2-hydroxypropane-1,2,3-tricarboxylic acid; 3-[(3R,4R)-4-methyl-3-[methyl({7H-pyrrolo[2,3-d]pyrimidin-4-yl})amino]piperidin-1-yl]-3-oxopropanenitrile

• ChemBase ID: 73080
• Molecular Formular: C22H28N6O8
• Molecular Mass: 504.49312
• Monoisotopic Mass: 504.19686189
• SMILES: n1cnc2c(c1N(C)[C@H]1CN(CC[C@H]1C)C(=O)CC#N)cc[nH]2.C(C(C(=O)O)(CC(=O)O)O)C(=O)O
• Canonical SMILES: OC(=O)CC(C(=O)O)(CC(=O)O)O.N#CCC(=O)N1CC[C@H]([C@H](C1)N(c1ncnc2c1cc[nH]2)C)C
• InChI: InChI=1S/C16H20N6O.C6H8O7/c1-11-5-8-22(14(23)3-6-17)9-13(11)21(2)16-12-4-7-18-15(12)19-10-20-16;7-3(8)1-6(13,5(11)12)2-4(9)10/h4,7,10-11,13H,3,5,8-9H2,1-2H3,(H,18,19,20);13H,1-2H2,(H,7,8)(H,9,10)(H,11,12)/t11-,13+;/m1./s1
• InChIKey: SYIKUFDOYJFGBQ-YLAFAASESA-N

NAMES AND DATABASE IDS

Names

• IUPAC name: 2-hydroxypropane-1,2,3-tricarboxylic acid; 3-[(3R,4R)-4-methyl-3-[methyl({7H-pyrrolo[2,3-d]pyrimidin-4-yl})amino]piperidin-1-yl]-3-oxopropanenitrile
• IUPAC Traditional name: citro; tofacitinib
• Synonyms: CP-690550; Tofacitinib citrate

Database IDs

• CAS Number: 540737-29-9
• PubChem SID: 162038000
• PubChem CID: 10174505

CALCULATED PROPERTIES

• Acid pKa: 12.405254
• H Acceptors: 5
• H Donor: 1
• LogD (pH = 5.5): -0.25029096
• LogD (pH = 7.4): 1.0526191
• Log P: 1.2447144
• Molar Refractivity: 87.8 cm^3
• Polarizability: 32.969494 Å^3
• Polar Surface Area: 88.91 Å^2
• Rotatable Bonds: 8
• Lipinski's Rule of Five: false

PROPERTIES

Physical Property

• Solubility: DMSO

Safety Information

• Storage Condition: -20°C

Pharmacology Properties

• Target: JAK

Product Information

• Salt Data: citrate

DETAILS

Selleck Chemicals - S5001

Protocol

• Protocol: Kinase Assay ^[1]
• Enzyme assays: The JAK1, JAK2, and JAK3 kinase assays utilize a protein expressed in baculovirus-infected SF9 cells (a fusion protein of GST and the catalytic domain of human JAK enzyme) purified by affinity chromatography on glutathione?Sepharose. The substrate for the reaction is polyglutamic acid-tyrosine [PGT (4:1)], coated onto Nunc Maxi Sorp plates at 100 μg/mL overnight at 37 °C. The plates are washed three times, and JAK enzyme is added to the wells, which contained 100 μL of kinase buffer (50 mM HEPES, pH 7.3, 125 mM NaCl, 24 mM MgCl₂) + ATP + 1 mM sodium orthovanadate). For Tofacitinib citrate, it is also added for kinase assay at different doses. After incubation at room temperature for 30 min, the plates are washed three times. The level of phosphorylated tyrosine in a given well is determined by standard ELISA assay utilizing an anti-phosphotyrosine antibody.
• Protocol: Cell Assay ^[2]
• Cell Lines: FDCP-EpoR JAK2^WT and JAK2^V617F cell lines
• Concentrations: 0-4 μM
• Incubation Time: 72 hours
• Methods: Determination of growth inhibition by Tofacitinib citrate is performed using identical culture conditions for both FDCP-EpoR JAK2^WT and JAK2^V617F cell lines. Briefly, 1 × 10^5 cells/mL are cultured in 96-well flat-bottom plates at 37 °C in a humidified 5% CO₂ atmosphere using RPMI 1640 supplemented with 1.25% FCS, and 5% WEHI supernatant. Decreased FCS concentration is necessary to prevent binding between Tofacitinib citrate and serum proteins. Growth inhibition assays are terminated by addition of 20 μL CellTiter96 One Solution Reagent. Flat-bottom plates are incubated for an additional 3 hours for MTT assay. Absorbance is determined at 595 nm on a BioTek Synergy-HT microplate reader. Results are the average ± standard deviation of three independent determinations.
• Protocol: Animal Study ^[2]
• Animal Models: Mauritius-origin adult cynomolgus monkeys
• Formulation: 0.5% methylcellulose in distilled water
• Doses: 10, 30 mg/kg/d
• Administration: Oral gavage

References

• References: [1] Flanagan ME, et al. J Med Chem, 2010, 53(24), 8468-8484.
• References: [2] Manshouri T, et al. Cancer Sci, 2008, 99(6), 1265-1273.
• References: [3] Conklyn M, et al. J Leukoc Biol, 2004, 76(6), 1248-1255.

REFERENCES

• Flanagan ME, et al. J Med Chem, 2010, 53(24), 8468-8484.
• Manshouri T, et al. Cancer Sci, 2008, 99(6), 1265-1273.
• Conklyn M, et al. J Leukoc Biol, 2004, 76(6), 1248-1255.