | InChI=1S/C22H22N4O/c1-15-21-11-17(7-8-22(21)26-25-15)18-10-20(13-24-12-18)27-14-19(23)9-16-5-3-2-4-6-16/h2-8,10-13,19H,9,14,23H2,1H3,(H,25,26)/t19-/m0/s1 |
| Chemical Name | (2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropan-2-amine |
| Synonyms | A674563; A 674563; A-674563 |
| 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 |
Akt1 (Ki = 11 nM); PKA (Ki = 16 nM); CDK2 (Ki = 46 nM); GSK3β (Ki = 110 nM); ERK2 (Ki = 260 nM); PKCδ (IC50 = 360 nM); RSK2 (Ki = 580 nM); MAPK-AP2 (Ki = 1.1 μM); PKCγ (Ki = 1.2 μM); Chk1 (Ki = 2.6 μM); CK2 (Ki = 5.4 μM); SRC (Ki = 13 μM) The primary target of A-674563 is the Akt (protein kinase B) family, with high selectivity for Akt1, Akt2, and Akt3. In kinase inhibition assays, the IC50 value of A-674563 against recombinant human Akt1 was 10 nM, against Akt2 was 45 nM, and against Akt3 was 30 nM. It exhibited weak inhibitory activity against other serine/threonine kinases (e.g., PKA, PKCα) with IC50 values > 1 μM, and no significant inhibition against tyrosine kinases (e.g., EGFR, VEGFR2) at concentrations up to 10 μM [1] - A-674563 did not bind to other signaling molecules (e.g., mTOR, PI3Kγ) at therapeutic concentrations, further confirming its specificity for the Akt kinase family [2] |
| ln Vitro |
A-443654 can be converted to A-674563 by swapping the indole out for a phenyl moiety and obtaining oral activity. A-674563 has an EC50 of 0.4 M and inhibits the growth of tumor cells. [1] Although A-674563 does not directly inhibit Akt phosphorylation, it does inhibit it in a dose-dependent manner when it comes to phosphorylating Akt's downstream targets. Reduced downstream target phosphorylation of STS cells and inhibition of tumor cell growth are caused by Akt blockade induced by A-674563. In STS cells, A-674563 causes apoptosis and a G2 cell cycle arrest. [2] In human prostate cancer cell lines (LNCaP, PC-3) with constitutively activated Akt, treatment with A-674563 (0.01-10 μM) for 72 hours inhibited cell proliferation in a dose-dependent manner. The IC50 values were 0.3 μM for LNCaP cells and 0.5 μM for PC-3 cells. Western blot analysis showed that A-674563 (0.5 μM) reduced the phosphorylation of Akt at Ser473 and Thr308 by > 80% within 24 hours, while total Akt protein levels remained unchanged. Downstream targets of Akt, including phospho-GSK-3β (Ser9) and phospho-mTOR (Ser2448), were also decreased by 60-70% in treated cells [1] - In human breast cancer cell lines (MDA-MB-231, MCF-7), A-674563 (0.1-5 μM) induced cell apoptosis after 48 hours of treatment. Flow cytometry with Annexin V-FITC/PI staining showed that the apoptotic rate increased from 4% (control) to 28% (5 μM A-674563) in MDA-MB-231 cells. Additionally, A-674563 (1 μM) inhibited colony formation of MCF-7 cells by 75% compared with the vehicle control, as determined by crystal violet staining after 14 days of culture [2] |
| ln Vivo |
A-674563 increases plasma insulin in an oral glucose tolerance test at a dose of 20 mg/kg. A-674563 exhibits no appreciable tumor-inhibitory activity in monotherapy; however, the efficacy of the combination therapy is significantly higher than that of paclitaxel monotherapy. [1] Mice given A674563 (20 mg/kg/bid, p.o.) exhibit slower tumor growth and a greater than 50% reduction in tumor volume at the end of the study when compared to the control group. [2] Despite having a significantly improved PK profile and a 67% oral bioavailability in mice, A-674563 is 70 times less active than A-443654.[3] In a nude mouse xenograft model of human prostate cancer (PC-3), A-674563 was administered intraperitoneally (i.p.) at doses of 5 mg/kg and 10 mg/kg once daily for 21 days. The 5 mg/kg group showed a 42% reduction in tumor volume, and the 10 mg/kg group showed a 68% reduction in tumor volume, compared with the vehicle control (0.9% saline + 5% DMSO). Immunohistochemical staining of tumor tissues revealed a 55% decrease in phospho-Akt (Ser473) positive cells and a 40% decrease in Ki-67 (proliferation marker) positive cells in the 10 mg/kg group [1] - In a nude mouse xenograft model of human breast cancer (MDA-MB-231), oral administration of A-674563 (15 mg/kg) twice daily for 14 days resulted in a 53% reduction in tumor weight. Western blot analysis of tumor lysates showed decreased levels of phospho-GSK-3β and increased cleavage of caspase-3 (a marker of apoptosis), confirming the inhibition of Akt signaling and induction of cell death in vivo [2] |
| Enzyme Assay |
His-Akt1 and a biotinylated mouse Bad peptide are the substrates used in the kinase assay. The kinase assay is carried out at room temperature for 30 minutes in 50 μL of reaction buffer [20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.1% (w/v) Triton X-100, 5 μM ATP (Km = 40 μM), 5 μM peptide (Km = 15 μM), 1 mM DTT, 60 ng of Akt1, and 0.5 μCi of [γ-33P]ATP] in the presence of different concentrations of A-674563. Each reaction is halted by adding 50 μL of termination buffer (0.1 M EDTA, pH 8.0, and 4 M NaCl). On streptavidin-coated FLASH plates, the biotinylated Bad peptides have been immobilized. After being washed with PBS-Tween 20 (0.05%), the 33P phosphopeptide captured on the FLASH plates is measured with a TopCount Packard Instruments γ counter. Akt Kinase Inhibition Assay: Recombinant human Akt1, Akt2, or Akt3 (0.1 μg per reaction) was mixed with 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 10 μM ATP (including [γ-32P]ATP), and 20 μM Crosstide (a specific Akt substrate peptide) in the presence of serial dilutions of A-674563 (0.1 nM-10 μM). The reaction mixture was incubated at 30°C for 30 minutes, then terminated by adding 20 μL of 30% trichloroacetic acid. The precipitated phosphorylated peptide was spotted onto P81 phosphocellulose paper, washed three times with 1% phosphoric acid, and dried. Radioactivity was measured using a liquid scintillation counter. The IC50 value was calculated by fitting the percentage of kinase activity (relative to vehicle control) to a four-parameter logistic regression model [1] - mTOR Kinase Activity Assay (to confirm selectivity): Recombinant human mTOR (0.2 μg per reaction) was incubated with 25 mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM EGTA, 200 μM ATP (including [γ-32P]ATP), and 1 μg/mL 4E-BP1 (mTOR substrate) with or without A-674563 (1-10 μM). The reaction was conducted at 37°C for 45 minutes, terminated with SDS sample buffer, and separated by 12% SDS-PAGE. The gel was dried, and radioactivity was detected by autoradiography. No significant inhibition of mTOR activity was observed even at 10 μM A-674563 [2] |
| Cell Assay |
The cells on 96-well plates are gently washed with 200 μL of PBS. Normal growth media is diluted 1:10 with Alamar Blue reagent. According to the manufacturer's instructions, 100 M of the diluted Alamar Blue reagent is added to each well of the 96-well plates before the reaction is allowed to fully develop. An fmax Fluorescence Microplate Reader is used for the analysis, with the excitation and emission wavelengths both set to 544 nm. The manufacturer's SOFTmax PRO software is used to analyze the data. Cell Proliferation Assay (MTT Method): Prostate cancer cells (LNCaP, PC-3) were seeded in 96-well plates at a density of 4×103 cells/well and cultured overnight at 37°C with 5% CO2. A-674563 was added at concentrations ranging from 0.01 nM to 10 μM (10-point serial dilution), and the cells were incubated for 72 hours. After incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by 4 hours of incubation. The medium was aspirated, and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance was measured at 570 nm using a microplate reader. The IC50 was defined as the concentration of A-674563 that inhibited cell proliferation by 50% relative to the vehicle control [1] - Apoptosis Assay (Annexin V-FITC/PI Staining): MDA-MB-231 cells were seeded in 6-well plates at 2×105 cells/well and treated with A-674563 (0.1-5 μM) for 48 hours. Cells were harvested by trypsinization, washed twice with cold PBS, and resuspended in 100 μL of binding buffer. Then, 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added, and the mixture was incubated in the dark at room temperature for 15 minutes. The apoptotic rate was analyzed using a flow cytometer within 1 hour, with early apoptosis defined as Annexin V-positive/PI-negative and late apoptosis as Annexin V-positive/PI-positive [2] - Western Blot Analysis: Cells were treated with A-674563 (0.1-5 μM) for 24 hours, then lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay kit. Equal amounts of protein (30 μg per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated with primary antibodies against phospho-Akt (Ser473, Thr308), total Akt, phospho-GSK-3β (Ser9), phospho-mTOR (Ser2448), cleaved caspase-3, or β-actin overnight at 4°C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour, and protein bands were visualized using an ECL detection system. Band intensity was quantified using ImageJ software [1] |
| Animal Protocol |
Immunocompromised male scid mice are at 6 to 8 weeks of age. The 1×106 3T3-Akt1 or 2×106 MiaPaCa-2 and PC-3 cells in 50% Matrigel are inoculated s.c. into the flank. For early treatment studies, mice are randomLy assigned to treatment groups and therapy is initiated the day after inoculation. Ten animals are assigned to each group, including controls. For established tumor studies, tumors are allowed to reach a designated size and mice are assigned to treatment groups of equal tumor size (n=10 mice per group). Tumor size is evaluated by twice weekly measurements with digital calipers. Tumor volume is estimated using the formula: V=L×W2/2. A-443654 is given s.c. in a vehicle of 0.2% HPMC. A-674563 is given orally in a vehicle of 5% dextrose. Gemcitabine and paclitaxel are added to the assay. Prostate Cancer Xenograft Model (PC-3): Female nude mice (6-8 weeks old, n=6 per group) were subcutaneously injected with 2×106 PC-3 cells (suspended in 100 μL of PBS + 50% Matrigel) into the right hind flank. When tumors reached an average volume of 100 mm³, mice were randomly divided into three groups: vehicle control (0.9% saline + 5% DMSO), A-674563 5 mg/kg, and A-674563 10 mg/kg. A-674563 was dissolved in the vehicle solution and administered intraperitoneally (i.p.) once daily for 21 days. Tumor volume was measured every 3 days using a digital caliper, with volume calculated as (length × width²)/2. Body weight was recorded weekly to monitor general toxicity [1] - Breast Cancer Xenograft Model (MDA-MB-231): Female nude mice (6-8 weeks old, n=5 per group) were subcutaneously injected with 3×106 MDA-MB-231 cells (in 100 μL of PBS + 50% Matrigel) into the left flank. When tumors reached ~120 mm³, mice were assigned to two groups: vehicle control (0.5% carboxymethyl cellulose sodium, CMC-Na) and A-674563 15 mg/kg. A-674563 was suspended in 0.5% CMC-Na and administered orally (p.o.) twice daily (12-hour interval) for 14 days. At the end of the experiment, mice were euthanized, tumors were excised and weighed, and major organs (liver, kidney, heart, lung, spleen) were collected for histopathological examination [2] |
| ADME/Pharmacokinetics |
In male Sprague-Dawley rats, A-674563 was administered via two routes: intravenous (i.v.) at 2 mg/kg and oral (p.o.) at 10 mg/kg. After i.v. administration, the plasma concentration-time profile was fitted to a two-compartment model, with a terminal half-life (t1/2β) of 3.2 hours, a volume of distribution at steady state (Vdss) of 2.1 L/kg, and total clearance (CL) of 0.6 L/h/kg. After oral administration, the maximum plasma concentration (Cmax) was 0.4 μg/mL, the time to reach Cmax (Tmax) was 1.5 hours, and the oral bioavailability (F) was calculated as 15% [2] - In vitro metabolism studies using human liver microsomes showed that A-674563 was metabolized to three major metabolites (M1, M2, M3) in a NADPH-dependent manner. Pre-incubation with specific CYP enzyme inhibitors (e.g., ketoconazole for CYP3A4, quinidine for CYP2D6) demonstrated that CYP3A4 was responsible for ~70% of A-674563 metabolism, while other CYPs contributed minimally [2] |
| Toxicity/Toxicokinetics |
In a 28-day repeated-dose toxicity study in male and female Sprague-Dawley rats, A-674563 was administered orally at doses of 5 mg/kg, 15 mg/kg, and 45 mg/kg once daily. At 45 mg/kg, both genders showed a 12-15% decrease in body weight, a 2.3-fold increase in serum ALT (alanine transaminase) and 1.9-fold increase in AST (aspartate transaminase) compared with controls, and mild hepatocellular vacuolation in histopathological examination. No significant toxicity (no body weight loss, no abnormal liver enzymes, no pathological changes) was observed at 5 mg/kg or 15 mg/kg [2] - In vitro plasma protein binding studies using equilibrium dialysis showed that A-674563 had high binding affinity to plasma proteins: 94% in human plasma, 92% in rat plasma, and 90% in dog plasma. The unbound fraction was < 10% across all species tested [2] - In the PC-3 xenograft model, A-674563 at doses up to 10 mg/kg (i.p. for 21 days) did not cause significant changes in body weight or gross pathological abnormalities in major organs (liver, kidney, heart) [1] |
| References |
[1]. Mol Cancer Ther. 2005 Jun;4(6):977-86. [2]. Mol Biosyst. 2010 Aug;6(8):1389-402. |
| Additional Infomation |
The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.[2] A-674563 is a small-molecule, ATP-competitive inhibitor of the Akt kinase family, developed for the treatment of solid tumors with dysregulated Akt signaling (e.g., prostate cancer, breast cancer, ovarian cancer). Its high selectivity for Akt over other kinases reduces the risk of off-target toxicities, making it a promising candidate for targeted cancer therapy [1] - Preclinical studies showed that A-674563 could enhance the efficacy of chemotherapy drugs (e.g., paclitaxel) in breast cancer models: combination treatment with A-674563 (5 mg/kg i.p.) and paclitaxel (10 mg/kg i.p.) resulted in a 78% reduction in MDA-MB-231 tumor volume, compared with 53% for A-674563 alone and 40% for paclitaxel alone [2] - A-674563 was shown to inhibit the growth of cancer cells with PTEN mutation (a common genetic alteration leading to Akt activation), such as PC-3 prostate cancer cells, indicating its potential to target tumors with specific genetic drivers of Akt hyperactivation [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 5.75 mg/mL (16.04 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 57.5 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 2: ≥ 2.08 mg/mL (5.80 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 3: Saline: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7899 mL | 13.9493 mL | 27.8987 mL | |
| 5 mM | 0.5580 mL | 2.7899 mL | 5.5797 mL | |
| 10 mM | 0.2790 mL | 1.3949 mL | 2.7899 mL |