4610 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Liu and Kodadek
were from Sigma. N-Acetylethylenediamine (technical grade),
N-hydroxysuccinimide (NHS), and anhydrous tetrahydrofuran
(THF) were from Sigma. N,N0-Dicyclohexylcarbodiimide (DCC)
was from Fluka. N-Acetyl-β-alanine (Ac-β-Ala-OH) was from
NOVAbiochem. Boc-β-alanine-(4-carbonylaminomethyl)benzyl
ester copoly(styrenedivinylbenzene) resin (Boc-β-Pam resin,
0.26 mmol/g) was from Peptide International. 4-Nitrobenzophe-
noneoxime copoly(styrenedivinylbenzene) resin (oxime resin LL,
0.85 mmol/g) was from NOVAbiochem. All chemical reagents
were of analytical grade unless were otherwise stated.
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine
serum (FBS), Dulbecco’s phosphate buffered saline, modified
Eagle’s medium (MEM) nonessential amino acids, penicillin-
streptomycin, reduced-serum medium (Opti-MEM 1), and
transfection reagent Lipofectamine Plus were from Invitrogen.
Dimethyl sulfoxide (DMSO, HYBRI-MAX) was from Sigma.
HeLa cells were from the American Type Culture Collection
(ATCC, CCL-2). Cell densities were determined on a Hausser
Scientific hemacytometer. Glucocorticoid receptor (GR) com-
petition assay kit was purchased from Invitrogen.
ImPy3-βDa-NH2 were purified by reversed-phase HPLC on
a preparatory C18 column (start at 15% acetonitrile, 0.25%
acetonitrile/min). The elution gradient for ImPy2-βDa-NH2
and ImPy-βDa-NH2 HPLC purification was 0.5% acetoni-
trile/min, starting at 0% acetonitrile. The collected fractions
were lyophilized. The freeze-dried samples were dissolved in
H2O and purified again by reversed-phase HPLC (2% acetoni-
trile/min) and lyophilized. MALDI-TOF-MS (MHþ): calcd for
ImPy7γβDa-NH2 1264.62, found 1265.22; calcd for ImPy6γβ-
Da-NH2 1142.58, found 1142.89; calcd for ImPy5γβDa-NH2
1020.53, found 1020.88; calcd for ImPy4γβDa-NH2 898.48,
found 899.05; calcd for ImPy4βDa-NH2 813.43, found 813.58;
calcd for ImPy3-βDa-NH2 691.38, found 691.68; calcd for
ImPy2-βDa-NH2 569.33, found 569.85; calcd for ImPy-βDa-
NH2 447.28, found 447.70.
ImPy7γβDa-Sdex (6), ImPy6γβDa-Sdex (7), ImPy5γβDaS-
dex (8), ImPy4γβDa-Sdex (9), ImPy4-βDa-Sdex (10), ImPy3-
βDa-Sdex (11), ImPy2-βDa-Sdex (12), and ImPy-βDa-Sdex
(13). Individual Py-Im amine (1 mg), Sdex-isothiocyanate (2 mg),
and triethylamine (7.5 μL) were added in anhydrous CH2Cl2
(500 μL). The mixture was vortexed at room temperature for 2 h,
and CH2Cl2 was completely removed on a SpeedVac system. The
residue was suspended in 50% acetonitrile and subjected to HPLC
purification on a semipreparatory C18 column, gradient elution of
3.3% acetonitrile/min. The retention times of imidazole-pyrrole
amines, the products, and Sdex-isothiocyanate were 11-14, 17, and
21 min, respectively. The fractions of the products were collected,
freeze-dried, and stored in a desiccate chamber at room tempera-
ture. MALDI-TOF-MS (MHþ): calcd for ImPy7γβDa-Sdex
1757.80, found 1757.21; calcd for ImPy6γβDa-Sdex 1635.75, found
1635.28; calcd for ImPy5γβDaSdex 1513.70, found 1513.76; calcd
for ImPy4γβDa-Sdex 1391.66, found 1392.01; calcd for ImPy4-
βDa-Sdex 1306.60, found 1306.40; calcd for ImPy3-βDa-Sdex
1184.55, found 1184.10; calcd for ImPy2-βDa-Sdex 1062.51, found
1062.95; calcd for ImPy-βDa-Sdex 940.46, found 940.91.
Ac-βDa-Sdex (14). A mixture of Ac-β-Ala-OH (500 mg,
3.8 mmol), N-hydroxysuccinimide (434 mg, 3.8 mmol), and
DCC (824 mg, 4 mmol) in anhydrous THF (10 mL) was stirred
at 0 °C for 1 h and then at room temperature for 12 h. The
solution was clarified by filtration, and THF was removed under
reduced pressure. The white gooey residue (Ac-β-Ala-CO-NHS
ester) was used in the subsequent reaction without purification.
Sdex-isothiocyanate (2 mg, 4 μmol), 3,30-diamino-N-methyldi-
propylamine (15 μL, 93 μmol), and triethylamine (7.5 μL) were
added in anhydrous CH2Cl2 (500 μL). The mixture was vortexed
at room temperature for 2 h. CH2Cl2 was completely removed
on a SpeedVac system. The residue was suspended in 50%
acetonitrile and subjected to HPLC purification on a semipre-
paratory C18 column. Lyophilization yielded a clear oil (NH2-
Da-Sdex). MALDI-TOF-MS (MHþ) calcd for NH2-Da-Sdex
639.34, found 639.52. All NH2-Da-Sdex was dissolved in anhy-
drous CH2Cl2 (500 μL). Triethylamine (7.5 μL) and Ac-β-Ala-
CO-NHS (10 mg) were added, and the mixture was vortexed at
room temperature for 2 h to yield Ac-βDa-Sdex. CH2Cl2 was
completely removed on a speedvac system. The residue was
suspended in 50% acetonitrile and purified by HPLC on a
semipreparatory C18 column. MALDI-TOF-MS (MHþ) calcd
for Ac-βDa-Sdex (14) 752.39, found 752.77.
Ac-Da-Sdex (28). NH2-Da-Sdex was synthesized as described
above and was dissolved in anhydrous CH2Cl2 (500 μL).
Triethylamine (5 μL) and acetic anhydride (10 μL) were added,
and the mixture was vortexed at room temperature for 2 h.
CH2Cl2 was completely removed on a SpeedVac system. The
residue was suspended in 50% acetonitrile and purified by
HPLC on a semipreparatory C18 column. MALDI-TOF-MS
(MHþ) calcd for Ac-Da-Sdex (28) 681.35, found 681.72.
Ac-Et-Sdex (15). Amixture ofN-acetylethylenediamine (15 μL),
triethylamine (7.5 μL), and Sdex-isothiocyanate (2 mg) in anhy-
drous CH2Cl2 (500 μL) was vortexed at room temperature for 2 h.
Purification was done following the procedures described above.
Thin layer chromatography (TLC) was performed on Sigma-
Aldrich silica gel 60 F254 precoated plates. Compounds were
1
visualized under 254 nm ultraviolet lights. H and 13C NMR
spectra were recorded on a Varian Inova 400 or 500 spectro-
meter as noted. UV spectra were measured on a Beckman
Coulter DU640 spectrophotometer. High-resolution electro-
spray ionization mass spectrometry (HRESI-MS) was per-
formed at Resource for Biomedical and Bio-Organic Mass
Spectrometry at Washington University, St Louis, MO. Matrix-
assisted laser desorption/ionization time-of-flight mass spectro-
metry (MALDI-TOF-MS) was carried out on a Voyager-DE
PRO mass spectrometer from PerSeptive Biosystems. High
performance liquid chromatograph (HPLC) was performed
on a Waters HPLC system. Preparatory HPLC was carried
˚
out using a Vydac 22 mm ꢀ 250 mm, 300 A, 10 μm C18 column
with a flow rate of 10 mL/min. Semipreparatory HPLC was
˚
carried out using a Vydac 10 mm ꢀ 250 mm, 300 A, 5 μm C18
column with a flow rate of 5 mL/min. Analytical HPLC
˚
was carried out using a Vydac 4.6 mm ꢀ 250 mm, 300 A, 5 μm
C18 column with a flow rate of 1 mL/min. The elution solu-
tions are H2O (0.1% trifluoroacetic acid (TFA), v/v) and
acetonitrile (0.1% TFA, v/v). HPLC was monitored at 254 nm
wavelength. Each product was analyzed by analytical HPLC.
The purity of products was evaluated as >95% by comparing
the integration of the product peak and the integrations of all
peaks in each chromatogram. Luciferase (Luc) activity was
measured on a Berthold Sirius single-tube luminometer. Fluor-
escence polarization was measured on a Panvera Beacon 2000
fluorometer.
Synthesis. Sdex-isothiocyanate (3, Dex-21-S(CH2)2NCS).
Synthesis was done as described previously.19,18 TLC (20%
ether in CHCl3) Rf = 0.2. Mp = 86-88 °C. 1H NMR (400
MHz, DMSO-d6): δ 7.27 (d, J=10.3 Hz, 1H), 6.20 (d, J=10.3
Hz, 1H), 5.98 (s, 1H), 5.28 (s, 1H), 5.09 (s, 1H), 4.11 (bs, 1H),
3.89 (d, J=16.8 Hz, 1H), 3.81-3.84 (m, 2H), 3.47 (d, J=16.8,
1H), 2.87-2.97 (m, 1H), 2.81-2.87 (m, 2H), 2.55-2.65 (m, 1H),
2.30-2.40 (m, 1H), 2.30 (d, J=11 Hz, 1H), 2.17 (d, J=11 Hz,
1H), 2.08 (dd, J=11, 6.5 Hz, 1H), 1.70-1.80 (m, 1H), 1.58 (dd,
J=11, 6.5 Hz, 1H), 1.47 (1H), 1.44 (s, 3H), 1.25-1.40 (m, 1H),
0.98-1.07 (m, 1H), 0.84 (s, 3H), 0.76 (d, J=6.9 Hz, 3H). 13C
NMR (125 MHz, DMSO-d6): δ 15.2, 16.7, 22.8, 27.1, 30.1, 31.6,
31.8, 33.4, 34.5, 35.9, 43.0, 44.5, 47.3, 47.8, 70.3, 70.6, 90.8,
100.4, 101.8, 124.0, 128.9, 152.5, 167.1, 185.2, 207.1. HRESI-
MS: calcd for C25H32FNNaO4S2 (MNaþ) 516.1654, found
516.1656.
ImPy7γβDa-NH2, ImPy6γβDa-NH2, ImPy5γβDa-NH2, Im-
Py4γβDa-NH2, ImPy4-βDa-NH2, ImPy3-βDa-NH2, ImPy2-
βDa-NH2, and ImPy-βDa-NH2 were synthesized as described
before22 on Boc-β-Pam resin. ImPy7γβDa-NH2, ImPy6γβDa-
NH2, ImPy5γβDa-NH2, ImPy4γβDa-NH2, ImPy4-βDa-NH2,