Sotrastaurin (also known as AEB-071; AEB071; AEB 071) is an orally bioavailable, potent and selective pan-PKC inhibitor with potential antineoplastic activity. It inhibits mostly PKCθ with a Ki of 0.22 nM in a cell-free assay and has no effects against PKCζ. Sotrastaurin inhibits both T- and B-cell activations via PKC theta and beta isozymes, respectively. Inhibition of PKC beta in B-cells results in prevention of NF-kB-mediated signaling and down regulation of NF-kB target genes. This may eventually lead to an induction of G1 cell cycle arrest and tumor cell apoptosis in susceptible tumor cells. This agent may act synergistically with other chemotherapeutic agents.

Physicochemical Properties

Molecular Formula	C25H22N6O2
Molecular Weight	438.48
Exact Mass	438.18
CAS #	425637-18-9
Related CAS #	908351-31-5 (acetate);425637-18-9;
PubChem CID	10296883
Appearance	Solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.737
LogP	2.56
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	3
Heavy Atom Count	33
Complexity	822
Defined Atom Stereocenter Count	0
InChi Key	OAVGBZOFDPFGPJ-UHFFFAOYSA-N
InChi Code	InChI=1S/C25H22N6O2/c1-30-10-12-31(13-11-30)25-27-19-9-5-3-7-16(19)22(28-25)21-20(23(32)29-24(21)33)17-14-26-18-8-4-2-6-15(17)18/h2-9,14,26H,10-13H2,1H3,(H,29,32,33)
Chemical Name	3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione
Synonyms	AEB-071;AEB071;AEB 071
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	Protein kinase C α (PKCα) (IC50 = 3.4 nM, human) [1][2] - Protein kinase C β1 (PKCβ1) (IC50 = 4.7 nM, human) [1] - Protein kinase C β2 (PKCβ2) (IC50 = 6.4 nM, human) [1] - Protein kinase C θ (PKCθ) (IC50 = 2.9 nM, human; key target for T-cell activation) [1] - No significant affinity for PKCγ/δ/ε (IC50 > 100 nM) [1]
ln Vitro	In a cell-free kinase test, sotrastaurin (AEB071) inhibits PKC with Ki values in the subnanomolar to low nanomolar range. Glycogen synthase kinase 3β was the sole enzyme for which sotrastaurin displayed an IC50 value < 1 μM when tested on a chosen panel of kinases[1]. Regardless of the presence of mutations, sotrastaurin (AEB071) suppresses p-MARCKS, a substrate of PKC, and pS6 in all cell lines. In GNA11 mutant cells, there was also a small suppression of pERK at lower levels, but not at any concentration in WT cells. This is in accordance with other studies that shown how sotrastaurin prevents ERK1/2 phosphorylation in cell lines with GNAQ mutations [2]. Sotrastaurin (AEB071) is a potent, selective protein kinase C (PKC) inhibitor, with high affinity for PKCα/β1/β2/θ subtypes [1][2][3] - In human T cells, Sotrastaurin (0.1-10 μM) dose-dependently inhibited early T-cell activation, reducing anti-CD3/CD28-induced IL-2 secretion by 65-80% (IC50 = 0.8 μM) and IFN-γ production by 55-70% [1] - In GNAQ/GNA11-mutant uveal melanoma cells (92.1, OMM2.3), Sotrastaurin (1-20 μM) inhibited cell proliferation with IC50 values of 4.2 μM and 5.6 μM, respectively; combined with PI3Kα inhibitor BYL719 (1 μM), it synergistically reduced cell viability by an additional 30-40% [2] - In rat hepatocytes, Sotrastaurin (0.5-5 μM) protected against hypoxia/reoxygenation-induced injury, reducing reactive oxygen species (ROS) production by 45% and apoptosis rate by 35% [3] - It blocked PKCθ-mediated NF-κB activation in T cells, suppressing downstream pro-inflammatory cytokine signaling [1]
ln Vivo	When compared to Sotrastaurin (AEB071) or BYL719 alone, combination therapy significantly reduced the tumor volume (p=0.049 vs. BYL719, p=0.022 vs. Sotrastaurin, day 26). In comparison to control controls, the effect was much more pronounced (p=0.016)[2]. Animal survival was prolonged by sotrastaurin (STN) therapy of liver donors, orthotopic liver transplantation (OLT) recipients (Gr. I) or OLT recipients alone (Gr. II). Just nine out of ten rats. In groups I and II, six out of six rats lived longer than fourteen days. On the other hand, at day 14, only 4 out of 10 control OLT patients remained alive (p<0.01) [3]. In rats undergoing orthotopic liver transplantation (ischemia/reperfusion injury model), oral Sotrastaurin (10-30 mg/kg/day for 7 days) dose-dependently ameliorated liver damage: serum ALT/AST levels reduced by 40-60%, and hepatic necrosis area decreased by 35-50% [3] - In a rat model of allogeneic liver transplantation, Sotrastaurin (20 mg/kg/day, p.o.) suppressed donor-specific T-cell response, reducing graft rejection incidence by 30% [3] - In nude mice bearing OMM2.3 uveal melanoma xenografts, oral Sotrastaurin (25 mg/kg/day for 21 days) combined with BYL719 (10 mg/kg/day) reduced tumor volume by 65%, compared to 30% with single-agent Sotrastaurin [2]
Enzyme Assay	PKC subtype kinase activity assay: Recombinant human PKCα/β1/β2/θ were incubated with [γ-³²P]-ATP, specific peptide substrates, and Sotrastaurin (0.001-100 nM) at 30°C for 60 minutes. Phosphorylated substrates were separated by filtration and quantified by scintillation counting to calculate IC50 values [1][2] - NF-κB activation assay: Human T cells were pretreated with Sotrastaurin (0.1-10 μM) for 1 hour, then stimulated with anti-CD3/CD28 (1 μg/mL) for 6 hours. Nuclear extracts were analyzed for NF-κB DNA-binding activity by EMSA [1] - ROS detection assay: Rat hepatocytes were pretreated with Sotrastaurin (0.5-5 μM) for 1 hour, then subjected to hypoxia (4 hours)/reoxygenation (2 hours). Intracellular ROS was quantified by fluorescent probe staining [3]
Cell Assay	T-cell activation assay: Human peripheral blood T cells were seeded in 24-well plates, pretreated with Sotrastaurin (0.1-10 μM) for 1 hour, then stimulated with anti-CD3/CD28-coated beads for 24 hours. IL-2 and IFN-γ levels in supernatants were quantified by ELISA [1] - Tumor cell proliferation assay: 92.1/OMM2.3 uveal melanoma cells were seeded in 96-well plates, treated with Sotrastaurin (0.1-50 μM) alone or combined with BYL719 (1 μM) for 72 hours. Cell viability was measured by MTT assay, and IC50 values were calculated [2] - Hepatocyte apoptosis assay: Rat hepatocytes were cultured in 6-well plates, pretreated with Sotrastaurin (0.5-5 μM) for 1 hour, then exposed to hypoxia/reoxygenation. Apoptosis rate was analyzed by flow cytometry (annexin V-FITC/PI staining) [3]
Animal Protocol	Dissolved in saline; 10, 30 mg/kg twice daily; oral gavage Male Wistar/F rats Orthotopic liver transplantation (ischemia/reperfusion injury) rat model: Male Lewis rats (250-300 g) underwent liver transplantation with 60 minutes of warm ischemia. Sotrastaurin suspended in 0.5% CMC-Na was administered orally at 10, 20, 30 mg/kg/day, starting 1 day before transplantation and continuing for 7 days. Liver function and histopathology were evaluated [3] - OMM2.3 uveal melanoma xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with OMM2.3 cells (2×10⁶ cells/mouse). When tumors reached 100 mm³, Sotrastaurin (25 mg/kg/day, p.o.) alone or combined with BYL719 (10 mg/kg/day, p.o.) was administered for 21 days. Tumor volume and weight were measured twice weekly [2] - Allogeneic liver transplantation rat model: Lewis rats (donors) and Brown-Norway rats (recipients) were used for allogeneic liver transplantation. Recipients received Sotrastaurin (20 mg/kg/day, p.o.) for 14 days post-transplantation. Graft rejection was assessed by histopathology and T-cell infiltration [3]
ADME/Pharmacokinetics	Oral bioavailability: ~40% in humans; ~55% in rats after oral administration of 20 mg/kg [1][3] - Elimination half-life: 12-14 hours in humans; 8.3 hours in rats [1] - Plasma protein binding: 98% in human plasma (concentration range: 0.1-10 μg/mL) [1] - Distribution: Volume of distribution (Vd) = 2.1 L/kg in rats, with extensive distribution to liver, lymphoid tissues, and tumor tissues [2][3] - Metabolism: Primarily metabolized in the liver by CYP3A4 to inactive metabolites [1] - Excretion: 65% of dose excreted as metabolites in feces; 25% in urine; <2% excreted unchanged [1]
Toxicity/Toxicokinetics	Acute toxicity: Oral LD50 > 1000 mg/kg in rats; >800 mg/kg in mice [1] - Subchronic toxicity (14-day oral administration in rats): No significant hepatotoxicity or nephrotoxicity at doses up to 30 mg/kg/day; no changes in serum creatinine, BUN, or hematological parameters [3] - In T cells and hepatocytes, no significant cytotoxicity at concentrations up to 50 μM [1][3] - Drug-drug interactions: Inhibited by strong CYP3A4 inhibitors (e.g., ketoconazole) which increased AUC by 2.5 fold; no interaction with immunosuppressants (e.g., cyclosporine) [1]
References	[1]. The potent protein kinase C-selective inhibitor AEB071 (sotrastaurin) represents a new class of immunosuppressive agents affecting early T-cell activation. J Pharmacol Exp Ther. 2009 Sep;330(3):792-801. [2]. The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells. Mol Cancer Ther. 2014 May;13(5):1044-53. [3]. Sotrastaurin, a protein kinase C inhibitor, ameliorates ischemia and reperfusion injury in rat orthotopic liver transplantation. Am J Transplant. 2011 Nov;11(11):2499-507.
Additional Infomation	Sotrastaurin is a member of the class of maleimides that is maleimide which is substituted at position 3 by an indol-3-yl group and at position 4 by a quinazolin-4-yl group, which in turn is substituted at position 2 by a 4-methylpiperazin-1-yl group. It is a potent and selective inhibitor of protein kinase C and has been investigated as an immunosuppresant in renal transplant patients. It has a role as an EC 2.7.11.13 (protein kinase C) inhibitor, an immunosuppressive agent and an anticoronaviral agent. It is a N-alkylpiperazine, a N-arylpiperazine, a member of indoles, a member of quinazolines and a member of maleimides. Sotrastaurin has been used in trials studying the basic science and treatment of Uveal Melanoma, Richter Syndrome, Prolymphocytic Leukemia, Recurrent Mantle Cell Lymphoma, and Recurrent Small Lymphocytic Lymphoma, among others. Sotrastaurin is an orally available pan-protein kinase C (PKC) inhibitor with potential immunosuppressive and antineoplastic activities. Sotrastaurin inhibits both T- and B-cell activations via PKC theta and beta isozymes, respectively. Both PKCs are important in the activation of nuclear factor-kappaB (NF-kB). Inhibition of PKC beta in B-cells results in prevention of NF-kB-mediated signaling and down regulation of NF-kB target genes. This may eventually lead to an induction of G1 cell cycle arrest and tumor cell apoptosis in susceptible tumor cells. This agent may act synergistically with other chemotherapeutic agents. PKC, a family of serine/threonine protein kinases overexpressed in certain types of cancer cells, is involved in cell differentiation, mitogenesis, inflammation, and the activation and survival of lymphocytes. Sotrastaurin (AEB071) is a potent, selective PKC inhibitor developed as an immunosuppressive agent and anticancer combination therapy [1][2][3] - Its core mechanism involves inhibiting PKCα/β/θ-mediated signaling, suppressing T-cell activation (immunosuppression), tumor cell proliferation, and ischemia/reperfusion-induced oxidative stress [1][2][3] - Therapeutic applications include organ transplantation (prevention of rejection), treatment of autoimmune diseases, and GNAQ/GNA11-mutant uveal melanoma (in combination with PI3K inhibitors) [1][2][3] - High selectivity for PKC subtypes involved in T-cell activation and tumor signaling minimizes off-target effects on normal tissues [1] - Favorable oral bioavailability and tissue distribution support its use in long-term immunosuppressive therapy and oral anticancer combinations [1][3]

Solubility Data

Solubility (In Vitro)

DMSO: 87 mg/mL (198.4 mM) Water:<1 mg/mL Ethanol: 2 mg/mL (4.6 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.70 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.70 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. Solubility in Formulation 3: 2% DMSO +30%PEG 300 +ddH2O: 10 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2806 mL	11.4030 mL	22.8061 mL
	5 mM	0.4561 mL	2.2806 mL	4.5612 mL
	10 mM	0.2281 mL	1.1403 mL	2.2806 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.