Journal of Medicinal Chemistry
Article
penicillin−streptomycin from Gibco (Grand Island NY), and fetal
bovine serum (FBS) from Atlanta Biologicals (Norcross, GA). CV-1
and COS cells were obtained from American Tissue Culture
Collection (Manassas, VA).
once daily. Intact (sham) rats served as controls and were not treated.
On the day of the last administration, the rats were euthanized by CO2
asphyxiation and blood was collected by cardiac puncture. Prostate,
seminal vesicles, and levator ani muscle tissues were collected and
weighed. Circulating LH concentrations in plasma were determined
with an LH ELISA kit from Endocrine Technologies, Newark, CA.
The concentration of test compounds in plasma, the levator ani
muscle, and prostate were determined in tissue samples solubilized in
water (4× volume per gram of tissue).
Cell-Based Assays. The binding and reporter assays were performed
on cells transfected in T225 cm2 flasks with the plasmids described
below, harvested, frozen, and replated in 96-well plates. For the AR
binding assay, COS cells were transfected with phAR, an expression
vector coding for full length human AR cDNA in pcDNA3
(Invitrogen). For the ARE-LUC reporter assay, CV-1 cells were
transfected with: (1) phAR and (2) pARE-Luciferase, a luciferase
reporter plasmid containing an androgen response element (ARE)
DNA sequence upstream of a firefly luciferase reporter in pGL3-Basic
vector (Promega, Madison, WI). For the 2-hybrid AR N/C-interaction
reporter assay CV-1 cells were transfected with three plasmids: (1)
pVP16-hAR-NTD, expressing human AR amino acids 1−561 in frame
to produce a fusion protein with VP16 in pVP16 vector (Clontech,
Mountain View, CA), (2) pGAL4-hAR-LBD, a plasmid containing the
human AR-LBD, AR amino acids 644−919, fused in frame with the
GAL4 DNA binding domain within the p133 vector (Promega), (3)
p5XGAL4-Luciferase reporter plasmid containing five copies of the
GAL4 upstream activation sequence followed by a minimal E1b
promoter controlling firefly luciferase expression in pGL3-Basic. Cells
were maintained in phenol red DMEM/high glucose, 10% non heat-
inactivated fetal bovine serum, 1% nonessential amino acids, 1%
penicillin−streptomycin at 37 °C in 5% CO2 and a humidified
atmosphere. For transfections, cells were plated at 11−12 million cells
per T225 cm2 flask one or two days before the transfection procedure.
Cells were transfected using Lipofectamine-2000 at a plasmid DNA to
Lipofectamine ratio of 1:3 (μg:μL) in phenol red free DMEM/high
glucose, 1% sodium pyruvate, 1% nonessential amino acids, and 1%
GlutaMAX-I (Gibco). After a 4 h incubation, cells were trypsinized,
frozen, and stored in cryovials at −150 °C.
For the luciferase reporter assays transfected, frozen CV-1 cells were
plated at 40000 cells per well in 96-well plates and incubated for 6 h.
Testosterone, DHT, and test compounds were resuspended and
serially diluted in DMSO and added to cells in phenol red free
DMEM/high glucose, 1% sodium pyruvate, 1% nonessential amino
acids, 1% GlutaMAX-I, 5% charcoal stripped fetal bovine serum, 1%
penicillin−streptomycin. After 17 h at 37 °C and 5% CO2, cells were
lysed in lysis reagent (Promega). Lysate samples were analyzed for
luciferase activity in Microlite microtiter plates (Thermo Labsystems)
using luciferase reagent (Promega) and a Victor 2 1420 multilabel
counter luminometer (Perkin-Elmer, Waltham, MA). All reporter
assays were performed in duplicate wells at least three times.
ASSOCIATED CONTENT
* Supporting Information
■
S
Supporting experimental details and characterization for
reported compounds. This material is available free of charge
AUTHOR INFORMATION
Corresponding Author
*Phone: 617-665-5621. Fax: 860-686-5499. E-mail: eugene.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Dr. K. Vishwanathan for conducting the in
vivo experiments. We would also like to thank Dr. B. Hu and
Dr. J. Wrobel for early synthetic contributions. We thank Dr. A.
Villalobos for supporting the project.
ABBREVIATIONS USED
■
HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
[4,5-b]pyridinium 3-oxid hexafluorophosphate; DIPEA, diiso-
propylethylamine; DHT, dihydrotestosterone; TPSA, total
polar surface area
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In Vivo Studies. All procedures conducted in this study were done
in accordance with the Pfizer Animal Care and Use Committee and
the National Institutes of Health Guide for the Care and Use of
Laboratory Animals. All animals were provided ad libitum with water
and standard rodent chow containing 28.5% protein, 13.5% fat, and
58% carbohydrate as a percentage of calories (Purina, LabDiet).
Orchidectomized Sprague−Dawley male rats were grouped six rats per
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propionate in 10% ethanol in corn oil administered subcutaneously
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