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.

Physicochemical Properties

Molecular Formula	C22H22FN5O3
Molecular Weight	423.44
Exact Mass	423.17
Elemental Analysis	C, 62.40; H, 5.24; F, 4.49; N, 16.54; O, 11.34
CAS #	1202757-89-8
Related CAS #	Spebrutinib besylate;1360053-81-1
PubChem CID	59174488
Appearance	White to khaki solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.655
LogP	2.95
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	10
Heavy Atom Count	31
Complexity	561
Defined Atom Stereocenter Count	0
InChi Key	KXBDTLQSDKGAEB-UHFFFAOYSA-N
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)
Chemical Name	N-[3-[[5-fluoro-2-[4-(2-methoxyethoxy)anilino]pyrimidin-4-yl]amino]phenyl]prop-2-enamide
Synonyms	Spebrutinib;CC-292;AVL292; AVL-292; AVL-292; cc-292; Btk inhibitor CC-292; AVL292; Spebrutinib [USAN:INN]; UNII-DRU6NG543J; CC292; CC 292; AVL 292;
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	Btk （IC50 < 0.5 nM)
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]
ln Vivo	In in vivo studies using the adoptive transfer TCL1 mouse model of CLL, CC-292 reduced tumor load and normalized tumor-associated expansion of T cells and monocytes, while not affecting T cell function. Importantly, the combination of CC-292 and bendamustine impaired CLL cell proliferation in vivo and enhanced the control of CLL progression. Our results demonstrate that CC-292 is a specific BTK inhibitor with promising performance in combination with bendamustine in CLL. Further clinical trials are warranted to investigate the therapeutic efficacy of this combination regimen.[3] CC-292/bendamustine treatment effectively controls CLL development in vivo. In order to assess the in vivo activity of CC-292 and to validate the in vitro results obtained from its combination with bendamustine, we used the TCL1 AT mouse model of CLL. Leukemic TCL1 AT splenocytes were transplanted into syngeneic immunocompetent C57BL/6 N mice. After 14 days, mice presented mean TL of 50% (Supporting Information Fig. S5a). They were then randomized in four groups (Supporting Information Fig. S5b) and treated for 11 days (Fig. 4a). During the treatment, absolute lymphocyte counts (ALC) in blood were monitored weekly. In all treatment conditions, a decrease was detected, being this more remarkable in the combination treatment (Supporting Information Fig. S6a). Furthermore, untreated mice exhibited low red blood cell and platelet counts, which were improved after treatment with CC-292, bendamustine, or the combination (Supporting Information Fig. S6b and S6c). Untreated mice showed severe splenomegaly and hepatomegaly (Fig. 4b), which were significantly less severe both in CC-292 and bendamustine-treated animals (spleen weight 2.2-fold lower in CC-292 cohort (p < 0.0001) and 2.5-fold lower in bendamustine cohort (p < 0.0001), liver weight 1.6-fold lower both in CC-292 (p < 0.0001) and bendamustine cohorts (p = 0.0002)). Mice treated with the combination showed a markedly lower spleen weight of up to 5-fold less (p < 0.0001) and a 1.7-fold lower liver weight (p < 0.0001) compared to untreated animals.[3] In a collagen-induced arthritis mouse model, AVL-292 (3-30 mg/kg, p.o.) dose-dependently inhibits the clinical signs of inflammatory disease, including reduction in joint and paw swelling and visible redness of the affected paws.
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.
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.
Animal Protocol	TCL1 adoptive transfer (AT) mouse model[3] Eμ-TCL1 (TCL1) mice on C57BL/6 background were used. In this model, the overexpression of TCL1 in B cells under the VH-promoter-IgH-Eμ-enhancer drives a clonal expansion of CD5+ B cells, representing an aggressive form of CLL.24 For treatment studies, adoptive transfer of TCL1 tumors were performed in C57BL/6 WT mice as described before.25 Briefly, 106 splenocytes with more than 95% of viable CD19+CD5+ cells from leukemic TCL1 mice were transplanted in 3-month-old female C57BL/6N wild-type mice via tail vein injection. Mice were housed in pathogen-free conditions, closely monitored for signs of illness. All experiments were performed according to the University of Barcelona animal experimental ethics committee guidelines. When PB tumor load (TL) reached mean values of 50% of CD19+ CD5+ (out of total CD45+ cells), animals were randomized into 4 groups (Vehicle, CC-292, bendamustine and Combination) with equal mean and standard deviation of TL percentage values. Fifteen milligram/kilogram CC-292 were administered twice daily via oral gavage, whereas 25 mg/kg bendamustine were administered intravenously once weekly. Mice were euthanized after 11 days of treatment and single cell suspensions were obtained from BM, inguinal LN and spleen.[3] 3-30 mg/kg; p.o Collagen-induced arthritis (CIA) mouse model
References	[1]. Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans. J Pharmacol Exp Ther. 2013 Aug;346(2):219-28.
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.

Solubility Data

Solubility (In Vitro)

DMSO: 85 mg/mL (200.7 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

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.