Journal of Medicinal Chemistry
Article
(2.45 g, 11.2 mmol). The solution was stirred at room temperature for
16 h and then washed with aqueous sodium bicarbonate (∼75 mL),
dried over sodium sulfate, and concentrated. The resulting solid was
purified by flash chromatography over silica (0−5% gradient of
methanol in methylene chloride) to provide tert-butyl (trans-4-((6-
chloropyrazin-2-yl)amino)cyclohexyl)carbamate as a tan solid (2.30 g,
75%): 1H NMR (CDCl3) δ 7.76 (s, 1H), 7.70 (s, 1H), 4.55 (d, J = 7.9
Hz, 1H), 4.50−4.28 (m, 1H), 3.77−3.61 (m, 1H), 3.61−3.39 (m, 1H),
2.20−2.12 (m, 2H), 2.12−2.02 (m, 2H), 1.45 (s, 9H), 1.36−1.19 (m,
4H) ppm.
excess EDTA (100 mM Hepes−NaOH pH 7.5, 0.02% Brij, 0.1% CR-
3, 0.36% DMSO, and 100 mM EDTA). The plate was run for one
cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, MA) in
an off-chip mobility shift assay with an upstream voltage of −2250 V, a
downstream voltage of −500 V, and a vacuum pressure of −1.6 psi.
The LabChip 3000 separates and measures the fluorescent signal of
fluorescein-labeled peptide substrate and fluorescein-labeled peptide
product present in each well. Results are expressed as percent
conversion by measuring peak heights for both the substrate and
product and dividing the product peak height by the sum of peak
heights for both substrate and product. On every plate 100% inhibition
(with a saturating concentration of staurosporine) and 0% inhibition
(substrate with enzyme and DMSO) controls were used to calculate
percent inhibition of tested compounds and a Z′-value.25
Crystallography. Purified human Pim-1 protein (aa 29−313) with
a C-terminal His tag was concentrated to 10−13 mg/mL in 20 mM
HEPES, pH 8, 120 mM NaCl, 5 mM DTT. Pim-1 was crystallized in
0.1 M imidazole, pH 6.4, anhydrous 1−1.4 M sodium acetate by sitting
drop vapor diffusion at 4 °C and reached a maximum size after about 5
days. Pim-1 crystals were soaked with 10 mM compound in the well
solution overnight at 4 °C and flash frozen with 30% glycerol in the
presence of the compound. Diffraction data were collected at home
and at ALS503 (Advanced Light Source at Lawrence Berkeley
National Laboratory, Berkeley, CA) and processed using HKL2000
and Scala. Crystals belong to space group P65, with a = b = 98 Å, c =
80 Å, α = β = 90.00°, γ = 120.00°. The structures were solved by
molecular replacement using the apo Pim-1 structure as a search
model.26 Iterative manual model building was carried out with Coot,27
coupled with refinement using Refmac5. The resolutions (Å) of the
structures elucidated were as follows: 2a, 2.48; 2b, 2.08; 2c, 2.03; 2d;
2.27; 2e, 2.23; 3, 2.23; 6, 1.85.
Step 3. Step 2 product (1.00 g, 3.07 mmol), 3-formylphenylboronic
acid (0.506 g, 3.37 mmol), and potassium carbonate (2.97 g, 21.5
mmol) were taken up in DMF (35 mL) and water (10 mL). Nitrogen
was bubbled through the stirred mixture for 15 min. Following
degassing, bis(triphenylphosphine)palladium(II) dichloride (0.054 g,
0.077 mmol) was added and the mixture was heated at 100 °C for 1.5
h. The reaction was then concentrated and the residue partitioned
between ethyl acetate (∼70 mL) and water (∼100 mL). The organic
layer was washed with additional water (2 × ∼100 mL), dried over
sodium sulfate, and concentrated. The resulting tan solid was purified
by flash chromatography over silica (0−5% gradient of methanol in
methylene chloride). tert-Butyl (trans-4-((6-(3-formylphenyl)pyrazin-
2-yl)amino)cyclohexyl)carbamate was afforded as a light yellow solid
1
(1.12 g, 92%): H NMR (400 MHz, CDCl3) δ 10.11 (s, 1H), 8.49−
8.46 (m, 1H), 8.32 (s, 1H), 8.25−8.21 (m, 1H), 7.96−7.91 (m, 1H),
7.84 (s, 1H), 7.67−7.61 (m, 1H), 4.62 (d, J = 7.6 Hz, 1H), 4.54−4.38
(m, 1H), 3.88−3.74 (m, 1H), 3.59−3.41 (m, 1H), 2.29−2.05 (m, 4H),
1.46 (s, 9H), 1.44−1.28 (m, 4H) ppm.
Step 4. To a stirred suspension of step 3 product (1.10 g, 2.77
mmol) and 2,4-thiazolidinedione (0.341 g, 2.91 mmol) in tert-butanol
(20 mL) was added piperidine (0.22 mL, 2.22 mmol). The mixture
was heated at reflux overnight, cooled slightly, and suction filtered to
remove suspended solid. The filter cake was rinsed with ethanol (2 ×
∼5 mL) and diethyl ether (2 × ∼15 mL). Drying on the filter frit
under house vacuum provided tert-butyl (trans-4-((6-(3-((Z)-(2,4-
dioxothiazolidin-5-ylidene)methyl)phenyl)pyrazin-2-yl)amino)-
ASSOCIATED CONTENT
■
S
* Supporting Information
IC50s and structures of all needle screening hits with a Pim-1
IC50 < 100 μM. This material is available free of charge via the
1
cyclohexyl)carbamate as a pale yellow solid (0.862 g, 63%): H NMR
(400 MHz, DMSO-d6) δ 12.67 (br s, 1H), 8.35−8.31 (m, 1H), 8.29 (s,
1H), 8.13−8.08 (m, 1H), 7.89 (s, 1H), 7.87 (s, 1H), 7.72−7.67 (m,
1H), 7.65−7.60 (m, 1H), 7.14 (d, J = 7.3 Hz, 1H), 6.52 (d, J = 8.0 Hz,
1H), 4.26−4.07 (m, 1H), 3.90−3.72 (m, 1H), 2.15−1.98 (m, 2H),
1.95−1.77 (m, 2H), 1.39 (s, 9H), 1.39−1.23 (m, 4H) ppm.
Accession Codes
†Crystal structure PDB IDs for inhibitors disclosed in this
paper: 3VBQ, 3VBT, 3VBV, 3VBW, 3VBX, 3VBY, and 3VC4.
Step 5. To a stirred suspension of step 4 product (0.300 g, 0.605
mmol) in methylene chloride (8 mL) was added trifluoroacetic acid
(2.0 mL, 26 mmol). After 45 min the reaction was concentrated. The
residue was further dried in a vacuum oven (60 °C) overnight. The
title compound was afforded as a bright yellow solid (0.317 g, 100%):
1H NMR (400 MHz, DMSO-d6) δ 12.70 (br s, 1H), 8.32−8.20 (m,
2H), 8.12−8.03 (m, 1H), 7.98−7.77 (m, 5H), 7.71−7.53 (m, 2H),
7.20 (br s, 1H), 3.87−3.66 (m, 1H), 3.15−2.95 (m, 1H), 2.24−1.88
(m, 4H), 1.58−1.22 (m, 4H) ppm; 13C NMR (100 MHz, DMSO-d6)
δ 167.7, 167.3, 158.3 (d, J = 34.3 Hz; TFA), 153.6, 146.9, 137.9, 133.5,
132.8, 131.5, 131.1, 129.7, 127.9, 127.4, 127.2, 124.0, 116.3 (q, J =
294.9 Hz; TFA); UPLC purity 100% (210 nm), 98% (254 nM);
retention time, 0.74 min; ESMS m/z 396.2 (M + H).
Pim-1 Dose−Response Assay. Reagents and consumables were
purchased from Sigma Aldrich or Caliper Life Sciences. Human Pim-1
was produced internally at Genzyme. All assay reaction conditions for
IC50 determinations were within the linear range with respect to time
and enzyme concentration. In a 384-well polypropylene plate, human
Pim-1 (1.2 nM) was preincubated with a compound in a 100 mM
Hepes−NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM
MgCl2, 0.1% BSA, 1 mM DTT, 10 μM sodium orthovanadate, 10 μM
β-glycerophosphate, and 2.5% DMSO for 15 min at room temper-
ature. The reaction was initiated with an equal volume of a peptide
substrate (5-FAM-RSRHSSYPAGT-CONH2, Caliper Life Sciences)
and ATP mixture in the aforementioned buffer. The final
concentrations in the reaction were 600 pM Pim-1, 1 μM peptide
substrate, and 150 μM ATP (ATP Km). The reaction was incubated at
room temperature for 45 min and terminated with a buffer containing
AUTHOR INFORMATION
■
Corresponding Author
781 290 5890.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
FBDD, fragment-based drug design; HTS, high-throughput
screening; LE, ligand efficiency; PDB, Protein Data Bank; SAR,
structure−activity relationship; SP, standard precision; SPR,
surface plasmon resonance; THZ, thiazolidinedione; XP, extra
precision
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dx.doi.org/10.1021/jm2014698 | J. Med. Chem. 2012, 55, 2641−2648