High-Throughput Screening of Cystathionine â-Lyase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 763
removal, the final product was precipitated with cold ether and
further purified using reversed-phase HPLC.
ing to the NCCLS guidelines for using both bacterial and fungal
test organisms in M9 medium with and without added methionine
and in synthetic complete medium without methionine, respectively.
N-Hydrazinocarbonylmethyl-4-nitrobenzamide (14, Method
B). Yield of 23% over two steps. ES-MS (C9H10N4O4 MW
238.20): (ES+) 239.0 [M + H]+, (ES-) 237.0 [M - H]-. 1H NMR
(d6-DMSO): 8.13 (m, 2H, Ar); 8.06 (m, 2H, Ar); 3.70 (s, 2H, CH2).
13C NMR (d6-DMSO): 177.26, 173.45, 136.28, 135.21, 134.10,
130.70, 47.89.
N-Hydrazinocarbonylmethyl-3,5-dinitrobenzamide (15, Method
B). Yield of 31% over two steps. ES-MS (C9H9N5O6 MW
283.20): (ES+) 284.0 [M + H]+, (ES-) 282.2 [M - H]-. 1H NMR
(d6-DMSO): 9.06 (s, 1H, Ar); 8.97 (s, 2H, Ar); 4.32 (s, 2H, CH2).
13C NMR (d6-DMSO): 176.20, 170.20, 155.52, 134.83, 128.26,
124.41, 48.01.
3,5-Diamino-N-hydrazinocarbonylmethyl-benzamide (16,
Method C). Yield of 12% over four steps. ES-MS (C9H13N5O2
MW 223.23): (ES+) 224.2 [M + H]+; (ES-) 222.2 [M - H]-. 1H
NMR (D2O): 7.47 (s, 2H, Ar); 7.24 (s, 1H, Ar); 3.79 (s, 2H, CH2).
13C NMR (D2O): 174.08, 169.47, 142.87, 139.29, 128.83, 127.89,
47.89.
N-Hydrazinocarbonylmethyl-2,6-dimethoxybenzamide (17,
Method B). Yield of 28% over two steps. ES-MS (C11H15N3O4
MW 253.25): (ES+) 254.2 [M + H]+; (ES-) 252.1 [M - H]-. 1H
NMR (D2O): 7.28 (m, 1H, Ar); 6.61 (d, 2H, J 8.5, Ar); 3.68 (s,
6H, CH3); 3.66 (s, 2H, CH2). 13C NMR (D2O): 165.80, 163.54,
161.20, 139.92, 138.94, 111.46, 62.82, 49.31.
Crystallization and X-Ray Structure Determination of CBL-
Inhibitor Complexes. Crystals of CBL-inhibitor complexes were
grown using the hanging-drop vapor diffusion method. The
crystallization conditions were similar to those previously reported
in Clausen et al.18 Specifically, CBL was concentrated to 5 mg/
mL in 5 mM HEPES (pH 7.5), and inhibitor was added to a final
concentration of 1 mM. Large (0.6 mm × 0.6 mm × 0.2 mm) single
crystals grew at room temperature within 1 week upon mixing 2
µL of the protein solution, 2 µL of the precipitant solution
containing 100 mM HEPES (pH 7.3), 150 mM CaCl2, and 26%
PEG400. To obtain the highest-quality diffraction data, the crystals
were soaked sequentially in the following two cryoprotecting
solutions for 14 h and 5 min, respectively: cryosolution I (100
mM HEPES (pH 7.3), 150 mM CaCl2, 30% PEG400, 8 mM
inhibitor) and cryosolution II (100 mM HEPES (pH 7.3), 150 mM
CaCl2, 30% PEG400, 16 mM inhibitor). The crystals were flash
frozen by transferring them directly into a nitrogen cold stream
(100 K).
A single X-ray diffraction data set was collected using an
RAXIS4++ image plate detector and Cu KR radiation from an
RU300 rotating anode (Rigaku MSC). The apparent focal-spot size
was set to 0.3 mm × 0.3 mm. Reflection data were processed,
scaled, and reduced using D*trek28 and truncated using CCP4
(Leslie, 1990). Because the CBL-inhibitor crystals grew in the same
space group (C2221) and with the same unit cell parameters (a )
59.6 Å, b ) 153.0 Å, c ) 151.2 Å) as native CBL (pdb code1CL1),
the native phases were used to solve the structure. Iterative cycles
of refinement and model building were performed with the programs
REFMAC29-32 and Coot,33 respectively. The final models each
contain two monomers of CBL in the asymmetric unit, each bound
to a single inhibitor molecule. Analysis of the Ramachandran plots
for these models calculated by PROCHECK34 indicated that in both
cases 92.6% of the residues are in the most-favored regions with
the remaining 7.4% located in the allowed regions. Crystallographic
data and final refinement statistics can be found in Table 1. Figures
illustrating the structure of CBL were generated using the PyMol
Molecular Graphics System.35
N-Hydrazinocarbonylmethyl-2-trifluoromethylbenzamide (18,
Method B). Yield of 20% over two steps. ES-MS (C10H10F3N3O2
MW 261.20): (ES+) 262.1 [M + H]+; (ES-) 260.0 [M - H]-. 1H
NMR (D2O-d3-acetonitrile ) 1:1): 7.68 (d, 1H, J 6.8, Ar); 7.48
(m, 3H, Ar); 3.91 (s, 2H, CH2).
13C NMR (D2O-d3-acetonitrile ) 1:1): 176.68, 173.92, 149.10,
139.52, 137.69, 135.35, 133.52, 127.51, 125.99, 46.46.
N-Hydrazinocarbonylmethyl-3-trifluoromethylbenzamide (19,
Method B). Yield of 15% over two steps. ES-MS (C10H10F3N3O2
MW 261.20): (ES+) 262.1 [M + H]+; (ES-) 260.0 [M - H]-. 1H
NMR (d6-DMSO): 9.07 (s, 1H, NH); 8.21 (s, 1H, Ar); 8.17 (d,
2H, J 7.5 Hz, Ar); 7.90 (d, 1H, J 7.4 Hz, Ar); 7.69 (t, 1H, J1 7.7
Hz, J2 7.6 Hz, Ar); 3.87 (d, 2H, J 5.7 Hz, CH2). 13C NMR (d6-
DMSO): 175.46, 172.43, 142.13, 138.80, 136.89, 135.25, 131.35,
125.91, 48.73.
N-Hydrazinocarbonylmethyl-4-trifluoromethylbenzamide (20,
Method B). Yield of 16% over two steps. ES-MS (C10H10F3N3O2
MW 261.20): (ES+) 262.1 [M + H]+; (ES-) 260.0 [M - H]-. 1H
NMR (d6-DMSO): 9.33 (s,1H, NH); 8.05 (d, 2H, J 5.7 Hz, Ar);
7.84 (d, 1H, J 8.4 Hz, Ar); 3.85 (d, 2H, J 5.7 Hz, CH2). 13C NMR
(d6-DMSO): 174.26, 170.41, 137.57, 136.81, 135.25, 131.35, 48.81.
2,4,5-Trifluoro-N-hydrazinocarbonylmethyl-3-methoxy-ben-
zamide (21, Method B). Yield of 31% over two steps. ES-MS
Acknowledgment. This work was supported by the Cana-
dian Bacterial Disease Network Centre of Excellence, the
Ontario Research and Development Fund, and by Canada
Research Chair awards to E.D.B. and G.D.W.
Acknowledgment. This Section Tagged Supporting In-
formation
1
(C10H10F3N3O3 MW 277.07): (ES+) 278.2 [M + H]+. H NMR
(d6-DMSO): 7.48 (s, 1H, Ar); 3.96 (s, 2H, CH2), 3.07 (s, 3H,
OCH3). 13C NMR (d6-DMSO): 175.03, 169.25, 166.47, 165.83,
127.30, 121.36, 117.90, 117.10, 69.51, 52.77.
Supporting Information Available: Addtional HPLC and
HRMS data. This material is available free of charge via the Internet
2-Amino-naphthalene-1-carboxylic Acid Hydrazinocarbon-
ylmethyl Amide (22, Method C). Yield of 10% over four steps.
References
1
ES-MS (C13H14N4O2 MW 258.28): (ES+) 259.2 [M + H]+. H
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13C NMR (D2O): 176.34, 173.92, 140.43, 138.01, 137.25,
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N-Hydrazinocarbonylmethyl-2-naphthalen-1-yl-acetamide (23,
Method C). Yield of 25% over 2 steps. ES-MS (C14H15N3O2 MW
257.27): (ES+) 258.2 [M + H]+.
1H NMR (D2O): 8.10 (s, 2H, Ar); 7.72 (m, 4H, Ar); 7.47 (m,
3H, Ar); 4.10 (s, 2H, CH2).
13C NMR (D2O): 176.34, 140.43, 138.01, 137.25, 136.65,
135.52, 134.87, 134.18, 132.67, 130.28, 129.73, 78.87, 48.73, 47.73.
Determination of Minimum Inhibitory Concentrations (MICs).
MICs of hit compounds were determined in duplicate over a test
range of 0.25-128 µg/mL by broth microdilution methods accord-