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| Targets |
BTK
(R)-Pirtobrutinib targets Bruton's tyrosine kinase (BTK) but is the less active enantiomer. The active enantiomer, Pirtobrutinib, is a highly selective and non-covalent next-generation BTK inhibitor that can suppress multiple BTK C481 substitution mutations. The (R)-enantiomer is used as a negative control in experiments to differentiate specific BTK inhibition from non-specific effects. It binds to the ATP-binding pocket of BTK but with significantly lower affinity and inhibitory activity compared to the active enantiomer. |
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| ln Vitro |
LOXO-305 is a highly selective, non-covalent, next generation BTK inhibitor. We previously showed that LOXO-305 potently inhibited both wild-type (WT) BTK and BTK C481S -mediated kinase activity in enzyme and cell-based assays with nanomolar potency, caused regression of BTK-dependent lymphoma mouse xenograft models, and was more than 300-fold selective for BTK over 98% of 370 other kinases tested and showed no significant inhibition of non-kinase off targets at 1 mM (Brandhuber et al. SOHO 2018) [1].
(R)-Pirtobrutinib is a less active enantiomer of Pirtobrutinib. In enzyme-based and cell-based assays, the active (S)-enantiomer (Pirtobrutinib) inhibits wild-type BTK and BTK C481S-mediated kinase activity with nanomolar potency. The (R)-enantiomer is expected to have significantly lower activity and is used as a control. Its IC50 for BTK inhibition is substantially higher than that of the active enantiomer, reflecting its reduced binding affinity and inhibitory potency. |
| ln Vivo |
In addition, ADME and pharmacokinetic experiments in two preclinical species predicted that LOXO-305 will have high human exposure and sustained BTK C481S target coverage in patients at clinically achievable doses[1].
(R)-Pirtobrutinib has lower in vivo activity compared to the active (S)-enantiomer. Preclinical ADME and pharmacokinetic experiments predicted that the active enantiomer, LOXO-305, would have high human exposure and sustained BTK C481S target coverage at clinically achievable doses. The (R)-enantiomer serves as a less active comparator. In vivo studies typically focus on the active enantiomer, with the (R)-enantiomer used as a control to confirm that observed effects are due to specific BTK inhibition. |
| Enzyme Assay |
The in vitro enzyme/receptor binding (non-cell-based) assay for (R)-Pirtobrutinib involves assessing its binding affinity and inhibitory activity against BTK. Surface plasmon resonance (SPR) is used to measure the equilibrium binding affinity of BTK inhibitors to BTK enzyme variants. Biotinylated BTK variants are immobilized on a sensor chip, and the compound's binding kinetics are analyzed. Kinase assays are performed to determine the IC50 for inhibition of BTK activity. The (R)-enantiomer is used as a less active control in these assays.
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| Cell Assay |
To assess cellular BTK inhibitor potency, HEK293T cell lines transiently expressing wild-type BTK and BTK C481 substitution mutations were serum starved and incubated with LOXO-305 overnight. Cells were next incubated with serum and orthovanadate for 5 min and the phosphorylated Y223 BTK was analyzed by immunoblot. Bands were quantified and the IC50 values calculated with GraphPad Prism. The equilibrium-binding affinities for targeted BTK inhibitors to BTK enzyme variants were determined by surface plasmon resonance (SPR) using the Biacore T200. Biotinylated BTK variants were immobilized on a docked streptavidin coated sensor chip. Five concentrations of each inhibitor plus blank controls were analyzed. Association/dissociation rate constants were calculated by global fitting of the data to a 1:1 binding interaction model[1].
The in vitro cell-based assay for (R)-Pirtobrutinib involves treating cells expressing wild-type BTK or BTK C481 substitution mutations with the compound. Cells are serum-starved and incubated with the compound overnight, then stimulated with serum and sodium orthovanadate. The phosphorylation of BTK at Y223 is analyzed by immunoblotting to assess the compound's cellular activity. The (R)-enantiomer is used as a less active control. IC50 values for inhibition of BTK phosphorylation are calculated from dose-response curves. |
| Animal Protocol |
In vivo animal experiments for (R)-Pirtobrutinib are typically conducted as part of the preclinical development of the active enantiomer. In mouse xenograft models, the active enantiomer, LOXO-305, leads to regression of BTK-dependent lymphoma tumors. The (R)-enantiomer may be used as a negative control to confirm that the observed effects are due to BTK inhibition. Pharmacokinetic studies may also compare the exposure of the two enantiomers.
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| ADME/Pharmacokinetics |
(R)-Pirtobrutinib has a molecular weight of 479.43 g/mol and a molecular formula of C22H21F4N5O3. It has a LogP of 3.3 and a tPSA of 125 Ų. The compound is soluble in DMSO. It is typically used for research purposes only. The compound is stored as a powder at -20°C.
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| Toxicity/Toxicokinetics |
As a research compound, its safety profile is evaluated in standard cytotoxicity and acute toxicity assays. The compound is classified for research use only and is not intended for human therapeutic use. Comprehensive toxicological characterization would be required prior to any clinical development. The compound is typically handled with standard laboratory safety precautions.
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| References | |
| Additional Infomation |
(R)-Pirtobrutinib ((R)-LOXO-305, CAS 2101700-14-3) is the less active enantiomer of Pirtobrutinib. It has a molecular weight of 479.43 and a molecular formula of C22H21F4N5O3. It is used as a research tool to study the activity of BTK inhibitors. It is not approved for clinical use and is available only for research purposes. The compound's chemical name is (R)-5-Amino-3-(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)-1-(1,1,1-trifluoropropan-2-yl)-1H-pyrazole-4-carboxamide.
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| Molecular Formula |
C22H21F4N5O3
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| Molecular Weight |
479.43
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| Exact Mass |
479.16
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| Elemental Analysis |
C, 55.12; H, 4.42; F, 15.85; N, 14.61; O, 10.01
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| CAS # |
2101700-14-3
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| Related CAS # |
Pirtobrutinib;2101700-15-4
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| PubChem CID |
129269918
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| Appearance |
Typically exists as light yellow to yellow solids at room temperature
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
34
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| Complexity |
719
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C[C@H](C(F)(F)F)N1C(=C(C(=N1)C2=CC=C(C=C2)CNC(=O)C3=C(C=CC(=C3)F)OC)C(=O)N)N
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| InChi Key |
FWZAWAUZXYCBKZ-LLVKDONJSA-N
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| InChi Code |
InChI=1S/C22H21F4N5O3/c1-11(22(24,25)26)31-19(27)17(20(28)32)18(30-31)13-5-3-12(4-6-13)10-29-21(33)15-9-14(23)7-8-16(15)34-2/h3-9,11H,10,27H2,1-2H3,(H2,28,32)(H,29,33)/t11-/m1/s1
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| Chemical Name |
5-amino-3-[4-[[(5-fluoro-2-methoxybenzoyl)amino]methyl]phenyl]-1-[(2R)-1,1,1-trifluoropropan-2-yl]pyrazole-4-carboxamide
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| Synonyms |
R)-Pirtobrutinib; 2101700-14-3; (R)-5-Amino-3-(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)-1-(1,1,1-trifluoropropan-2-yl)-1H-pyrazole-4-carboxamide; SCHEMBL19014260; (R)-LOXO-305; FWZAWAUZXYCBKZ-LLVKDONJSA-N; AKOS040754869;
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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 |
| 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) |
DMSO: 200 mg/mL (417.16 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (10.43 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 50.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0858 mL | 10.4291 mL | 20.8581 mL | |
| 5 mM | 0.4172 mL | 2.0858 mL | 4.1716 mL | |
| 10 mM | 0.2086 mL | 1.0429 mL | 2.0858 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.
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