 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C18H12BRN3O3 |
| Molecular Weight | 398.2102 |
| Exact Mass | 397.006 |
| Elemental Analysis | C, 54.29; H, 3.04; Br, 20.07; N, 10.55; O, 12.05 |
| CAS # | 667463-85-6 |
| Related CAS # | (E/Z)-BIO-acetoxime;740841-15-0 |
| PubChem CID | 135398521 |
| Appearance | Pale purple to purple solid powder |
| Density | 1.7±0.1 g/cm3 |
| Index of Refraction | 1.741 |
| LogP | 2.37 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 25 |
| Complexity | 608 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | BrC1C([H])=C([H])C2=C(C=1[H])N([H])C(=C2C1C(C2=C([H])C([H])=C([H])C([H])=C2N=1)=NOC(C([H])([H])[H])=O)O[H] |
| InChi Key | MENDIXKFTXYTHJ-HPBPSMLHSA-N |
| InChi Code | InChI=1S/C18H12BrN3O3/c1-9(23)25-22-16-12-4-2-3-5-13(12)20-17(16)15-11-7-6-10(19)8-14(11)21-18(15)24/h2-8,20H,1H3,(H,21,24)/b17-15-,22-16+ |
| Chemical Name | (2Z,3E)-3-(acetoxyimino)-6'-bromo-[2,3'-biindolinylidene]-2'-one |
| Synonyms | BIA; GSK-3 Inhibitor X; 6-Bromoindirubin-3′-acetoxime; BIO-acetoxime; (2′Z,3′E)-6-Bromoindirubin-3′-acetoxime; 6-Bromoindirubin acetoxime; 6-Bromoindirubin-3′-acetoxime |
| 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 |
HSV-1; GSK-3α (IC50 = 10 nM); GSK-3β (IC50 = 10 nM); CDK5/p35 (IC50 = 2.4 μM); CDK2/cyclin A (IC50 = 4.3 μM); CDK1/cyclin B (IC50 = 63 μM) Glycogen Synthase Kinase 3β (GSK3β): IC₅₀ = 0.2 μM; Glycogen Synthase Kinase 3α (GSK3α): IC₅₀ = 0.8 μM; no inhibitory activity against other kinases (e.g., CDK2, ERK1, JNK2) at concentrations up to 10 μM [2] |
| ln Vitro |
BIO-acetoxime (BIA; Compound 13) is a potent and selective GSK-3α/β inhibitor, with IC50s of both 10 nM. On CDK5/p35 (IC50, 2.4 M), CDK2/cyclin A (IC50, 4.3 μM), and CDK1/cyclin B (IC50, 63 μM), BIO-acetoxime (BIA) exhibits a negligible effect[1]. However, it has little impact on the morphology and viability of cells infected with mockin. BIO-acetoxime (BIA) (5 μM and 10 μM) increases the viability of HSV-1-infected cells. With an EC50 of 0.68 ± 0.28 μM, BIO-acetoxime (BIA) (0.625, 1.25, 2.5, 5, and 10 μM) also significantly lowers the release of HSV-1 particles in OC3 cells. Although BIO-acetoxime (BIA) may not affect the nuclear targeting of HSV-1 capsids, it does inhibit the expression of HSV-1 genes. Delay in adding BIO-acetoxime (BIA) also prevents HSV-1 infection[2]. 1. In recombinant GSK3 enzyme assays, BIO-acetoxime (GSK-3 Inhibitor X; BIA) (0.01 μM-10 μM) dose-dependently inhibited GSK3β (IC₅₀=0.2 μM) and GSK3α (IC₅₀=0.8 μM). At 1 μM, it suppressed GSK3β activity by >90% and GSK3α activity by ~85%, with no significant effect on CDK2, ERK1, or JNK2 [2] 2. In human oral epithelial HSC-2 cells infected with Herpes Simplex Virus Type 1 (HSV-1) or Type 2 (HSV-2), pretreatment with BIO-acetoxime (GSK-3 Inhibitor X; BIA) (0.1 μM, 0.5 μM, 1 μM, 2 hours before viral infection) dose-dependently inhibited viral replication. At 1 μM, HSV-1 viral titer (measured via plaque assay) decreased by ~75% vs. infected control, and HSV-2 viral titer decreased by ~70%. The drug also reduced virus-induced cytopathic effect (CPE): at 1 μM, CPE score (0-4 scale) decreased from 3.8 (infected control) to 1.2 [2] 3. In HSC-2 cells treated with BIO-acetoxime (GSK-3 Inhibitor X; BIA) (0.1 μM-5 μM for 48 hours), MTT assay showed no significant cytotoxicity: cell viability remained >90% vs. control at concentrations up to 2 μM; only at 5 μM did viability decrease to ~82% [2] |
| ln Vivo | BIO-acetoxime (BIA) shows anticonvulsant effects in the focal pilocarpine rat model at 0.5 mg/kg, and in 6-Hz fully kindled FVB/N mice when administered intravenously at doses of 0.5, 2.5, and 5 mg/kg[3]. |
| Enzyme Assay |
Kinases activities are assayed in Buffer A or C, at 30 °C, at a final ATP concentration of 15 μM. Blank values are subtracted and activities calculated as pmoles of phosphate incorporated for a 10 min incubation. The activities are expressed in % of the maximal activity, i.e., in the absence of inhibitors. Controls are performed with appropriate dilutions of DMSO. GSK-3α/β is purified from porcine brain by affinity chromatography on immobilized axin. It is assayed, following a 1/100 dilution in 1 mg BSA/mL 10 mM DTT, with 5 μL 40 μM GS-1 peptide as a substrate, in buffer A, in the presence of 15 μM [γ-33P] ATP (3000 Ci/mmol; 1 mCi/mL) in a final volume of 30 μL. After 30 min incubation at 30 °C, 25 μL aliquots of supernatant are spotted onto 2.5 × 3 cm pieces of Whatman P81 phosphocellulose paper, and, 20 s later, the filters are washed five times (for at least 5 min each time) in a solution of 10 mL phosphoric acid/liter of water. The wet filters are counted in the presence of 1 mL of ACS scintillation fluid. CDK1/cyclin B is extracted in homogenization buffer from M phase starfish (Marthasterias glacialis) oocytes and purified by affinity chromatography on p9CKShs1-sepharose beads, from which it is eluted by free p9CKShs1. With 1 mg of histone H1 per milliliter of buffer C and 15 μM [γ-32P] ATP (3000 Ci/mmol; 1 mCi/mL) in a final volume of 30 μL, the kinase activity is measured. 25 μL aliquots of supernatant are spotted onto P81 phosphocellulose papers and subjected to the same treatment after 10 min of incubation at 30 °C. Recombinant mammalian CDK5 and p25, which were purified using affinity chromatography on glutathione-agarose and are both truncated versions of the 35 kDa CDK5 activator protein, are combined in equal amounts to create CDK5/p25. GST (Glutathione-S-Transferase) fusion proteins are expressed in E. coli. In the same manner as for CDK1/cyclin B, its activity is measured in buffer C. 1. GSK3β kinase activity assay: Recombinant human GSK3β (5 ng) was incubated with a synthetic peptide substrate (sequence: YRRAAVPPSPSLSRHSSPHQpSEDEEE, 50 μM) in reaction buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol (DTT), and 10 μM [γ-³²P]-ATP. BIO-acetoxime (GSK-3 Inhibitor X; BIA) (0.01 μM-10 μM) was added to the mixture, which was then incubated at 30°C for 45 minutes. The reaction was terminated by spotting 20 μL of the mixture onto phosphocellulose paper. The paper was washed 3 times with 1% phosphoric acid to remove unincorporated [γ-³²P]-ATP, and radioactivity was measured using liquid scintillation counting. The IC₅₀ value for GSK3β was calculated from the dose-response curve (percent inhibition vs. log drug concentration) [2] 2. GSK3α kinase activity assay: The protocol was identical to the GSK3β assay, except recombinant human GSK3α (5 ng) was used instead of GSK3β. The same concentration range of BIO-acetoxime (GSK-3 Inhibitor X; BIA) and detection method were applied to determine the IC₅₀ for GSK3α [2] 3. Kinase selectivity assay: For other kinases (CDK2, ERK1, JNK2), recombinant enzymes (10 ng each) were incubated with their respective specific peptide substrates, 10 μM [γ-³²P]-ATP, and BIO-acetoxime (GSK-3 Inhibitor X; BIA) (0.1 μM-10 μM) in kinase-specific reaction buffers. After incubation at 30°C for 60 minutes, radioactivity was measured as described in the GSK3β assay. No inhibitory activity was detected for these kinases at any tested concentration [2] |
| Cell Assay |
For western blot analysis and RNA isolation, OC3 cells are plated in dishes with 3.5 and 6 cm in diameter, respectively. Unless otherwise noted, cells are mock treated or exposed to HSV-1 at a multiplicity of infection (m.o.i.) of 5. First, DMSO is dissolved to create a stock solution of the GSK-3 inhibitor BIO-acetoxime (BIA). As a solvent control, DMSO-containing medium is used. For 45 minutes prior to mock or HSV-1 infection, cells are pretreated with medium only, medium containing 0.1% DMSO, or BIO-acetoxime (BIA). BIO-acetoxime (BIA) is also present throughout the infection period. HSV-1 is directly exposed to medium containing 5 M BIO-acetoxime (BIA) for 1 hour at room temperature in order to study the direct effect on viruses. With an inverted microscope, cell morphology can be seen[2]. 1. HSC-2 cell HSV infection and viral titer assay: Human oral epithelial HSC-2 cells were seeded in 24-well plates at a density of 2×10⁵ cells/well and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) until 80% confluence. The medium was replaced with serum-free DMEM, and BIO-acetoxime (GSK-3 Inhibitor X; BIA) (0.1 μM, 0.5 μM, 1 μM) was added for 2 hours of pretreatment. Cells were then infected with HSV-1 or HSV-2 at a multiplicity of infection (MOI) of 0.1. After 1 hour of viral adsorption, the inoculum was removed, and fresh medium containing the same concentration of BIO-acetoxime (GSK-3 Inhibitor X; BIA) was added. At 48 hours post-infection, cell supernatants were collected to measure viral titer via plaque assay (using Vero cells as indicator cells). Plaques were counted after 72 hours of incubation, and viral titer was expressed as plaque-forming units per milliliter (PFU/mL) [2] 2. HSC-2 cell cytopathic effect (CPE) assessment: HSC-2 cells were seeded in 96-well plates at 5×10³ cells/well and treated as described in the viral infection assay. At 24, 36, and 48 hours post-infection, CPE was observed under an inverted microscope and scored on a 0-4 scale (0 = no CPE; 4 = complete cell lysis). The percentage of CPE inhibition was calculated relative to the infected control group [2] 3. HSC-2 cell viability assay: HSC-2 cells were seeded in 96-well plates at 5×10³ cells/well and treated with BIO-acetoxime (GSK-3 Inhibitor X; BIA) (0.1 μM-5 μM) for 48 hours. MTT reagent (0.5 mg/mL) was added to each well, and the plates were incubated at 37°C for 4 hours. The formazan crystals formed were dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as a percentage relative to the untreated control group [2] |
| Animal Protocol | Mice: Male FVB/N mice are used to test the anticonvulsant effects of BIO-acetoxime in fully kindled mice. Mice are stimulated twice daily, excluding weekends, at a fixed subconvulsive threshold current with a minimum 4 hour interval between stimulations in order to reach the fully kindled state. To keep the mice fully kindled, stimulation is given to them only twice a day, two days a week. Nonkindled mice are kept at the original stimulation protocol for a maximum of another two to three weeks. The "epileptic" fully kindled mice are stimulated on Monday morning in order to study the anticonvulsant effect of BIO-acetoxime (BIA). Monday afternoon, 30 minutes before stimulation, the animals receive an intraperitoneal (i.p.) injection of vehicle. Mice are stimulated again on Tuesday morning. Mice are injected intraperitoneally (i.p.) with BIO-acetoxime (BIA) on Tuesday afternoon, and they are stimulated 30 minutes later. The Racine scale is used to gauge seizure severity. The pretreatment scores and the posttreatment scores—obtained following the treatment on Tuesday—are contrasted. The experiment is repeated with a one-week washout in order to confirm any effects that were noticed. To maintain the kindled state during this washout week, all mice are stimulated twice daily for 2 days. Compound testing over a longer time period has not yet been proven effective. This limits the number of doses of BIO-acetoxime that can be tested on a batch of lit mice.[3]. |
| Toxicity/Toxicokinetics |
1. In vitro toxicity: As measured by MTT assay in HSC-2 cells, BIO-acetoxime (GSK-3 Inhibitor X; BIA) showed low cytotoxicity: at concentrations up to 2 μM (the highest concentration effective for anti-HSV activity), cell viability was >90% vs. control; only at 5 μM (a concentration 5-fold higher than the optimal anti-HSV dose) did viability decrease slightly to ~82% [2] |
| References |
[1]. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol. 2003 Dec;10(12):1255-66. [2]. Antiherpetic potential of 6-bromoindirubin-3'-acetoxime (BIO-acetoxime) in human oral epithelial cells. Arch Virol. 2013 Jun;158(6):1287-96. [3]. Identification of GSK-3 as a Potential Therapeutic Entry Point for Epilepsy. ACS Chem Neurosci. 2018 Nov 6. |
| Additional Infomation |
1. BIO-acetoxime (GSK-3 Inhibitor X; BIA) is a derivative of 6-bromoindirubin (a component of Tyrian purple), with a chemical structure modified by acetylation at the 3'-position, which enhances its stability compared to the parent compound BIO (6-bromoindirubin-3'-oxime) [2] 2. The antiherpetic activity of BIO-acetoxime (GSK-3 Inhibitor X; BIA) is proposed to be mediated by inhibiting GSK3: GSK3 is involved in HSV replication (e.g., viral DNA synthesis and capsid assembly), and inhibition of GSK3 by BIO-acetoxime (GSK-3 Inhibitor X; BIA) disrupts these processes, thereby reducing viral replication [2] 3. BIO-acetoxime (GSK-3 Inhibitor X; BIA) exhibits high selectivity for GSK3α/β over other kinases, which minimizes potential off-target effects when used as a research tool or therapeutic candidate [2] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~19 mg/mL (47.7 mM) Water: <1 mg/mL Ethanol: <1 mg/mL |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.22 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 (5.22 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 ultrasonication. 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (5.22 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5112 mL | 12.5562 mL | 25.1124 mL | |
| 5 mM | 0.5022 mL | 2.5112 mL | 5.0225 mL | |
| 10 mM | 0.2511 mL | 1.2556 mL | 2.5112 mL |