Identification and optimization of novel partial agonists of Neuromedin B receptor using parallel synthesis

Bioorganic and Medicinal Chemistry Letters

Volumn 14, Issue 12, 2004, Pages 3037-3042

  Shuttleworth, Stephen J ^a   Lizarzaburu, Mike E ^a   Chai, Anne ^a   Coward, Peter ^a  

^a AMGEN INC   (United States)

Author keywords

Lead optimization; Neuromedin B receptor; Parallel synthesis; Partial agonist

Indexed keywords

3 AMINO 2,3,4,9 TETRAHYDRO 1H CARBAZOLE 3 CARBOXYLIC ACID AMIDE; AMIDE; CARBOXYLIC ACID DERIVATIVE; NEUROMEDIN B RECEPTOR; RECEPTOR; UNCLASSIFIED DRUG;

AGONIST; ARTICLE; DRUG DESIGN; DRUG IDENTIFICATION; DRUG POTENCY; DRUG SYNTHESIS; MOLECULAR SIZE;

CHEMISTRY, PHARMACEUTICAL; DOSE-RESPONSE RELATIONSHIP, DRUG; HELA CELLS; HUMANS; INDOLES; RECEPTORS, BOMBESIN; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 2442442870     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2004.04.045     Document Type: Article

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15. 6 transfected cells in Dulbeco's Modified Eagle Media (Mediatech, Inc.) in 100 μL in 96 well plates. Bound ligand was separated from unbound ligand by filtration using a Filtermate (Packard) and total counts bound determined on a TopCount NTX reader (Packard)
16. Typical synthetic procedure for 2 and analogues thereof: A solution of N-Fmoc-amino-4-(ethylene ketal)cyclohexylcarboxylic acid, A (1.0 wt) in N, N-dimethylformamide was treated with amine (1.0 equiv), HBTU (1.2 equiv) and Hünig's base (1.2 equiv), and the reaction mixture was agitated at room temperature for 16 h. The solvent was removed using a Genevac centrifugal evaporator, and the residue partitioned between brine and dichloromethane. The organic phase containing B was isolated, and treated with piperidine (20% of overall volume); the reaction mixture was agitated for 1 h at room temperature, and was then concentrated to dryness. The residue, C, was then treated with 1 M HCl (aq), and the solution washed with dichloromethane. The aqueous phase was isolated and phenylhydrazine (1.0 equiv) was added, together with a few drops of concentrated HCl, and the reaction mixture was heated to 85°C for 2 h. The solution was cooled, neutralized with saturated sodium bicarbonate solution, and the product, D, was extracted with dichloromethane. The overall yields for the synthesis of D typically exceeded 60%. For the final step, stock solutions of the primary amine D were treated with equal mole quantities of isocyanate in N, N-dimethylformamide, in the presence of Hünig's base. After 16 h stirring at room temperature, conversions in excess of 90% to the desired ureas E were determined by HPLC at 220 and 254 nm using an Agilent 1100 LC/MSD VL ESI system. The products were then purified directly using automated preparative HPLC (see Ref. 19). Note that for several examples, step 3 involved the use of 1, 4, -dioxane as the solvent to circumvent problems with poor aqueous solubility of a number of phenylhydrazines used in the library synthesis
17. Typical protocol for cytotoxicity assay: HeLa cells were seeded at 5k/well in a 96 well plate (6 plates-for triplicate 0 and 72 h readings). A three-fold dilution series of each compound was generated, starting at 10 mM. The solution was diluted 8 times to prepare 9 concentrations ranging from 10 mM to 150 nM. Compound was added to cells (1-100 μL total volume), and the final concentration of compound was 100 μM to 15 nM. Zero and 72 h time points were recorded as follows: (a) 10 μL Alamar Blue reagent was added to the wells, and the samples were incubated at 37°C for 3 h; (b) fluorescence intensity was then recorded on an LJL Analyst. The relative growth was compared to a DMSO control well for each compound
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21. 3OH))
22. 3 requires 635.38
23. 3H]Inositol (82.0 Ci/mmol, Amersham Pharmacia Biotech) overnight at 37°C. Test compounds were then added in inositol-free DMEM containing 0.3% BSA (Sigma) and 10 mM LiCl (Sigma) for 60 min at 37°C. The cells were lysed with 20 mM formic acid for 2 h at 4°C and added to RNA-binding Ysi scintillation proximity assay beads (Amersham Pharmacia Biotech) for 30 min. Plates were stored overnight at room temperature in the dark and read the next day on a TopCount NTX