Sodium Chloride → Camp

HRID 1677207Notes

Reaction equation

HRID 1677207 reaction equation
[P:1]([O:13][CH2:14][C@H:15]1[O:19][C@@H:18]([N:20]2[C:30]3[N:29]=[C:27](N)[NH:26][C:24](=O)[C:23]=3[N:22]=[CH:21]2)[C@H:17]([OH:31])[C@@H:16]1[OH:32])(OP(OP(O)(O)=O)(O)=O)(=[O:3])[OH:2].[Na+].[Cl-].[Mg+2].[Cl-].[Cl-].C[C@@H](N)CC1NC=[N:43]C=1>C1N(CCO)CCN(CCS(O)(=O)=O)C1>[CH:27]1[N:26]=[C:24]([NH2:43])[C:23]2[N:22]=[CH:21][N:20]([C@@H:18]3[O:19][C@@H:15]4[CH2:14][O:13][P:1]([OH:2])([O:32][C@H:16]4[C@H:17]3[OH:31])=[O:3])[C:30]=2[N:29]=1

Participants

6

Reactant 4

HCID 5234Sodium ChlorideHCID 156615alpha-methylhistamine, (R)-HCID 5360315Magnesium ChlorideHCID 135398633Guanosine Triphosphate3×

Product 1

HCID 6076Camp

Reagents, catalysts & solvents 1

HCID 238314-(2-Hydroxyethyl)-1-piperazineethanesulfonic acidSolvent

Procedure

Antagonist activity of selected compounds was tested for inhibition of [35S] GTP γ [S] binding to H3R membranes in the presence of agonists. Assays were run at room temperature in 20 mM HEPES, 100 mM NaCl, 5 mM MgCl2 and 10 uM GDP at pH 7.4 in a final volume of 200 ul in 96-well Costar plates. Membranes isolated from H3R8-expressing HEK293 cell line (20 ug/well) and GDP were added to each well in a volume of 50 μl assay buffer. Antagonist was then added to the wells in a volume of 50 μl assay buffer and incubated for 15 minutes at room temperature. Agonist R(−)alpha methylhistamine (RBI) at either a dose response from 1×10−10 to 1A1-5 M or fixed concentration of 100 nM were then added to the wells in a volume of 50 μl assay buffer and incubated for 5 minutes at room temperature. GTP γ [35S] was added to each well in a volume of 50 μl assay buffer at a final concentration of 200 pM, followed by the addition of 50 μl of 20 mg/ml WGA coated SPA beads (Amersham). Plates were counted in Wallac Trilux 1450 Microbeta scintillation counter for 1 minute. Compounds that inhibited more than 50% of the specific binding of radioactive ligand to the receptor were serially diluted to determine a K[i](nM). The results are given below for the indicated compound.

Workup

  1. customMembranes isolated from H3R8-expressing HEK293 cell line (20 ug/well) and GDP
  2. additionwere added to each well in a volume of 50 μl assay buffer
  3. additionAntagonist was then added to the wells in a volume of 50 μl assay buffer and
  4. waitincubated for 5 minutes at room temperature
  5. additionwas added to each well in a volume of 50 μl assay buffer at a final concentration of 200 pM
  6. additionfollowed by the addition of 50 μl of 20 mg/ml WGA coated SPA beads (Amersham)
  7. waitPlates were counted in Wallac Trilux 1450 Microbeta scintillation counter for 1 minute
  8. additionwere serially diluted

Sources & evidence

ORD record
ord-64a6136d4fc249fc820ce516f9d657b7
Source dataset
uspto-grants-2011_08
Patent number
US08008301B2

Reaction notes

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