| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| Other Sizes |
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Purity: ≥98%
| Targets |
Akt1 (Ki = 11 nM); PKA (Ki = 16 nM); CDK2 (Ki = 46 nM); GSK3β (Ki = 110 nM); ERK2 (Ki = 260 nM); PKCδ (Ki = 360 nM); RSK2 (Ki = 580 nM); MAPK-AP2 (Ki = 1.1 μM nM); PKCγ (Ki = 1.2 μM); Chk1 (Ki = 2.6 μM); CK2 (Ki = 5.4 μM); SRC (Ki = 13 μM)
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| ln Vitro |
A-674563 slows proliferation of tumor cells with an EC50 of 0.4 μM[1]. A563 (0-10 µM) significantly decreases GSK3 and MDM2 phosphorylation in STS cells. All STS cell lines are inhibited by A563, with IC50 values ranging from 0.22 0.034 M (SW684) to 0.35 ±0.06 µM (SKLMS1) at 48 hours. In STS cells, A563 causes apoptosis and a G2 cell cycle arrest. Independent of p53, A563 (1 µM/12 hr) increases the expression of GADD45A[2]. In cultured human melanoma cells, A-674563 (10–1000 nM) is cytotoxic and anti-proliferative. It also induces melanoma cell apoptosis, which is inhibited by caspase inhibitors, and it inhibits melanoma cells via Akt-dependent and Akt-independent mechanisms[3]. A-674563 is cytotoxic and anti-proliferative when added to U937 and AmL progenitor cells, activates caspase-3/9 and apoptosis in U937 and AmL progenitor cells, and manipulates other signalings in AmL cells whiling blocking Akt[4].
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| ln Vivo |
A-674563 (40 mg/kg/d, p.o.) exhibits negligible monotherapy activity, but A-674563 plus paclitaxel significantly increases the effectiveness of treatment in the PC-3 prostate cancer xenograft model. In an oral glucose tolerance test, A-674563 (20, 100 mg/kg) increases plasma insulin levels[1]. A563 (20 mg/kg/bid; p.o.) causes mice to lose only a small amount of weight while showing slow tumor growth and a significant difference in tumor volume. Tumors that have received treatment with A563 express higher levels of GADD45 and lower levels of PCNA (a nuclear marker for proliferation). Additionally, the apoptosis marker TUNEL assay staining levels rise in the A563-treated specimens[2]. A-674563 (25, 100 mg/kg, lavage daily) significantly reduces the growth of the A375 xenograft in mice[3]. A-674563 (15, 40 mg/kg) injection increases mouse survival while inhibiting U937 xenograft in vivo growth[4].
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| Enzyme Assay |
Recombinant CK2, PKCγ, and PKCδ; PKA; cyclin-dependent kinase 2/CyclinA, GSK3β, MAPK-AP2, Src, and RSK2; extracellular signal–regulated kinase 2, cKit were commercially obtained. His-tagged Akt1 (S378A, S381A, T450D, S473D; 139-480), Chk1 (1-269), KDR (789-1354), Flt-1 (786-1338), and phosphatidylinositide-dependent kinase 1 (1-396), were expressed using the FastBac bacculovirus expression system and purified using either nickel (his-tag) or glutathione S-transferase affinity chromatography. Peptides substrates had the general structure biotin-Ahx-peptide with the following sequences: Akt, EELSPFRGRSRSAPPNLWAAQR; PKA, LRRASLG; PKCγ and PKCδ, ERMRPRKRQGSVRRRV; CK2, RRADDSDDDDD; cyclin-dependent kinase 2, LPPCSPPKQGKKENGPPHSHTLKGRRAAFDNQL; GSK3β, YRRAAVPPSPSLSRHSSPHQS(p)EDEEE; MAPK-AP2, KKLNRTLSVA; RSK2, KKKNRTLSVA; extracellular signal–regulated kinase 2, KRELVEPLTPSGEAPNQALLR; Chk1, AKVSRSGLYRSPSMPENLNRPR; phosphatidylinositide-dependent kinase 1, KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC; KDR, Flt-1, and cKit, AEEEYFFLFA-amide. For Src assays, the biotinylated substrate PTK-2 was used. Inhibition of kinase activity was assessed using a radioactive FlashPlate-based assay platform as previously described[1].
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| 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.
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| 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.
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| References |
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| Additional Infomation |
The present study aims to investigate the anti-melanoma activity by an Akt1 specific inhibitor A-674563. We showed that A-674563 was anti-proliferative and cytotoxic when added to human melanoma cells (A375, WM-115 and SK-Mel-2 lines). A-674563 induced caspase-dependent apoptotic death of human melanoma cells, and its cytotoxicity was inhibited with pre-treatment of caspase inhibitors. Further, A-674563 treatment blocked Akt and its downstream S6 Kinase 1 (S6K1) activation in A375 melanoma cells. Significantly, restoring Akt-S6K1 activation via introduction of constitutively-active Akt1 (ca-Akt1) only partially attenuated A-674563's cytotoxicity against A375 cells. Further, A-674563 induced pro-apoptotic ceramide production in A375 cells. Significantly, sphingosine-1-phosphate (S1P) inhibited A-674563-induced ceramide production and subsequent A375 cell apoptosis. On the other hand, co-treatment with the glucosylceramide synthase (GCS) inhibitor PDMP or the cell permeable short-chain ceramide (C6) potentiated A-674563's cytotoxicity against A375 cells. In vivo, A-674563 oral gavage inhibited A375 xenograft growth in severe combined immunodeficiency (scid) mice. Akt inactivation, caspase-3 activation and ceramide production were also observed in A-674563-treated A375 xenografts. Together, these results suggest that A-674563 exerts potent anti-melanoma activity, involving Akt-dependent and Akt-independent mechanisms[4].
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| Molecular Formula |
C22H23CLN4O
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|---|---|
| Molecular Weight |
394.8972
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| Exact Mass |
394.156
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| Elemental Analysis |
C, 66.91; H, 5.87; Cl, 8.98; N, 14.19; O, 4.05
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| CAS # |
2070009-66-2
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| Related CAS # |
2070009-66-2 (HCl);552325-73-2;
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| PubChem CID |
73357690
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| Appearance |
Light yellow to khaki solid
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
28
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| Complexity |
456
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC1=C2C=C(C=CC2=NN1)C3=CC(=CN=C3)OC[C@H](CC4=CC=CC=C4)N.Cl
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| InChi Key |
HLNHYVLLEFHBJD-FYZYNONXSA-N
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| InChi Code |
InChI=1S/C22H22N4O.ClH/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);1H/t19-;/m0./s1
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| Chemical Name |
(2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropan-2-amine;hydrochloride
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| Synonyms |
A-674563 hydrochloride; A-674563 hcl; A674563 hydrochloride; A674563 hcl; A 674563 hydrochloride; A 674563 hcl
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
H2O: ~100 mg/mL (~253.2 mM)
DMSO: ~50 mg/mL (~126.6 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.27 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 (5.27 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 (5.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 100 mg/mL (253.23 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C). |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5323 mL | 12.6614 mL | 25.3229 mL | |
| 5 mM | 0.5065 mL | 2.5323 mL | 5.0646 mL | |
| 10 mM | 0.2532 mL | 1.2661 mL | 2.5323 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.
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