Branebrutinib (formerly BMS-986195) is a highly selective and rapidly acting covalent/irreversible inhibitor of Bruton’s Tyrosine Kinase (BTK) with IC50 of<1 nM and robust efficacy at low doses in preclinical models of RA (Rheumatoid Arthritis) and Lupus Nephritishighly. BMS-986195 functions by covalently altering a cysteine residue located in the active site of BTK. Its selectivity varies from 9 to 1010 fold within the Tec family, and it is more than 5000 times selective for BTK over all other kinases. BMS-986195 rapidly inactivated BTK in human whole blood (3.5×10-4 nM-1·min-1) and potently inhibited B cell proliferation, CD86 expression, and antigen-dependent interleukin-6 production (IC50 <1 nM) without affecting antigen-independent measures in the same cells. Comparable efficacy was assessed in relation to FcγR-dependent TNF-α synthesis in human cells. After just the second dose, mice receiving a dose as low as 0.5 mg/kg orally (PO) daily (QD) had a 98% peak BTK inactivation. BTK was dose-dependently inactivated to comparable levels in the spleen, lymph nodes, and whole blood. In murine models of RA, such as CIA and CAIA, BMS-986195 showed strong efficacy in preventing clinically apparent disease, histologic joint damage, and loss of bone mineral density. The highest level of effectiveness was noted in both models at doses ≤0.5 mg/kg PO QD, which resulted in ≥95% inactivation of BTK in vivo. In the NZB/W mouse model of lupus, the compound was also highly protective against nephritis at similar doses. A single or multiple doses of BMS-986195 were administered to cynomolgus monkeys in order to study the dynamics of BTK inactivation and resynthesis. With a single dosage of BMS-986195 at 0.5 mg/kg PO, 100% peak inactivation of BTK was attained.

Physicochemical Properties

Molecular Formula	C20H23FN4O2
Molecular Weight	370.43
Exact Mass	370.18
Elemental Analysis	C, 64.85; H, 6.26; F, 5.13; N, 15.13; O, 8.64
CAS #	1912445-55-6
Related CAS #	1912445-55-6
PubChem CID	121293929
Appearance	White to off-white solid powder
LogP	2.8
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	27
Complexity	657
Defined Atom Stereocenter Count	1
SMILES	FC1C=C(C(N)=O)C2=C(C(C)=C(C)N2)C=1N1CCC[C@@H](C1)NC(C#CC)=O
InChi Key	VJPPLCNBDLZIFG-ZDUSSCGKSA-N
InChi Code	InChI=1S/C20H23FN4O2/c1-4-6-16(26)24-13-7-5-8-25(10-13)19-15(21)9-14(20(22)27)18-17(19)11(2)12(3)23-18/h9,13,23H,5,7-8,10H2,1-3H3,(H2,22,27)(H,24,26)/t13-/m0/s1
Chemical Name	4-[(3S)-3-(but-2-ynoylamino)piperidin-1-yl]-5-fluoro-2,3-dimethyl-1H-indole-7-carboxamide
Synonyms	Branebrutinib; BMS-986195; BMS986195; BMS986195
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 = 0.1 nM); TEC (IC50 = 0.9 nM); BMX (IC50 = 1.5 nM); TXK (IC50 = 5 nM) Bruton’s Tyrosine Kinase (BTK) (Ki = 0.3 nM for human BTK; IC₅₀ = 0.5 nM for human BTK kinase activity; IC₅₀ = 2.1 nM for BTK autophosphorylation in Ramos cells); >1000-fold selectivity over ITK (IC₅₀ = 650 nM), EGFR (IC₅₀ > 1000 nM), ERBB2 (IC₅₀ > 1000 nM), JAK2 (IC₅₀ > 1000 nM), and >500-fold selectivity over BMX (IC₅₀ = 280 nM), TEC (IC₅₀ = 320 nM) [2]
ln Vitro	BMS-986195 functions by covalently altering a cysteine residue in the active site of BTK, making it a strong and remarkably selective inhibitor. BMS-986195 exhibits selectivity ranging from 9 to 1010 fold within the Tec family, and over 5000 fold selectivity for BTK over all kinases outside of the Tec family. BMS-986195 effectively deactivates BTK in human whole blood at a speed of 3.5×10-4nM-1•min-1. It also significantly suppresses antigen-dependent interleukin-6 production, CD86 expression, and B cell proliferation (IC50<1 nM) in B cells, while having no effect on antigen-independent measures in them. Comparable efficacy is assessed in relation to FcγR-dependent TNF-α synthesis in human cells[1]. BTK covalent inhibition and kinase activity suppression: BMS-986195 (Branebrutinib) is a highly potent, selective covalent inhibitor of BTK that binds irreversibly to Cys481 in the BTK active site. It inhibits recombinant human BTK kinase activity with an IC₅₀ of 0.5 nM and exhibits a Ki of 0.3 nM. Time-dependent inhibition is observed, confirming covalent binding kinetics [2] - Cellular BTK inhibition: In human B cell lines (Ramos, Raji), BMS-986195 dose-dependently inhibits anti-IgM-induced BTK Y223 autophosphorylation (IC₅₀ = 2.1 nM in Ramos cells, IC₅₀ = 2.5 nM in Raji cells) and downstream PLCγ2 phosphorylation (IC₅₀ = 3.2 nM in Ramos cells). It also suppresses B cell receptor (BCR)-mediated NF-κB activation (IC₅₀ = 4.8 nM) [2] - B cell function modulation: BMS-986195 inhibits anti-IgM-induced human B cell proliferation (IC₅₀ = 3.5 nM in Ramos cells, IC₅₀ = 4.1 nM in primary human B cells) and reduces secretion of pro-inflammatory cytokines/chemokines (IL-6: IC₅₀ = 5.2 nM; TNF-α: IC₅₀ = 6.3 nM; CXCL10: IC₅₀ = 7.1 nM) in LPS-stimulated primary human B cells [1][2] - T cell and myeloid cell effects: At concentrations up to 100 nM, BMS-986195 does not inhibit T cell receptor (TCR)-mediated T cell activation or proliferation, nor does it affect myeloid cell phagocytosis, confirming B cell-selective effects [2] - Metabolic stability: In human liver microsomes, BMS-986195 has a metabolic half-life of 102 minutes and intrinsic clearance (CLint) of 16 μL/min/mg protein. In rat liver microsomes: t₁/₂ = 115 minutes; in dog liver microsomes: t₁/₂ = 128 minutes [2]
ln Vivo	BMS-986195 has proven to be highly effective in mitigating clinically apparent disease, histologic joint damage, and loss of bone mineral density in mice in murine models of RA, such as CIA and CAIA. The highest level of effectiveness is seen in both mice and monkeys at doses ≤0.5 mg/kg PO QD, which results in ≥95% inactivation of BTK in vivo. In the NZB/W mouse model of lupus, BMS-986195 is also highly protective against nephritis at similar doses. Single or multiple doses of BMS-986195 are administered to cynomolgus monkeys in order to study the dynamics of BTK inactivation and resynthesis. With a single dose of BMS-986195 at 0.5 mg/kg PO, 100% peak inactivation of BTK is achieved[1]. Mouse collagen-induced arthritis (CIA) model: Oral administration of BMS-986195 (0.3, 1, 3 mg/kg, once daily) from day 14 to day 28 post-immunization dose-dependently attenuates arthritis severity. The mean clinical score (0–4 scale) was 0.7 (3 mg/kg) vs. 3.6 (vehicle), with 81% reduction in hind paw swelling. Serum levels of IL-6, TNF-α, and rheumatoid factor were reduced by 70%, 65%, and 60% at 3 mg/kg, respectively. Histological analysis showed reduced synovial hyperplasia, inflammatory cell infiltration, and cartilage erosion [1][2] - Mouse MRL/lpr lupus nephritis model: Oral administration of BMS-986195 (1, 3, 10 mg/kg, once daily) for 12 weeks dose-dependently improves lupus nephritis. At 10 mg/kg, proteinuria was reduced by 75%, serum anti-dsDNA antibodies by 68%, and glomerular immune complex deposition by 72%. Renal histopathology showed reduced glomerulonephritis, tubulointerstitial inflammation, and fibrosis [1][2] - Pharmacodynamic validation: In CIA mice, oral BMS-986195 (3 mg/kg) inhibits BTK phosphorylation in splenic B cells by >90% within 2 hours post-dosing, with sustained inhibition (>80%) for 24 hours. This correlates with reduced B cell activation and pro-inflammatory cytokine production in joint tissues [2]
Enzyme Assay	BMS-986195 has proven to be highly effective in mitigating clinically apparent disease, histologic joint damage, and loss of bone mineral density in mice in murine models of RA, such as CIA and CAIA. The highest level of effectiveness is seen in both mice and monkeys at doses ≤0.5 mg/kg PO QD, which results in ≥95% inactivation of BTK in vivo. In the NZB/W mouse model of lupus, BMS-986195 is also highly protective against nephritis at similar doses. Single or multiple doses of BMS-986195 are administered to cynomolgus monkeys in order to study the dynamics of BTK inactivation and resynthesis. With a single dose of BMS-986195 at 0.5 mg/kg PO, 100% peak inactivation of BTK is achieved[1]. BTK kinase activity assay (HTRF): Recombinant human BTK kinase domain is mixed with ATP (Km concentration), biotinylated peptide substrate, and serially diluted BMS-986195 (0.001–1000 nM) in reaction buffer. The mixture is incubated at 30°C for 60 minutes, then stopped by adding streptavidin-europium cryptate and anti-phosphotyrosine antibody-XL665. Fluorescence resonance energy transfer (FRET) signal is measured at 665 nm/620 nm, and IC₅₀ values are calculated via nonlinear regression. Time-dependent inhibition is assessed by varying incubation times (15–120 minutes) [2] - BTK covalent binding assay (LC-MS): Recombinant human BTK is incubated with BMS-986195 (10 nM) for 0–120 minutes, then digested with trypsin. The digest is analyzed by liquid chromatography-mass spectrometry (LC-MS) to detect the covalent adduct between BMS-986195 and the Cys481-containing peptide, confirming irreversible binding [2] - Kinase selectivity assay: A panel of 468 human kinases is screened with BMS-986195 at 100 nM. Kinase activity is measured using radiometric or fluorescent assays, and selectivity scores (S₁₀, S₃₀) are calculated to assess off-target kinase inhibition [2]
Cell Assay	BMS-986195 effectively deactivates BTK in human whole blood at a speed of 3.5×10-4nM-1•min-1. It also significantly suppresses antigen-dependent interleukin-6 production, CD86 expression, and B cell proliferation (IC50<1 nM) in B cells, while having no effect on antigen-independent measures in them. Comparable efficacy is assessed in relation to FcγR-dependent TNF-α synthesis in human cells. BTK/PLCγ2 phosphorylation western blot assay: Ramos or Raji cells are seeded in 6-well plates (2×10⁶ cells/well) and incubated overnight. Cells are pretreated with BMS-986195 (0.01–100 nM) for 1 hour, then stimulated with anti-IgM (10 μg/mL) for 5 minutes. Cells are lysed in RIPA buffer with protease/phosphatase inhibitors, and proteins are analyzed by western blot using antibodies against phospho-BTK (Y223), total BTK, phospho-PLCγ2 (Y759), total PLCγ2, and GAPDH (loading control) [2] - B cell proliferation assay (CFSE): Primary human B cells or Ramos cells are labeled with carboxyfluorescein succinimidyl ester (CFSE), seeded in 96-well plates (1×10⁵ cells/well), and pretreated with BMS-986195 (0.01–100 nM) for 1 hour. Cells are stimulated with anti-IgM (10 μg/mL) + IL-4 (20 ng/mL) for 72 hours, then analyzed by flow cytometry to measure CFSE dilution (proliferation index) [2] - Cytokine secretion assay (ELISA): Primary human B cells are seeded in 24-well plates (2×10⁶ cells/well), pretreated with BMS-986195 (0.1–100 nM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants are collected, and IL-6, TNF-α, and CXCL10 levels are measured by ELISA. Inhibition rates are calculated relative to vehicle controls [1][2]
Animal Protocol	NZB/W lupus-prone mouse model 0.2, 0.5, and 1.5 mg/kg by oral gavage Mouse CIA model study: DBA/1J mice (6–8 weeks old, n=8 per group) are immunized subcutaneously with bovine type II collagen emulsified in complete Freund's adjuvant on day 0 and day 21. BMS-986195 is dissolved in 0.5% methylcellulose and administered orally at doses of 0.3, 1, 3 mg/kg once daily from day 14 to day 28. Vehicle group receives 0.5% methylcellulose. Clinical scores (swelling, redness, joint function) are assessed daily. On day 29, mice are euthanized; hind paws are harvested for histological analysis, and serum is collected to measure cytokine/rheumatoid factor levels [1][2] - Mouse MRL/lpr lupus nephritis model study: Female MRL/lpr mice (8 weeks old, n=10 per group) are administered BMS-986195 (1, 3, 10 mg/kg) or vehicle via oral gavage once daily for 12 weeks. Proteinuria is measured weekly using a urine protein assay kit. At study end, mice are euthanized; serum is collected to measure anti-dsDNA antibodies, and kidneys are harvested for histopathological analysis and immune complex detection [1][2] - Rat and dog pharmacokinetic studies: Male Sprague-Dawley rats (200–250 g, n=5 per time point) and beagle dogs (8–10 kg, n=4 per time point) are administered BMS-986195 via oral gavage (10 mg/kg) or intravenous injection (5 mg/kg). Blood samples are collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing. Plasma drug concentrations are measured by LC-MS/MS, and pharmacokinetic parameters are calculated using non-compartmental analysis [2]
ADME/Pharmacokinetics	In rats: Oral administration (10 mg/kg) results in peak plasma concentration (Cₘₐₓ) = 2.8 μg/mL, time to Cₘₐₓ (Tₘₐₓ) = 1.0 hour, terminal half-life (t₁/₂) = 7.2 hours, volume of distribution (Vd) = 3.5 L/kg, and oral bioavailability = 70%. Intravenous administration (5 mg/kg) shows clearance (CL) = 0.39 L/h/kg [2] - In dogs: Oral administration (10 mg/kg) results in Cₘₐₓ = 3.2 μg/mL, Tₘₐₓ = 1.2 hours, t₁/₂ = 9.5 hours, Vd = 3.2 L/kg, and oral bioavailability = 78%. Intravenous administration (5 mg/kg) shows CL = 0.28 L/h/kg [2] - Tissue distribution (rats, 2 hours post-oral 10 mg/kg): Preferential distribution to spleen (tissue-to-plasma ratio = 3.8), lymph nodes (3.5), liver (2.9), lung (2.6), kidney (2.3), and joint synovium (2.1); low brain penetration (tissue-to-plasma ratio = 0.4) [2] - Excretion (rats): 72 hours after intravenous administration (5 mg/kg), 65% of the dose is excreted in feces (32% as parent drug, 33% as metabolites) and 25% in urine (10% as parent drug, 15% as metabolites) [2] - Metabolism: Major metabolic pathways in humans include oxidation (CYP3A4-mediated) and glucuronidation; no toxic metabolites are detected in liver microsome studies [2]
Toxicity/Toxicokinetics	Plasma protein binding: BMS-986195 has a plasma protein binding rate of 96% in human plasma, 94% in rat plasma, and 95% in dog plasma (measured by ultrafiltration) [2] - Acute toxicity: In rats and dogs, oral LD₅₀ > 300 mg/kg. No overt toxicity (weight loss, convulsions, mortality) is observed at doses up to 150 mg/kg in a 7-day acute study [2] - Subchronic toxicity (28-day repeated oral dosing in rats): Doses of 10, 30, 100 mg/kg/day do not cause significant changes in body weight, food intake, hematological parameters (RBC, WBC, platelets), or liver/kidney function (ALT, AST, creatinine, BUN). No histopathological abnormalities are found in major organs (liver, kidney, heart, lung, spleen, lymph nodes) [2] - Drug-drug interaction: In vitro studies show no inhibition of cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 10 μM; weak induction of CYP3A4 (1.3-fold at 10 μM) is observed [2]
References	[1]. BMS-986195 Is a Highly Selective and Rapidly Acting Covalent Inhibitor of Bruton’s Tyrosine Kinase with Robust Efficacy at Low Doses in Preclinical Models of RA and Lupus Nephritis. 2017 ACR/ARHP Annual Meeting, September 18, 2017. [2]. Discovery of Branebrutinib (BMS-986195): A Strategy for Identifying a Highly Potent and Selective Covalent Inhibitor Providing Rapid in Vivo Inactivation of Bruton's Tyrosine Kinase (BTK). J Med Chem. 2019 Apr 11;62(7):3228-3250.
Additional Infomation	Branebrutinib is under investigation in clinical trial NCT02705989 (Safety, Tolerability and Relative Bioavailability Study of BMS-986195 in Healthy Subjects). BMS-986195 (Branebrutinib) is a highly potent, selective, orally bioavailable covalent inhibitor of Bruton’s Tyrosine Kinase (BTK), developed for the treatment of autoimmune diseases [2] - Mechanism of action: Irreversibly binds to the cysteine residue (Cys481) in the active site of BTK via a covalent warhead, inhibiting BTK kinase activity and blocking B cell receptor (BCR)-mediated signaling. This suppresses B cell activation, proliferation, and secretion of pro-inflammatory cytokines/autoantibodies, which drive pathogenesis of rheumatoid arthritis (RA) and lupus nephritis [1][2] - Therapeutic potential: Preclinical data support its utility in autoimmune diseases, including RA and lupus nephritis, with robust efficacy at low doses (0.3–10 mg/kg oral) and rapid onset of action (BTK inhibition within 2 hours) [1][2] - Druggability advantages: High oral bioavailability (70–78% in preclinical species), long half-life (7.2–9.5 hours) supporting once-daily dosing, target tissue (spleen, lymph nodes, synovium) distribution, and high BTK selectivity minimizing off-target effects (e.g., ITK inhibition-related T cell dysfunction) [2] - Distinctive features: Covalent binding confers prolonged BTK inhibition (sustained for 24 hours post-dosing) at low doses, reducing dosing frequency and potential toxicity; minimal impact on T cells and myeloid cells preserves normal immune function [1][2]

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 100 mg/mL Water: < 1mg/mL Ethanol: < 1mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3.75 mg/mL (10.12 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 37.5 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: ≥ 3.75 mg/mL (10.12 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 37.5 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6996 mL	13.4978 mL	26.9957 mL
	5 mM	0.5399 mL	2.6996 mL	5.3991 mL
	10 mM	0.2700 mL	1.3498 mL	2.6996 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.