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Purity: ≥98%
Spebrutinib (formerly AVL-292; CC-292; SBT) is a covalent/irreversible, orally bioactive, and highly selective inhibitor of Bruton's agammaglobulinemia tyrosine kinase/BTK inhibitor with potential antitumor activity. It inhibits BTK with an IC50 of<0.5 nM, and exhibits > 1400-fold higher selectivity for BTK over the other kinases.
| Targets |
Btk （IC50 < 0.5 nM)
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| ln Vitro |
Spebrutinib (CC-292) is an oral Btk inhibitor that is covalent, highly selective, and has an IC50 of 0.5 nM. Moreover, spebrutinib exhibits mild inhibitory effects on Yes, c-Src, Brk, Lyn, and Fyn, with corresponding IC50s of 723 nM, 1.729 μM, 2.43 μM, 4.4 μM, and 7.15 μM. A further investigation revealed a close correlation between the cellular EC50 of Spebrutinib inhibition of Btk kinase (EC50=8 nM) and the EC50 of Btk occupancy in Ramos cells in response to the drug's dosage response (EC50=6 nM). Moreover, 35 nM of spebrutinib is the concentration that suppresses 90% of Btk activity in Ramos cells, whereas 39 nM is the concentration needed for 90% Btk occupancy [1].
In this study, researchers first investigated the antitumor effects of CC-292 in five MCL cell lines (REC-1, MINO, UPN-1, MAVER-1 and Z138) after 72 h of treatment. CC-292 (10–1000 nM) had a cytostatic effect in a subset of cell lines, with REC-1, MINO and UPN-1 appearing to be the most sensitive, while MAVER-1 and Z138 were the most resistant to CC-292, following a trend similar to that for ibrutinib (Figure 1A,B). CC-292 induced marginal apoptosis (10–15%) in the most sensitive cell lines (UPN-1 and REC-1) (Online Supplementary Figure S1). Identification of Tyr223 pBTK is considered a surrogate marker for kinase activity.6 MCL cell lines pre-incubated with CC-292 were IgM-stimulated to mimic BCR activation. As displayed in Figure 1C, CC-292 significantly reduced both constitutive and IgM-induced BTK phosphorylation at the Y223 residue in MCL cell lines and primary cells, independently of their sensitivity to the inhibitor.[2] |
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| ln Vivo |
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| Enzyme Assay |
Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro.[1]
ELISA cytokine quantification [2] CCL3 and CCL4 levels were assessed in duplicate using ELISA kits in supernatants harvested from cells that had been pretreated with 1µM CC-292 at 37ºC for 1h and subsequently stimulated with 10µg/ml of anti-IgM for 24h. |
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| Cell Assay |
Cell proliferation assay and apoptosis quantification [2]
MCL cells (5x104 ) were treated with Spebrutinib (CC-292), lenalidomide or NIK inhibitors for the times indicated and 0.5 mg/mL MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) reagent was added for 2–6 additional hrs before spectrophotometric measurement. Each measurement was made in triplicate. Values were represented using untreated control cells as reference. Apoptosis induction was evaluated by flow cytometry in an Attune acoustic focusing cytometer after staining MCL cells with Annexin V-FIT and co-stained with CD19-PE in the case of primary cells. Flow cytometry [2] Cells were pretreated with 1µM Spebrutinib (CC-292) at 37ºC for 1h and subsequently stimulated with 10µg/ml anti-IgM for 24h. Cellular activation was evaluated by co-staining of MCL cells with CD69-PC7/CD86-FITC, including CD19-PE and Annexin V-Pacific Blue in the case of primary cells, followed by cytofluorimetric evaluation in an Attune cytometer. Migration assay [2] SDF-1α/CXCL12-induced migration was evaluated using 24-well chemotaxis chambers containing 5 µm pore size inserts and coated with 1 µg/ml VCAM-1. The lower chamber contained 200ng/ml CXCL12. The cells were pretreated with 1µM Spebrutinib (CC-292) at 37ºC for 1h and deposited in the upper compartment allowing them to migrate for 3h at 37ºC and enumerated by flow cytometry. |
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| References | |||
| Additional Infomation |
Spebrutinib has been used in trials studying the treatment of Rheumatoid Arthritis, Lymphoma, Large B-Cell, Diffuse, and Leukemia Lymphocytic Chronic B-Cell.
Spebrutinib is an orally bioavailable, selective inhibitor of Bruton's agammaglobulinemia tyrosine kinase (BTK), with potential antineoplastic activity. Upon administration, spebrutinib targets and covalently binds to BTK, thereby preventing its activity. By irreversibly inhibiting BTK, administration of this agent may lead to an inhibition of B cell receptor (BCR) signaling and may inhibit cell proliferation of B-cell malignancies. BTK, a cytoplasmic tyrosine kinase and member of the Tec family of kinases, plays an important role in B lymphocyte development, activation, signaling, proliferation and survival. |
| Molecular Formula |
C22H22FN5O3
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| Molecular Weight |
423.44
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| Exact Mass |
423.17
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| Elemental Analysis |
C, 62.40; H, 5.24; F, 4.49; N, 16.54; O, 11.34
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| CAS # |
1202757-89-8
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| Related CAS # |
Spebrutinib besylate;1360053-81-1
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| PubChem CID |
59174488
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| Appearance |
White to khaki solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.655
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| LogP |
2.95
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
31
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| Complexity |
561
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
KXBDTLQSDKGAEB-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H22FN5O3/c1-3-20(29)25-16-5-4-6-17(13-16)26-21-19(23)14-24-22(28-21)27-15-7-9-18(10-8-15)31-12-11-30-2/h3-10,13-14H,1,11-12H2,2H3,(H,25,29)(H2,24,26,27,28)
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| Chemical Name |
N-[3-[[5-fluoro-2-[4-(2-methoxyethoxy)anilino]pyrimidin-4-yl]amino]phenyl]prop-2-enamide
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.90 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 (5.90 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 25.0 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 | 2.3616 mL | 11.8080 mL | 23.6161 mL | |
| 5 mM | 0.4723 mL | 2.3616 mL | 4.7232 mL | |
| 10 mM | 0.2362 mL | 1.1808 mL | 2.3616 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01975610 | Completed | Drug: CC-292 Drug: CC-292 |
Rheumatoid Arthritis | Celgene | October 2013 | Phase 2 |
| NCT02031419 | Terminated | Drug: CC-122 Drug: CC-223 |
Lymphoma, Large B-Cell, Diffuse |
Celgene | December 18, 2013 | Phase 1 |
Btk occupancy and Btk protein resynthesis can be detected in mice in vivo. Mice were treated orally once with 50 mg/kg CC-292 to inhibit all Btk protein.J Pharmacol Exp Ther.2013 Aug;346(2):219-28. |
CC-292 is efficacious in an established collagen-induced arthritis model.J Pharmacol Exp Ther.2013 Aug;346(2):219-28. |
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