Supplementary Methods
Purmorphamine activates the Hedgehog pathway by targeting Smoothened
Surajit Sinha1 and James K. Chen1
Address: 1Department of Molecular Pharmacology, Stanford University School of Medicine,
Stanford, CA 94305, USA
Correspondence: James K. Chen
Email: jameschen@stanford.edu
Telephone: (650) 725-3582
Running Head: Purmorphamine targets Smoothened
Nature Chemical Biology Reference Number: 2005-09-00577B
Filename: Chen_Supplement.doc
Synthesis of purmorphamine
Purmorphamine was synthesized according to the procedure reported by Wu et al. with
slight modifications1.
2,6-Dichloro-9-cyclohexylpurine:
2,6-Dichloropurine
(250
mg,
1.3
mmol),
triphenylphosphine (350 mg, 1.3 mmol) and cyclohexanol (126 L, 1.19 mmol) were dissolved
in anhydrous tetrahydrofuran (5 mL) under nitrogen atmosphere. To this was added diethyl
azodicarboxylate (570 L of a 40% solution in toluene, d 0.956 g/mL, 3.12 mmol) in an ice cold
condition. The reaction mixture was stirred at room temperature for 12 h. The solvent was
removed in vacuo. The resulting yellow gum was purified by column chromatography over
silica gel with methanol/chloroform (3:97, v/v). The crude product (275 mg) was contaminated
with triphenylphosphine oxide and diethyl dihydroazodicarboxylate byproducts and was used in
the next step without further purification.
2-Chloro-6-(4-morpholinoanilino)-9-cyclohexylpurine: The above crude mixture (275 mg),
4-morpholinoaniline (80 mg, 0.45 mmol) and diisopropylethylamine (0.5 mL, 2.87 mmol) were
dissolved in n-butanol (3 mL). The reaction mixture was stirred at 105 °C for 16 h. The solvent
was removed in vacuo and the resulting residue was azeotroped with methanol (3 x 2 mL). The
crude product was purified by column chromatography over silica gel with acetone/chloroform
(20:80, v/v) to give 110 mg as a sticky brown solid contaminated with 4-morpholinoaniline. The
product was further purified by reverse-phase HPLC (Varian Microsorb MV 300-5 C18 column,
250 x 4.6 mm) with trifluoroacetic acid (0.1% in water) and acetonitrile as solvents. A linear
gradient of acetonitrile (10 to 98%) over 30 min. was used in which 4-morpholinoaniline has a
retention time of 9.9 min and product has a retention time of 24 min. The corresponding fraction
was lyophilized to yield the pure product as a powder (6 mg). 1H NMR (200 MHz, CDCl3): 
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(ppm) 1.50 – 2.0 (m, 8H), 2.35 (m, 2H), 3.29 (t, 4H, J = 4.8 Hz), 3.97 (t, 4H, J = 5 Hz), 4.57 (m,
1H), 7.18 (d, 2H, J = 9 Hz), 7.83 (d, 2H, J = 9.2 Hz), 8.16 (s, 1H), 9.55 (s, 1H). MS (ES): [MH+]
413.1.
Purmorphamine: 2-Chloro-6-(4-morpholinoanilino)-9-cyclohexylpurine (6 mg, 14.5 mol),
1-napthol (4 mg, 27.7 mol), Pd2(dba)3 (1 mg, 7.5 mol%), 2-(di-t-butylphosphino)biphenyl
(1.2 mg, 4 mol) and K3PO4 (22 mg, 104 mol) were dissolved in anhydrous toluene (1 mL)
under nitrogen atmosphere. The reaction mixture was then heated at 80 °C for 16 h. The solvent
was removed under reduced pressure and was purified by pipette column over silica gel with
acetone/chloroform (20:80, v/v) to yield the product (5.5 mg, 72%) as a pale yellow solid.
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H NMR (200 MHz, CDCl3):  (ppm) 1.50 – 2.0 (m, 8H), 2.20 (m, 2H), 3.0 (t, 4H, J = 4.8 Hz),
3.83 (t, 4H, J = 5 Hz), 4.47 (m, 1H), 6.46 (d, 2H, J = 8.5 Hz), 6.96 (d, 2H, J = 8.0 Hz), 7.33 –
7.60 (m, 4H), 7.75 – 8.10 (m, 4H). MS (ES): [MH+] 521.2.
Cell-based assays for Hh pathway activation
Assays for Hh pathway activation in Shh-LIGHT2 cells, a clonal NIH-3T3 cell line stably
incorporating Gli-dependent firefly luciferase and constitutive Renilla luciferase reporters, were
conducted as previously described2. Shh-LIGHT2 cells were cultured to confluency in 96-well
plates and then treated with various concentrations of purmorphamine and/or KAADcyclopamine in DMEM containing 0.5% bovine calf serum. The treated cells were then cultured
for 30 h under standard conditions, and firefly and Renilla luciferase activities were determined
using a dual luciferase kit (Promega) according to the manufacturer’s protocols.
Assays for Hh pathway activation in Ptch1–/– cells, fibroblasts derived from mouse
embryos in which each Ptch1 allele has been replaced with -galactosidase reporter, were
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conducted as previously described2. Ptch1–/– cells were cultured to confluency in 96-well plates
and then treated with 100 nM KAAD-cyclopamine and various concentrations of
purmorphamine in DMEM containing 0.5% fetal bovine serum. The treated cells were cultured
for another 30 h under standard conditions. Cell Titer 96 reagent (Promega) was then added to
the culture medium (20 L/well) and the cells were incubated with the viability dye for 2 h at 37
°C. Cell Titer 96 reagent was similarly added to DMEM containing 0.5% fetal bovine serum in
L/well) was then
transferred to another 96-well plate and the 485-nm absorbance of each well was determined to
assess cell growth. The treated Ptch1–/– cells were then washed with 1X PBS buffer (1 x
L) and -galactosidase activities were measured using a chemiluminescence kit
(Tropix) according to the manufacturer’s protocols.
To study Hh pathway activation in the absence of Smo, fibroblasts were derived from Smo–
/–
mouse embryos3. Generation and characterization of this cell line will be described elsewhere
(M. Varjosalo, S.-P. Li, and J. Taipale, manuscript in preparation). The Smo–/– cells were
cultured in 24-well plates until 50% confluency and then co-transfected with Gli-dependent
firefly luciferase and SV40 promoter-driven Renilla luciferase reporters (125 ng/well; 100:1
plasmid ratio) and a mixture of pEGFP-C1 (Clontech) and a CMV promoter-driven Smo-Myc3
(murine Smo containing three consecutive Myc epitopes at the C-terminus) expression construct
(125 ng/well; pEGFP-C1/Smo-Myc3 plasmid ratios of 1:0 and 3:1). FuGene 6 (Roche) was used
as the transfection reagent according to the manufacturer’s protocols. Two days after
transfection, the confluent Smo–/– cells were treated with 0.5 M purmorphamine in DMEM
containing 0.5% fetal bovine serum for 30 h under standard conditions. Firefly and Renilla
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luciferase activities were determined using a dual luciferase kit (Promega) according to the
manufacturer’s protocols.
Smo binding assays
Smo binding assays were conducted with BODIPY-cyclopamine and Smo-overexpressing
cells as previously described4,5, using CMV promoter-based, SV40 origin-containing expression
constructs for Smo-Myc3, the deletion mutant SmoCRD (deletion of amino acids 68 to 182),
and SmoCT (deletion of amino acids 556 to 793). HEK 293T cells were grown on poly-Dlysine-treated glass coverslips in 12-well plates until 70% confluency and then transfected with
the appropriate expression construct (0.5 g/well) using FuGene 6 according the manufacturer’s
protocols.
Two days after transfection, the HEK 293T cells were incubated with DMEM
containing 0.5% bovine calf serum, 5 nM BODIPY-cyclopamine, and varying concentrations of
purmorphamine (0, 1.5, or 5 M) (1 mL/well) for 1 h at 37 °C. The Smo-overexpressing cells
were then washed with 1X PBS buffer (1 mL/well), mounted with DAPI-containing medium,
and visualized using a Leica DM4500B fluorescence microscope. For binding assays using fixed
cells, the Smo-overexpressing HEK 293T cells were fixed with 3% paraformaldehyde in 1X PBS
buffer for 10 min at room temperature (1 mL/well), treated with 1X PBS containing 10 mM
glycine and 0.2% sodium azide for 5 min (1 mL/well), washed with 1X PBS buffer (1 mL/well),
and treated with the compound-containing media described above for 4 h at room temperature.
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Supplementary References
1. Wu, X., et al. J. Am. Chem. Soc. 124, 14520-14521 (2002).
2. Taipale, J., et al. Nature 406, 1005-1009 (2000).
3. Ma, Y., et al. Cell 111, 63-75 (2002).
4. Chen, J. K., et al. Proc. Natl. Acad. Sci. U. S. A. 99, 14071-14076 (2002).
5. Chen, J. K., Taipale, J., Cooper, M. K. & Beachy, P. A. Genes Dev. 16, 2743-2748 (2002).
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