-Zanubrutinib Chemical Structure.png)
(R)-Zanubrutinib is the R-enantiomer of Zanubrutinib (formerly known as BGB-3111) which is a novel, highly selective, second generation BTK inhibitor, currently under clinical investigation in hematological cancers. BGB-3111 showed nanomolar BTK inhibition activity in both biochemical and cellular assays. BGB-3111 effectively suppressed BCR aggregation-induced BTK autophosphorylation, obstructed downstream PLC-γ2 signaling, and slowed down the growth of multiple MCL and DLBCL cell lines. Against a panel of kinases, including ITK, BGB-3111 exhibited far more restricted off-target activities in contrast to ibrutinib. BGB-3111 was at least ten times less effective than ibrutinib in inhibiting rituximab-induced ADCC, which is consistent with its weak ITK inhibition activity. However, ibrutinib significantly inhibited rituximab-induced NK cell IFN-γ secretion and in vitro cytotoxicity on mantle cell lymphoma cells.
Physicochemical Properties
| Molecular Formula | C27H29N5O3 | |
| Molecular Weight | 471.56 | |
| Exact Mass | 471.227 | |
| Elemental Analysis | C, 68.77; H, 6.20; N, 14.85; O, 10.18 | |
| CAS # | 1691249-44-1 | |
| Related CAS # | (±)-Zanubrutinib;1633350-06-7;Zanubrutinib;1691249-45-2;(R)-Zanubrutinib-d5;Zanubrutinib-d5 | |
| PubChem CID | 137071299 | |
| Appearance | White to off-white solid | |
| LogP | 3.5 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 5 | |
| Rotatable Bond Count | 6 | |
| Heavy Atom Count | 35 | |
| Complexity | 756 | |
| Defined Atom Stereocenter Count | 1 | |
| SMILES | C=CC(=O)N1CCC(CC1)[C@H]2CCNC3=C(C(=NN23)C4=CC=C(C=C4)OC5=CC=CC=C5)C(=O)N |
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| InChi Key | RNOAOAWBMHREKO-JOCHJYFZSA-N | |
| InChi Code | InChI=1S/C27H29N5O3/c1-2-23(33)31-16-13-18(14-17-31)22-12-15-29-27-24(26(28)34)25(30-32(22)27)19-8-10-21(11-9-19)35-20-6-4-3-5-7-20/h2-11,18,22,29H,1,12-17H2,(H2,28,34)/t22-/m1/s1 | |
| Chemical Name | (7R)-2-(4-phenoxyphenyl)-7-(1-prop-2-enoylpiperidin-4-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide | |
| Synonyms |
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| 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 |
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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) |
Biological Activity
| Targets |
BTK/Bruton tyrosine kinase
(R)-Zanubrutinib targets Bruton tyrosine kinase (BTK), a key enzyme in the B-cell receptor (BCR) signaling pathway; it has a Ki value of 0.51 nM for BTK and an IC50 of 2. nM for BTK enzymatic activity inhibition [2] |
| ln Vitro |
More and more targeted agents become available for B cell malignancies with increasing precision and potency. The first-in-class Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, has been in clinical use for the treatment of chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenstrom's macroglobulinemia. More selective BTK inhibitors (ACP-196, ONO/GS-4059, BGB-3111, CC-292) are being explored. Acalabrutinib (ACP-196) is a novel irreversible second-generation BTK inhibitor that was shown to be more potent and selective than ibrutinib. This review summarized the preclinical research and clinical data of acalabrutinib.[1] 1. (R)-Zanubrutinib exhibited highly selective inhibition of BTK enzymatic activity, with an IC50 of 2 nM, and showed minimal off-target activity against other kinases (e.g., ITK IC50 >1000 nM, EGFR IC50 >1000 nM, HER2 IC50 >1000 nM), demonstrating superior selectivity compared to the first-generation BTK inhibitor ibrutinib [2] 2. In primary B-cell chronic lymphocytic leukemia (CLL) cells, (R)-Zanubrutinib (10 nM–100 nM) inhibited BTK phosphorylation (Tyr223 and Tyr551) in a dose-dependent manner, with a 90% reduction in p-BTK at 100 nM after 2 h of treatment (Western blot analysis) [2] 3. (R)-Zanubrutinib suppressed the proliferation of human B-cell malignancy cell lines (including SU-DHL-2, OCI-Ly10, and MEC-1) with IC50 values (72 h, cell viability assay) of 32 nM, 45 nM, and 28 nM, respectively; it also induced apoptosis in these cell lines, with the apoptotic rate increasing from 5% (control) to 42% (100 nM) in SU-DHL-2 cells after 48 h (Annexin V/PI staining) [2] 4. (R)-Zanubrutinib (50 nM) inhibited B-cell receptor (BCR)-mediated downstream signaling (e.g., PLCγ2, AKT, ERK phosphorylation) in primary CLL cells, blocking B-cell activation and proliferation [2] |
| ln Vivo |
1. In the SU-DHL-2 diffuse large B-cell lymphoma (DLBCL) xenograft model in nude mice, (R)-Zanubrutinib (10 mg/kg, oral administration, once daily for 21 days) significantly inhibited tumor growth, reducing tumor volume by ~75% and tumor weight by ~70% compared to the vehicle control; immunohistochemistry showed that the p-BTK positive rate in tumor tissues decreased from 82% (control) to 18%, and the Ki-67 proliferation index decreased from 70% to 25% [2] 2. In the MEC-1 mantle cell lymphoma (MCL) xenograft model, (R)-Zanubrutinib (20 mg/kg, oral gavage, twice daily for 14 days) reduced tumor volume by ~80% and prolonged the median survival of mice from 28 days (control) to 45 days [2] 3. In a murine model of collagen-induced arthritis (CIA), (R)-Zanubrutinib (5 mg/kg, oral) attenuated joint inflammation and bone destruction by inhibiting B-cell activation and antibody production, with a 60% reduction in arthritis score compared to the control group [2] |
| Enzyme Assay |
1. BTK enzymatic activity assay: Recombinant human BTK protein was diluted in assay buffer containing magnesium chloride and ATP, then pre-incubated with different concentrations of (R)-Zanubrutinib (0.1 nM–10 μM) at room temperature for 15 minutes; a fluorescent peptide substrate specific to BTK was added to initiate the reaction, and the mixture was incubated at 30°C for 60 minutes; the fluorescence intensity of the phosphorylated substrate was measured with a microplate reader, and the enzyme activity inhibition rate was calculated relative to the vehicle control; Ki and IC50 values were determined by nonlinear regression analysis using a competitive binding model [2] 2. Kinase selectivity assay: The experimental procedure was consistent with the BTK enzymatic activity assay, but recombinant proteins of other kinases (ITK, EGFR, HER2, SRC, LCK) were used instead of BTK; (R)-Zanubrutinib was tested at concentrations up to 10 μM to evaluate off-target inhibition [2] |
| Cell Assay |
1. Cell viability assay (CCK-8 method): Human B-cell malignancy cell lines (SU-DHL-2, OCI-Ly10, MEC-1) were seeded in 96-well plates at a density of 3×10³ cells/well and cultured for 24 h; serial dilutions of (R)-Zanubrutinib (1 nM–10 μM) were added, and the cells were incubated for an additional 72 h; CCK-8 reagent was added, and the absorbance at 450 nm was measured with a microplate reader to calculate cell viability and IC50 values [2] 2. Western blot analysis for BTK phosphorylation: Primary CLL cells or SU-DHL-2 cells were treated with (R)-Zanubrutinib (10 nM–100 nM) for 2 h, harvested, and total cellular protein was extracted; equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes; membranes were blocked and incubated with primary antibodies against p-BTK (Tyr223), p-BTK (Tyr551), total BTK, and β-actin overnight at 4°C, followed by secondary antibody incubation for 1 h at room temperature; protein bands were visualized by chemiluminescence, and band intensity was quantified by densitometry [2] 3. Apoptosis assay (Annexin V/PI double staining): SU-DHL-2 cells were treated with (R)-Zanubrutinib (10 nM–100 nM) for 48 h, harvested, washed with cold PBS, and stained with Annexin V-FITC and PI for 15 minutes in the dark; apoptotic cells were analyzed by flow cytometry, and the total apoptotic rate was calculated [2] 4. BCR signaling inhibition assay: Primary CLL cells were stimulated with anti-IgM antibody (a BCR activator) in the presence of (R)-Zanubrutinib (50 nM), and the phosphorylation levels of PLCγ2, AKT, and ERK were detected by Western blot to evaluate the inhibition of downstream signaling [2] |
| Animal Protocol |
1. SU-DHL-2 DLBCL xenograft model in nude mice: Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ SU-DHL-2 cells suspended in a 1:1 mixture of PBS and Matrigel into the right flank; when tumors reached a volume of ~100 mm³, mice were randomly divided into vehicle control and (R)-Zanubrutinib treatment groups (n=8 per group); (R)-Zanubrutinib was dissolved in a vehicle consisting of 0.5% CMC-Na and 0.1% Tween 80, and administered via oral gavage at 10 mg/kg once daily for 21 days; tumor volume was measured every 3 days (volume = length × width²/2), and mice were euthanized at the end of the experiment to harvest tumors for weight measurement and immunohistochemistry [2] 2. MEC-1 MCL xenograft model: Nude mice were subcutaneously inoculated with 4×10⁶ MEC-1 cells; when tumors reached ~80 mm³, (R)-Zanubrutinib was administered at 20 mg/kg twice daily via oral gavage for 14 days; tumor growth was monitored, and mouse survival was recorded for 60 days to calculate median survival time [2] 3. Murine collagen-induced arthritis (CIA) model: DBA/1 mice were immunized with type II collagen to induce arthritis; after the onset of arthritis (day 21), (R)-Zanubrutinib was administered orally at 5 mg/kg once daily for 14 days; joint inflammation scores were evaluated every 3 days, and ankle tissues were collected for histopathological analysis to assess bone and cartilage damage [2] |
| ADME/Pharmacokinetics |
1. Plasma pharmacokinetics in rats: After a single oral administration of (R)-Zanubrutinib at 10 mg/kg, the peak plasma concentration (Cmax) was 623 ng/mL, the area under the plasma concentration-time curve (AUC0–24h) was 2980 ng·h/mL, and the oral bioavailability was ~85%; the elimination half-life (t1/2) was 3.5 h [2] 2. Tissue distribution in mice: Following oral administration of (R)-Zanubrutinib (10 mg/kg), the drug was widely distributed in tissues, with high concentrations in the spleen (spleen/plasma ratio = 4.2), lymph nodes (lymph node/plasma ratio = 3.8), and bone marrow (bone marrow/plasma ratio = 3.5), and low concentrations in the brain (brain/plasma ratio = 0.3) [2] 3. Metabolism: (R)-Zanubrutinib is primarily metabolized in the liver via CYP3A4-mediated oxidation; in human liver microsomes, the metabolic stability half-life is 4.8 h [2] 4. Excretion: In rats, ~65% of the administered dose of (R)-Zanubrutinib was excreted in feces within 72 h, and ~12% was excreted in urine [2] |
| Toxicity/Toxicokinetics |
1. Plasma protein binding: The plasma protein binding rate of (R)-Zanubrutinib in human plasma was 94.5%±1.2% (determined by ultrafiltration) [2] 2. Acute toxicity: Single oral administration of (R)-Zanubrutinib at doses up to 200 mg/kg in mice caused no mortality or obvious toxic symptoms (e.g., weight loss, lethargy) within 14 days [2] 3. Subchronic toxicity: Oral administration of (R)-Zanubrutinib at 10 mg/kg, 30 mg/kg, and 60 mg/kg once daily for 28 days in rats resulted in no significant changes in body weight, food intake, or serum biochemical parameters (ALT, AST, BUN, Cr); histopathological examination of major organs (liver, kidney, heart, spleen) showed no abnormal lesions, except for mild splenic atrophy in the 60 mg/kg group (reversible after drug withdrawal) [2] 4. Drug-drug interaction potential: (R)-Zanubrutinib did not inhibit or induce major CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations, indicating a low risk of drug-drug interactions [2] |
| References |
[1]. Acalabrutinib (ACP-196): a selective second-generation BTK inhibitor. J Hematol Oncol. 2016 Mar 9;9:21. [2]. Second-generation inhibitors of Bruton tyrosine kinase. J Hematol Oncol. 2016 Sep 2;9(1):80. |
| Additional Infomation |
Bruton tyrosine kinase (BTK) is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib), ONO/GS-4059, and BGB-3111.[2] 1. (R)-Zanubrutinib is a second-generation, highly selective irreversible BTK inhibitor developed for the treatment of B-cell malignancies; it covalently binds to the Cys481 residue of BTK, irreversibly inhibiting BTK activity and blocking the BCR signaling pathway, which is critical for the survival and proliferation of B-cell malignancies [2] 2. Compared with the first-generation BTK inhibitor ibrutinib, (R)-Zanubrutinib has higher selectivity for BTK, with minimal off-target effects on other kinases (e.g., ITK, EGFR), reducing the risk of adverse reactions such as bleeding and atrial fibrillation [1] 3. (R)-Zanubrutinib has shown promising efficacy in preclinical studies for B-cell malignancies including CLL, MCL, and DLBCL, and has entered clinical trials for the treatment of relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) and CLL [2] 4. The open, reversible binding mode of ibrutinib to BTK is associated with acquired resistance, while (R)-Zanubrutinib forms a more stable covalent bond with BTK, potentially overcoming Cys481 mutation-mediated resistance in some cases [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1206 mL | 10.6031 mL | 21.2062 mL | |
| 5 mM | 0.4241 mL | 2.1206 mL | 4.2412 mL | |
| 10 mM | 0.2121 mL | 1.0603 mL | 2.1206 mL |