Journal of Medicinal Chemistry
Article
radiofrequency (Rf) tag associated to a unique ID number. In a split-
and-mix approach, Kans undergoing identical reactions were pooled
together in a normal glass vessel that was filled with solvent and
reagents. Upon completion of the reaction, MacroKans were washed
three times sequentially with DMF, MeOH, and DCM, separated,
sorted, and pooled accordingly for next reactions. Cleavage of the final
peptide from the resin with simultaneous removal of protection from
amino acid side chains was performed by treatment with
TFA:H2O:TES (95:2.5:2.5 v/v/v) for 2 h. The resin was filtered
and washed with TFA, and the filtrate and washings were concentrated
to an approximate volume of 1 mL before dilution with Et2O for
precipitation of the azapeptides. The ether layer was decanted after
centrifugation, and the resulting peptide was dissolved in MeCN:H2O
(1:1) and freeze-dried to yield a white foam in average yields of 44%
for peptides 1−15 and 5.1% for peptides 15−49 after purification
using RP-HPLC.
Analytical HPLC analyses were performed on a Gemini column
(4.6 mm × 150 mm, 5 μM, C18) with a flow rate of 0.5 mL/min using
either a 2−40%, 10−50%, or 20−80% gradient from water (0.1% FA)
to CH3CN (0.1% FA) or MeOH (0.1% FA). Azapeptides were
purified using semipreparative LC-MS (Previal C18 column, 22 mm ×
250 mm, particle size 5 μm) with solvent A, H2O (0.1% FA), and
solvent B, acetonitrile (0.1% FA) using a gradient over 20 min at a
flow rate of 10.6 mL/min.
Purity of azapeptides was assessed using a GEMINI column from
Phenomenex (4.6 mm × 150 mm, 5 μM, C18) with a flow rate of 0.5
mL/min using a 2−40%, 10−50%, or 20−80% gradient of water (0.1%
FA) in CH3CN (0.1% FA) or in MeOH using (0.1% FA), and high
resolution mass spectrometry. Azapeptides were purified to 93−99%
purity.
General Procedure for the Synthesis of 9H-Fluoren-9-
ylmethyl carbazate and N′-Alkyl-Fluorenylmethyl carbazates.
9H-Fluoren-9-ylmethyl carbazate (51),40 N′-2-isobutyl-fluorenyl meth-
yl carbazate,18 N′-benzyl-fluorenylmethyl carbazate,18 N′-(4-(t-butyldi-
methylsilyloxy)-benzyl)-fluorenyl-methyl carbazate,18 N′-(1-naphthyl-
methyl)-fluorenylmethyl carbazate,32 and N′-(biphenyl-4-ylmethyl)-
fluorenylmethyl carbazate32 all were synthesized according to literature
procedures.
Synthesis of N′-2-Phenylethyl-fluorenylmethyl Carbazate. A
suspension of 9H-fluoren-9-ylmethyl carbazate in EtOH (0.2 M, 2.5
mmol) was treated with 100 mol % of phenylacetaldehyde (2.5 mmol),
heated at reflux for 2 h, and concentrated in vacuo. The hydrazone was
dissolved in THF (0.2 M) and treated successively with 110 mol % of
AcOH and 110 mol % of NaBH3CN, stirred for 1 h, and treated with
additional NaBH3CN if necessary until completion of the reaction was
observed by TLC. The mixture was concentrated in vacuo. The
residue was dissolved in EtOAc, and the organic solution was washed
with aqueous KHSO4 (1M) and brine, dried over Na2SO4, and
concentrated under reduced pressure to yield a white solid, which was
dissolved in EtOH and heated at reflux for 1 h. The mixture was
concentrated under reduced pressure to yield a residue that was
purified by flash chromatography using 50% EtOAc in hexane as eluant
and isolated as white foam in 76% yield: Rf = 0.26 (50% EtOAc in
hexanes). 1H NMR (DMSO) δ 2.67 (t, J = 7.2 Hz, 2H), 2.94 (m, 2H),
4.24 (t, J = 6.8 Hz, 1H), 4.34 (d, J = 6.8 Hz, 2H), 4.63 (m, 1H), 7.14−
7.36 (m, 7H), 7.42 (t, J = 7.6 Hz, 2H), 7.71 (d, J = 7.2 Hz, 2H), 7.90
(d, J = 7.6 Hz, 2H), 8.78 (brs, 1H). 13C NMR (DMSO) δ 33.9, 46.8,
52.5, 65.6, 120.2 (2C), 125.3 (2C), 125.9, 127.2 (2C), 127.8 (2C),
128.3 (2C), 128.8 (2C), 140.2, 140.8 (2C), 143.9 (2C), 157.0. LRMS
(EI) 359.1 (M + H)+, 381.2 (M + Na)+. HRMS (EI) m/e for
C23H23N2O2 (M + H)+, calcd 396.1918, found 396.1919.
Evaporation of the collected fractions afforded 1-(9H-fluoren-9-
yl)methyl 2-tert-butylpyrazolidine-1,2-dicarboxylate as a low melting
white solid in a 94% yield. Pyrazolidine-1,2-dicarboxylate (1.34 g, 3.4
mmol) was treated with 25 mL of a 1:1 TFA:DCM solution and
stirred for 1 h. Removal of the volatiles by rotary evaporation gave a
residue, which was dissolved in 1N HCl, stirred for 1 h, and freeze-
dried to yield (9H-fluoren-9-pyrazolidine-1-yl)methyl-1-carboxylate
hydrochloride: mp 143.2−148.1 °C, lit. mp 142 °C.30 Spectral
characterization data was identical with that in ref 29.
Membrane Preparation for CD36. Animal use was in accordance
with the Institutional Animal Ethics Committee and the Canadian
Council on Animal Care guidelines for the use of experimental
animals. Sprague−Dawley (275−350 g) rats were necrotized with CO2
until complete loss of consciousness, and their hearts were promptly
removed in ice-cold saline and the cardiac membranes were prepared
according to Harigaya and Schwartz.42
Competitive Covalent CD36 Binding Assay Using Photo-
activatable [125I]-Tyr-Bpa-Ala-Hexarelin as Radioligand. The
radioiodination procedure to prepare the photoactivatable ligand and
the receptor binding assays were performed as previously described by
Bodart et al.35 Briefly, the rat cardiac membranes (200 μg) as source of
CD36 were incubated in the dark in 525 μL of 50 mM Tris-HCl pH
7.4 containing 2 mM EGTA (buffer A) in the presence of a fixed
concentration of [125I]-Tyr-Bpa-Ala-hexarelin (750 000 cpm) in buffer
B (50 mM Tris-HCl pH 7.4 containing 2 mM EGTA and 0.05%
bacitracin) and increasing concentrations of competitive ligand
(ranging from 0.1 to 50 μM). Nonspecific binding was defined as
binding not displaced by 50 μM peptide. After an incubation period of
60 min at 22 °C, membranes were submitted to UV irradiation at 365
nm for 15 min at 4 °C. After centrifugation at 12000g for 15 min, the
pellets were suspended in 100 μL of sample buffer consisting of 62
mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 15% 2-mercapto-
ethanol, and 0.05% bromophenol blue and boiled for 5 min prior to be
subjected to electrophoresis on 7.5% SDS-PAGE. The SDS/PAGE
gels were fixed, colored in Coomassie Brilliant Blue R-250, dried,
exposed to a storage phosphor intensifying screen (Amersham
Biosciences), and analyzed by using a Typhoon PhosphorImager
(Amersham Biosciences) and ImageQuant 5.0 software to establish
competition curves. The band corresponding to the specifically labeled
protein of 87 kDa was quantified by densitometry analysis.
Membrane Preparation for GHS-R1a Receptor. Transfection.
LLC-PK1 cells were seeded at 1.5 × 106 cells/10 cm in Petri dishes
and grown for 24 h in DMEM high-glucose (4.5 g/L) with 10% fetal
bovine serum supplemented with penicillin (10000 units/mL) and
streptomycin (10000 μg/mL), and cultured at 37 °C, under 5% of
CO2. The medium was then replaced for another 4−5 h, before
CaPO4 calcium phosphate transfection. The DNA solution consisted
of 40 μg of DNA in a volume of 500 μL in which was added 500 μL of
2 mM Tris-HCl pH 8.0, and 0.2 mM EDTA pH 8.0 containing 500
mM CaCl2, to a final volume of 1 mL. Then 1 mL of 50 mM Hepes,
280 mM NaCl, 1.5 mM Na2HPO4 pH to 7.1 (HBSS) was added by
alternating 1 drop/2 air bubbles. The transfection mixture was
incubated at RT for 30 min. After the incubation period, 1 mL of the
mix was added to each plate and distributed evenly for incubation. The
media was then replaced with standard DMEM-high glucose media for
another 24 h, and cells were collected for membrane preparation.
Membrane Preparation. The experiment was carried out at 4 °C
unless specified. Cells were washed twice with PBS and with the
homogenization buffer (HB) consisting of 50 mM Tris, 5 mM MgCl2,
2.5 mM EDTA, and 30 μg/mL bacitracin at pH 7.3, and were scraped
into Eppendorf tubes. Cells were lysed with two cycles of freeze/
thawing using liquid nitrogen and were then centrifuged at 4 °C for 20
min at 10000g to collect the membranes. The membranes were
suspended in a small volume of HB, aliquoted, and stored at −80 °C.
GHS-R1a Receptor Binding Assay. The competitive binding assay
employed 200 μL HB, 100 μL 125I-ghrelin (40,000 cpm), 100 μL
competitive ligand (from 10−12 to 10−5 M), and 100 μL of GHS-R1a
transiently transfected in LLC-PK1 cells as source of binding sites (10
μg protein/tube). The nonspecific binding was determined using
excess of competitive ligand at 10−5 M. The reaction was performed at
Synthesis of Fluoren-9-yl-methyl pyrazolidine-1-carboxy-
late hydrochloride. tert-Butyl pyrazolidine-1-carboxylate41 (1.39 g,
8.08 mmol) was treated with Fmoc succinimide (3.27 g, 9.69 mmol,
1.2 equiv) in dry dichloromethane (20 mL) with stirring overnight.
The volatiles were removed using a rotary evaporator. The residue was
dissolved in EtOAc. The organic solution was washed three times,
respectively, with 5% citric acid, 5% NaHCO3 and brine, dried over
Na2SO4, filtered, and concentrated to a white foam, which was purified
by column chromatography using 7:3 EtOAc:hexane as eluant.
6509
dx.doi.org/10.1021/jm300557t | J. Med. Chem. 2012, 55, 6502−6511
Proulx, Caroline
