-Zanubrutinib Chemical Structure.png)
(±)-Zanubrutinib is a racemic mixture 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 # | 1633350-06-7 | |
| Related CAS # | Zanubrutinib;1691249-45-2;(R)-Zanubrutinib;1691249-44-1;Zanubrutinib-d5; 1633350-06-7 (racemic); 1691249-45-2 (S-isomer); 1691249-44-1 (R-isomer) | |
| PubChem CID | 135905454 | |
| Appearance | White to light yellow solid | |
| Density | 1.3±0.1 g/cm3 | |
| Boiling Point | 713.4±60.0 °C at 760 mmHg | |
| Flash Point | 385.2±32.9 °C | |
| Vapour Pressure | 0.0±2.3 mmHg at 25°C | |
| Index of Refraction | 1.680 | |
| LogP | 3.64 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 5 | |
| Rotatable Bond Count | 6 | |
| Heavy Atom Count | 35 | |
| Complexity | 756 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C(C([H])=C([H])[H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])C1([H])C([H])([H])C([H])([H])N([H])C2=C(C(N([H])[H])=O)C(C3C([H])=C([H])C(=C([H])C=3[H])OC3C([H])=C([H])C([H])=C([H])C=3[H])=NN12 |
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| InChi Key | RNOAOAWBMHREKO-UHFFFAOYSA-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) | |
| Chemical Name | 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
(±)-Zanubrutinib (BGB-3111) targets Bruton's tyrosine kinase (BTK), a core component of the B-cell receptor (BCR) signaling pathway; it exhibits nanomolar-level inhibitory activity against BTK, [1] |
| ln Vitro |
(±)-Zanubrutinib ((±)-BGB-3111) exhibits nanomolar Btk inhibition activity in both biochemical and cellular assays. (±)-Zanubrutinib inhibits BCR aggregation-triggered Btk autophosphorylation, blocks downstream PLC-γ2 signaling, and potently inhibits cell proliferation in a number of MCL and DLBCL cell lines. When it comes to a panel of kinases, including ITK, (±)-Zanubrutinib exhibits far more limited off-target activities when compared to PCI-32765[1]. 1. In biochemical and cellular assays, (±)-Zanubrutinib showed nanomolar potency in inhibiting BTK activity, demonstrating highly selective inhibition of BTK compared to other kinases (e.g., ITK) [1] 2. In multiple mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cell lines, (±)-Zanubrutinib inhibited BTK autophosphorylation triggered by BCR aggregation, blocked downstream PLC-γ2 signaling transduction, and potently suppressed cancer cell proliferation [1] 3. Unlike ibrutinib (the first-generation BTK inhibitor), (±)-Zanubrutinib had significantly reduced off-target activity against a panel of kinases including ITK; it was at least 10-fold weaker than ibrutinib in inhibiting rituximab-induced antibody-dependent cell-mediated cytotoxicity (ADCC), as evidenced by its weaker suppression of rituximab-induced NK cell IFN-γ secretion and in vitro cytotoxicity against MCL cells [1] |
| ln Vivo |
(±)-Zanubrutinib produces dose-dependent anti-tumor effects against REC-1 MCL xenografts that are injected into mice's tail veins and engrafted subcutaneously or systemically. in the xenografts placed beneath the skin. An initial 14-day rat toxicity study reveals that (±)-Zanubrutinib is highly well tolerated, and doses up to 250 mg/kg/day do not result in the maximal tolerated dose (MTD) being reached[1]. 1. In mouse BTK occupancy assays, treatment with (±)-Zanubrutinib resulted in dose-dependent BTK occupancy in target organs (PBMC and spleen), and its potency in these organs was approximately 3-fold higher than that of ibrutinib [1] 2. (±)-Zanubrutinib induced dose-dependent anti-tumor effects in mice bearing REC-1 MCL xenografts (both subcutaneous and systemic models via tail vein injection): in subcutaneous xenografts, (±)-Zanubrutinib at 2.5 mg/kg twice daily (BID) exhibited similar anti-tumor activity to ibrutinib at 50 mg/kg once daily (QD, its clinically relevant dose) [1] 3. In the systemic REC-1 MCL xenograft model, the median survival of mice treated with (±)-Zanubrutinib at 25 mg/kg BID was significantly longer than those treated with ibrutinib at 50 mg/kg QD or BID [1] 4. In a subcutaneous xenograft model of ABC-subtype DLBCL (TMD-8), (±)-Zanubrutinib demonstrated superior anti-tumor activity compared to ibrutinib [1] |
| Enzyme Assay | In both biochemical and cellular assays, BGB-3111 demonstrated nanomolar BTK inhibition activity. In several MCL and DLBCL cell lines, BGB-3111 inhibited BCR aggregation-triggered BTK autophosphorylation, blocked downstream PLC-γ2 signaling, and potently inhibited cell proliferation. In comparison with ibrutinib, BGB-3111 showed much more restricted off-target activities against a panel of kinases, including ITK. While ibrutinib significantly inhibited rituximab-induced NK cell IFN-γ secretion and in vitro cytotoxicity on mantle cell lymphoma cells, BGB-3111 was at least 10-fold weaker than ibrutinib in inhibiting rituximab induced ADCC, consistent with its weak ITK inhibition activity.[1] |
| Cell Assay |
1. BTK signaling inhibition assay: MCL and DLBCL cell lines were cultured and stimulated with BCR aggregation in the presence of (±)-Zanubrutinib at different concentrations; the cells were then lysed, and the levels of BTK autophosphorylation and downstream PLC-γ2 signaling were detected using relevant biochemical methods to evaluate the inhibitory effect of (±)-Zanubrutinib on the BCR-BTK signaling pathway [1] 2. Cell proliferation assay: MCL and DLBCL cell lines were seeded in culture plates and treated with (±)-Zanubrutinib; after a period of incubation, cell proliferation was assessed using appropriate cell viability detection methods to determine the anti-proliferative effect of the drug [1] 3. ADCC inhibition assay: NK cells and MCL target cells were co-cultured with rituximab in the presence of (±)-Zanubrutinib or ibrutinib; NK cell IFN-γ secretion was measured, and the in vitro cytotoxicity of NK cells against MCL cells was evaluated to compare the impact of (±)-Zanubrutinib and ibrutinib on rituximab-induced ADCC [1] |
| Animal Protocol |
In mouse BTK occupancy assays, treatment with BGB-3111 resulted in a dose-dependent BTK occupancy and showed about 3-fold more potency than ibrutinib in target organs, including PBMC and spleen. BGB-3111 induced dose-dependent anti-tumor effects against REC-1 MCL xenografts engrafted either subcutaneously or systemically via tail vein injection in mice. In the subcutaneous xenografts, BGB-3111 at 2.5 mg/kg BID showed similar activity as ibrutinib at 50 mg/kg QD, its clinical relevant dose. In the systemic model, the median survival of BGB-3111 25 mg/kg BID group was significantly longer than those of both ibrutinib 50 mg/kg QD and BID groups. In an ABC-subtype DLBCL (TMD-8) subcutaneous xenograft model, BGB-3111 also demonstrated better anti-tumor activity than ibrutinib. Preliminary 14-day toxicity study in rats showed that BGB-3111 was very well tolerated and maximal tolerate dose (MTD) was not reached when it was dosed up to 250mg/kg/day.[1] 1. Mouse BTK occupancy assay: (±)-Zanubrutinib was administered to mice at different doses; at specific time points, PBMC and spleen tissues were collected, and the level of BTK occupancy in these tissues was detected to assess the target engagement of the drug [1] 2. REC-1 MCL subcutaneous xenograft model: REC-1 MCL cells were subcutaneously engrafted into mice; when tumors reached a certain volume, (±)-Zanubrutinib was administered at 2.5 mg/kg BID, and ibrutinib was administered at 50 mg/kg QD as a control; tumor growth was monitored regularly to evaluate the anti-tumor activity of the drug [1] 3. REC-1 MCL systemic xenograft model: REC-1 MCL cells were injected into mice via tail vein to establish a systemic tumor model; (±)-Zanubrutinib was administered at 25 mg/kg BID, and ibrutinib at 50 mg/kg QD/BID as controls; the survival time of mice was recorded to compare the anti-tumor efficacy of different treatments [1] 4. TMD-8 DLBCL subcutaneous xenograft model: TMD-8 DLBCL cells were subcutaneously engrafted into mice; (±)-Zanubrutinib and ibrutinib were administered respectively, and tumor growth was monitored to evaluate the anti-tumor activity of (±)-Zanubrutinib [1] 5. Rat toxicity study: (±)-Zanubrutinib was administered to rats at doses up to 250 mg/kg/day for 14 consecutive days; the general condition of rats was observed, and the maximum tolerated dose (MTD) was evaluated [1] |
| Toxicity/Toxicokinetics |
1. In a 14-day preliminary toxicity study in rats, (±)-Zanubrutinib was well tolerated at doses up to 250 mg/kg/day, and the maximum tolerated dose (MTD) was not reached during the study [1] |
| References |
[1]. BGB-3111 is a novel and highly selective Bruton's tyrosine kinase (BTK) inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): A. |
| Additional Infomation |
See also: Zanubrutinib (annotation moved to). 1. BTK is an essential component of the BCR pathway and a novel therapeutic target for B-cell malignancies; the first-generation irreversible BTK inhibitor ibrutinib has promising clinical activity but can antagonize rituximab-induced ADCC by inhibiting ITK kinase activity, which limits its clinical application [1] 2. (±)-Zanubrutinib is a novel, highly selective second-generation BTK inhibitor, currently under clinical investigation for the treatment of hematological cancers [1] 3. (±)-Zanubrutinib does not affect rituximab-induced ADCC and has better anti-tumor efficacy than ibrutinib in xenograft models, supporting further clinical investigation of this compound as a single agent or in combination with anti-CD20 antibodies for hematological cancers [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.41 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 20.8 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: 2.08 mg/mL (4.41 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (4.41 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 20.8 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 | 2.1206 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 |