4,5-Disubstituted cis-pyrrolidinones as inhibitors of type II 17β-hydroxysteroid dehydrogenase. Part 2. SAR

Bioorganic and Medicinal Chemistry Letters

Volumn 15, Issue 12, 2005, Pages 3053-3057

  Gunn, David ^a   Akuche, Christiana ^a   Baryza, Jeremy ^a   Blue, Marie Louise ^a   Brennan, Catherine ^a   Campbell, Ann Marie ^a   Choi, Soongyu ^a   Cook, James ^a   Conrad, Patricia ^a   Dixon, Brian ^a   Dumas, Jacques ^a   Ehrlich, Paul ^a   Gane, Todd ^a   Joe, Ted ^a   Johnson, Jeffrey ^a   Jordan, Jerold ^a   Kramss, Richard ^a   Liu, Peying ^a   Levy, Joan ^a   Lowe, Derek ^a   McAlexander, Ian ^a   Natero, Reina ^a   Redman, Anikó M ^a   Scott, William ^a   Seng, Thomas ^a   Sibley, Robert ^a   Wang, Ming ^a   Wang, Yamin ^a   Wood, Jill ^a   Zhang, Zhonghua ^a   more..

^a BAYER CORPORATION   (United States)

Author keywords

17 HSD; Hydroxysteroid dehydrogenase; Pyrrolidinone

Indexed keywords

2 PYRROLIDONE DERIVATIVE; TESTOSTERONE 17BETA DEHYDROGENASE;

ARTICLE; DRUG ACTIVITY; DRUG MECHANISM; DRUG POTENCY; ENZYME INHIBITION; STRUCTURE ACTIVITY RELATION; SUBSTITUTION REACTION;

17-HYDROXYSTEROID DEHYDROGENASES; ENZYME INHIBITORS; HUMANS; PYRROLIDINONES; STEREOISOMERISM; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 19944363836     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2005.04.025     Document Type: Article

References (20)

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18. 1H NMR and LC-MS analysis (>95% purity)
19. 16 was diluted to the appropriate concentration (generally 1/15 dilution) and 55 μL of assay buffer (50 mM Hepes, pH 8.0) was added to all wells of a 96-well plate. DMSO (5 μL, 5%) were added to wells A1-C1, A2-C2, F1-H1, and F2-H2. A 10 μL aliquot of a 10 mM DMSO stock of each compound well was diluted in 190 μL of assay buffer. The plate with these compound dilutions was then mixed for 15 min on a shaker and a 5 μL aliquot from each well was added to the test plate. This was followed by 10 μL of HSD 2 enzyme (1/15 dilution)/estradiol mix in assay buffer containing 1% Triton X-100. The estradiol mix was prepared from a 20 mM estradiol DMSO stock solution, which was diluted 1/20 (50 μg/mL) in 50 mM Hepes buffer containing 1% Triton X-100. All wells, except F1-H1 and F2-H2, also received 10 μL of 25 mg/mL NAD (Sigma) in 100 mM Hepes, pH 9. The blank wells F1-H1 and F2-H2 received 10 μL of NAD buffer and 20 μL of PMS-MTS solution (100 μL PMS into 1800 μL MTS, Promega) was added to all wells. The plate was covered with aluminum foil and incubated at room temperature for 1.5 h. The plate was analyzed at 490 nm on a Hewlett-Packard 8453 spectrophotometer and results were calculated using an in-house analysis program and the Hill slope method
20. Type II 17β-HSD protein purification: The method for purification of baculovirus-expressed HSD II was standardized as follows. The cell pellet containing recombinant HSD II was lysed in 2.5 vol of lysis buffer (40 mM Tris, pH 7.5, 1 mM EDTA, 2 mM DTT, 10 μM NAD) containing 500 mM AEBSF, 10 μg/mL aprotinin, 1 μM pepstatin A, and 100 μM leupeptin. The pellet was nutated for 30 min in this buffer. Subsequently, the pellet was homogenized with 15 strokes using a glass Teflon potter. The volume was doubled with a 2× sucrose/NaCl solution (500 mM sucrose, 300 mM NaCl in lysis buffer). After centrifugation at 10,000g for 30 min, the supernatant was decanted and stored at -20°C. The pellet was resuspended in 2.5 vol of lysis buffer containing 250 mM sucrose and 150 mM NaCl and 1% Triton X-100. The suspension was nutated for 20 min and then homogenized with five strokes. Subsequently, the suspension was centrifuged at 30,000g for 1 h and the supernatant was decanted and poured into a chromatographic column. Sephadex G-25 (fine) was added in small amounts, allowing approximately 15 min after each addition for complete swelling. Sephadex addition was continued until all liquid was absorbed. The protein was eluted with one void volume of lysis buffer and the eluate was monitored at 280 nm optical density. The chromatography was repeated and an equal volume of glycerol was added to the final eluate