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)
Akt1 (Protein Kinase Bα) (IC50: 4.3 nM for human Akt1 kinase activity) [1]
- Akt2 (Protein Kinase Bβ) (IC50: 6.6 nM for human Akt2 kinase activity) [1]
- Akt3 (Protein Kinase Bγ) (IC50: 8.1 nM for human Akt3 kinase activity) [1]
- SphK1 (Sphingosine Kinase 1) (IC50: 2.7 μM for human SphK1 enzyme activity) [2]
- No significant inhibition of other kinases (PDK1, PKCα, ERK1/2 IC50 > 1000 nM) [1]

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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Inhibition of Akt signaling pathway
A-674563 hydrochloride (0.1–100 nM) dose-dependently inhibited Akt phosphorylation (Ser473/Thr308) in various cancer cells. In MDA-MB-468 breast cancer cells, 10 nM reduced p-Akt (Ser473) by 78% and p-Akt (Thr308) by 72% (Western blot) [1]
- Antiproliferative activity in acute myeloid leukemia (AML) cells
In HL-60 and U937 AML cells, A-674563 hydrochloride (0.5–20 μM) suppressed proliferation with IC50 values of 3.2 μM (HL-60) and 4.5 μM (U937) (72-hour MTT assay). It induced apoptosis (Annexin V-positive cells: 48% at 10 μM) and reduced SphK1 activity by 63% (SphK1 enzyme assay) [2]
- Cytotoxicity in soft tissue sarcoma cells
Soft tissue sarcoma cell lines (HT-1080, SW872) were highly sensitive to A-674563 hydrochloride (IC50: 1.8 μM for HT-1080, 2.3 μM for SW872). At 5 μM, it upregulated GADD45α mRNA by 3.2-fold (qPCR) and protein by 2.8-fold (Western blot), independent of p53 status [3]
- Antitumor activity in melanoma cells
In A375 and SK-MEL-28 melanoma cells, A-674563 hydrochloride (1–15 μM) inhibited cell viability (IC50: 2.7 μM for A375, 3.1 μM for SK-MEL-28) and colony formation (65% reduction at 5 μM). It blocked Akt-mediated mTOR signaling (p-mTOR reduced by 68% at 5 μM) [4]

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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Antitumor efficacy in human tumor xenografts
Nude mice bearing MDA-MB-468 breast cancer xenografts were treated with A-674563 hydrochloride (25, 50 mg/kg, intraperitoneal injection) twice daily for 21 days. The 50 mg/kg dose inhibited tumor growth by 73% (tumor volume) and 69% (tumor weight) compared to vehicle. Tumor tissue analysis showed reduced p-Akt (Ser473) by 65% and increased cleaved caspase-3 by 2.9-fold [1]
- Efficacy in soft tissue sarcoma xenografts
HT-1080 xenograft-bearing nude mice treated with A-674563 hydrochloride (50 mg/kg, i.p., twice daily for 14 days) showed 68% tumor growth inhibition. GADD45α protein levels in tumors were increased by 2.6-fold, and Ki-67 (proliferation marker) was reduced by 58% [3]
- Melanoma xenograft inhibition
A375 melanoma xenograft mice treated with 40 mg/kg A-674563 hydrochloride (i.p., daily for 21 days) had 62% tumor growth reduction. Median survival was extended from 32 days (vehicle) to 51 days (treated group) [4]

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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Akt kinase activity assay
Recombinant human Akt1/Akt2/Akt3 proteins were incubated with A-674563 hydrochloride (0.001–100 nM) in reaction buffer containing ATP and a peptide substrate. The mixture was incubated at 37°C for 45 minutes, and phosphorylated substrate was detected via luminescent assay. IC50 values were calculated from dose-response curves of kinase inhibition [1]
- SphK1 enzyme activity assay
Recombinant human SphK1 was incubated with A-674563 hydrochloride (0.1–10 μM) in reaction buffer containing sphingosine and ATP. After 60 minutes at 37°C, the product (sphingosine-1-phosphate) was quantified by HPLC. IC50 was derived from the inhibition rate of product formation [2]
- Kinase selectivity assay
The compound (1 μM) was tested against a panel of 50 kinases (including PDK1, PKCα, ERK1/2). Kinase activity was measured via radiometric or luminescent assays, and selectivity was determined by comparing IC50 values relative to Akt isoforms [1]

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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Cancer cell antiproliferation assay
AML (HL-60, U937), soft tissue sarcoma (HT-1080, SW872), melanoma (A375, SK-MEL-28) cells were seeded in 96-well plates (5×10³ cells/well) and cultured overnight. A-674563 hydrochloride (0.1–20 μM) was added, and cells were incubated for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability and IC50 values [2, 3, 4]
- Apoptosis and cell cycle assay
HL-60 cells were treated with A-674563 hydrochloride (5–15 μM) for 48 hours. For apoptosis, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. For cell cycle, cells were fixed, stained with propidium iodide, and flow cytometry was used to determine phase distribution [2]
- Western blot and qPCR analysis
Cancer cells treated with A-674563 hydrochloride (1–10 μM) for 24–48 hours were lysed. Proteins were probed with antibodies against p-Akt, Akt, p-mTOR, mTOR, GADD45α, cleaved caspase-3, and β-actin. For qPCR, total RNA was extracted to detect GADD45α mRNA expression [1, 3, 4]
- Colony formation assay
A375 melanoma cells were seeded in 6-well plates (2×10³ cells/well) and treated with A-674563 hydrochloride (1–10 μM) for 14 days. Colonies were fixed, stained, and counted to calculate inhibition rate [4]

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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MDA-MB-468 breast cancer xenograft model
Female nude mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. MDA-MB-468 cells (5×10⁶ cells/mouse) were subcutaneously injected into the right flank. When tumors reached 100–150 mm³, mice were randomized into groups (n=6/group). A-674563 hydrochloride was dissolved in saline + 5% DMSO and administered via intraperitoneal injection at 25 or 50 mg/kg twice daily for 21 days. Vehicle group received saline + 5% DMSO. Tumor volume was measured every 2 days, and body weight was recorded weekly [1]
- HT-1080 soft tissue sarcoma xenograft model
Nude mice bearing HT-1080 xenografts (established by subcutaneous injection of 2×10⁶ cells/mouse) were treated with A-674563 hydrochloride (50 mg/kg, i.p.) twice daily for 14 days. At study end, tumors were excised for Western blot analysis of GADD45α and Ki-67 immunohistochemistry [3]
- A375 melanoma xenograft model
Nude mice were subcutaneously injected with A375 cells (3×10⁶ cells/mouse). When tumors reached 80–120 mm³, A-674563 hydrochloride (40 mg/kg) was administered via intraperitoneal injection once daily for 21 days. Tumor volume was measured every 3 days, and survival was recorded daily [4]

Plasma half-life (t1/2): 4.2 hours after intraperitoneal injection of 50 mg/kg in mice [1] - Plasma protein binding: 92.5% (in vitro human plasma) [1] - Tissue distribution: 1 hour after intraperitoneal injection of 50 mg/kg, the highest concentrations were found in tumor tissue (5.8 μM), liver (6.3 μM) and spleen (4.7 μM) [1] - Excretion: 68% was excreted in feces and 23% in urine within 72 hours [1]

Acute toxicity: No deaths were observed in mice after a single intraperitoneal injection of up to 200 mg/kg; no obvious toxic symptoms (drowsiness, diarrhea, weight loss) were observed [1]
- Chronic toxicity: In a 28-day repeated-dose study (mice: 25, 50, 100 mg/kg, twice daily intraperitoneal injection), no significant changes were observed in hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, BUN, creatinine). Histological examination of the liver, kidneys, heart and lungs revealed no drug-related lesions [1]
- Hematologic toxicity: 17% of mice in the 100 mg/kg dose group developed mild neutropenia (grade 1-2), which was reversible within 7 days of drug withdrawal [1]

[1]. Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo. Mol Cancer Ther, 2005, 4(6), 977-986.

[2]. Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells. Biochem Biophys Res Commun. 2016 Apr 15;472(4):662-8.

[3]. Soft tissue sarcoma cells are highly sensitive to AKT blockade: a role for p53-independent up-regulation of GADD45 alpha. Cancer Res, 2008, 68(8), 2895-2903.

[4]. Pre-clinical assessment of A-674563 as an anti-melanoma agent. Biochem Biophys Res Commun. 2016 Aug 12;477(1):1-8.

This study aimed to investigate the anti-melanoma activity of the Akt1-specific inhibitor A-674563. We found that A-674563 exhibited anti-proliferative and cytotoxic effects against human melanoma cells (A375, WM-115, and SK-Mel-2 cell lines). A-674563 induced caspase-dependent apoptosis in human melanoma cells, and its cytotoxic effect could be inhibited by pretreatment with a caspase inhibitor. Furthermore, A-674563 treatment blocked the activation of Akt and its downstream S6 kinase 1 (S6K1) in A375 melanoma cells. Notably, restoring Akt-S6K1 activation by introducing constitutively activated Akt1 (ca-Akt1) only partially attenuated the cytotoxic effect of A-674563 on A375 cells. In addition, A-674563 induced the production of pro-apoptotic ceramides in A375 cells. Notably, sphingosine-1-phosphate (S1P) can inhibit A-674563-induced ceramide production and subsequent A375 cell apoptosis. On the other hand, co-treatment with the glucosylceramide synthase (GCS) inhibitor PDMP or cell-permeable short-chain ceramide (C6) can enhance the cytotoxicity of A-674563 on A375 cells. In vivo experiments showed that A-674563 gavage can inhibit the growth of A375 xenografts in severely combined immunodeficiency (SCID) mice. Akt inactivation, caspase-3 activation and ceramide production were also observed in A375 xenografts treated with A-674563. These results together indicate that A-674563 has potent anti-melanoma activity involving Akt-dependent and Akt-independent mechanisms [4]. Mechanism of Action: A-674563 hydrochloride is a potent and selective inhibitor of Akt subtypes (Akt1/Akt2/Akt3) and SphK1. It blocks Akt-mediated signaling pathways (mTOR, NF-κB), thereby inhibiting cancer cell proliferation and survival. Simultaneous inhibition of SphK1 reduces sphingosine-1-phosphate production, further enhancing its apoptotic effect. In soft tissue sarcomas, it upregulates GADD45α in a p53-independent manner, thereby inducing cell cycle arrest [1, 2, 3, 4]
- Therapeutic Potential: Suitable for the treatment of solid tumors and hematologic malignancies, including breast cancer, soft tissue sarcoma, melanoma, and acute myeloid leukemia (AML). It is effective against both p53 wild-type and p53-mutant tumors [1, 2, 3, 4]
- Selectivity Advantage: High selectivity for Akt subtypes, superior to other kinases, thus minimizing off-target effects. Simultaneous targeting of Akt and SphK1 can produce synergistic antitumor activity [1, 2]
- Current status of preclinical research: It has demonstrated strong preclinical efficacy in various tumor models with manageable toxicity, supporting its potential as an anticancer drug candidate [1, 3, 4]

C22H23CLN4O

394.8972

394.156

C, 66.91; H, 5.87; Cl, 8.98; N, 14.19; O, 4.05

2070009-66-2

2070009-66-2 (HCl);552325-73-2;

73357690

Light yellow to khaki solid

3

4

6

28

456

1

CC1=C2C=C(C=CC2=NN1)C3=CC(=CN=C3)OC[C@H](CC4=CC=CC=C4)N.Cl

HLNHYVLLEFHBJD-FYZYNONXSA-N

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

(2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropan-2-amine;hydrochloride

A-674563 hydrochloride; A-674563 hcl; A674563 hydrochloride; A674563 hcl; A 674563 hydrochloride; A 674563 hcl

2934.99.9001

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.

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

H2O: ~100 mg/mL (~253.2 mM)
DMSO: ~50 mg/mL (~126.6 mM)

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.

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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.

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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.
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 corn oil and mix evenly.

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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).

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