F. Leonetti et al. / Bioorg. Med. Chem. 16 (2008) 7450–7456
7455
7.1.2. Synthesis of 3,30-(butane-1,4-diylbis(oxy))dibenzenamine
(17)
graphic complex coded 1B41 in the PDB. Using the Protein Prepara-
tion module of Maestro software,26
light relaxation was
a
Compound 18 (332 mg, 1.0 mmol) was dissolved in 60 mL of a
1:1 mixture of ethanol/dioxane, and then Pd ‘black’ (60 mg) was
added. The mixture was stirred for 7 h at room temperature under
H2 pressure (4 bar). The catalyst was removed by filtration through
a pad of CeliteÒ, and the solvent was removed under vacuum
yielded the desired product as a yellow oil with acceptably high
purity (248 mg, 91% yield). MS (ESI) m/z 318 (M+2Na)+; 1H NMR
(CDCl3) d 1.93 (br, 4H), 3.98 (br, 4H), 3.50 (br, 4H), 6.24–6.33 (m,
6H), 7.02–7.07 (m, 2H).
performed for optimizing first hydroxyl and thiol torsion angles
followed by an all-atom constrained minimization to remove steric
clashes until the RMSD reached a value of 0.18 Å.
GOLD 2.2, a genetic algorithm-based software, was used in
the docking study selecting the GOLDScore as the fitness func-
tion. GOLDScore is made up of four components that account
for protein–ligand binding energy: protein–ligand hydrogen bond
energy (external H-bond), protein–ligand van der Waals energy
(external vdw), ligand internal vdw energy (internal vdw), and
ligand torsional strain energy (internal torsion). Empirical param-
eters used in the fitness function (hydrogen bond energies, atom
radii and polarizabilities, torsion potentials, hydrogen bond
directionalities, and so forth) were taken from the GOLD
parameter file. The fitness score is taken as the negative of the
sum of the energy terms, so that larger fitness scores indicated
a better binding. The fitness function has been optimized for
the prediction of ligand binding positions rather than the predic-
tion of binding affinities, although some correlation with the lat-
ter can also be found. The protein input file may be the entire
protein structure or a part of it comprising only the residues
which are in the region of the ligand binding site. In this study,
GOLD was allowed to calculate interaction energies within a
sphere of a 14 Å centered on the middle of the four-methylene
spacer.
7.1.3. Synthesis of 3,30-(butane-1,4-diylbis(oxy))bis(N,N,N-
trimethylbenzenaminium iodide) (16)
Diamine 17 (100 mg, 0.37 mmol) was dissolved in ethanol
(3.0 mL), and then anhydrous K2CO3 (102 mg, 0.74 mmol) and
methyl iodide (230 lL, 3.7 mmol) were added. The mixture was re-
fluxed for 2 h, filtered, and the filtrate was concentrated to dryness.
The resulting crude solid was crystallized from dry ethanol yield-
ing 84 mg (37%) of the title ammonium salt. Mp 172–175 °C dec.
Found: C, 43.50; H, 5.60; N, 4.63. C22H34I2N2O2 requires C, 43.15;
H, 5.60; N, 4.57%; 1H NMR (DMSO-d6) d 1.90 (br, 4H), 3.56 (s,
18H), 4.13 (br, 4H), 7.15–7.17 (m, 2H), 7.45–7.56 (m, 6H).
7.2. Cholinesterase inhibition assay
The inhibition assays on AChE, from bovine erythrocytes
(0.36 U/mg), and BChE from equine serum (13 U/mg) were run in
phosphate buffer 0.1 M, at pH 8.0. Acetyl- and butyryl-thiocoline
iodides were used as substrates and 5,50-dithiobis(2-nitrobenzoic
acid) (DTNB) as the chromophoric reagent. Inhibition assays were
carried out on an Agilent 8453E UV–visible spectrophotometer
equipped with a cell changer. AChE inhibitory activity was deter-
Acknowledgment
The authors gratefully thank MUR, Rome, Italy (PRIN, 2006) for
financial support.
References and notes
mined in a reaction mixture containing 200
AChE (0.415 U/mL in 0.1 M phosphate buffer, pH 8.0), 100
3.3 mM solution of DTNB in 0.1 M phosphate buffer (pH 7.0) con-
l
L of a solution of
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lL of a
taining 6 mM NaHCO3, 100 lL of a solution of the inhibitor (five
to seven concentrations ranging from 1 ꢁ 10ꢀ11 to 1 ꢁ 10ꢀ4 M),
and 500 lL of phosphate buffer, pH 8.0. After incubation for
20 min at 25 °C, acetylthiocholine iodide (100 lL of 0.05 mM water
6. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
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solution) was added as the substrate, and AChE-catalyzed hydroly-
sis was followed by measuring the increase of absorbance at
412 nm for 3.0 min at 25 °C. The concentration of compound which
determined 50% inhibition of the AChE activity (IC50) was calcu-
lated by non-linear regression of the response–concentration
(log) curve, using GraphPad Prism v. 4.0. BChE inhibitory activity
was assessed similarly using butyrylthiocholine iodide (0.05 mM)
as the substrate.
9. Camps, P.; Munoz-Torrero, D. Mini-Rev. Med. Chem. 2001, 1, 163.
10. (a) Pang, Y. P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin, S. J. Biol. Chem. 1996,
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7.3. Computational studies
Computational analyses were conducted on a 16 nodes Linux
Cluster employing an openMosixÒ architecture composed by
AMD Athlon XP 2400+ and Intel Xeon 2600 cpus. All the molecules
were built from the Sybyl fragment libraries.31 Geometrical optimi-
zation and charge calculation were carried out by means of a quan-
tum mechanical method with the PM3 Hamiltonian. Molecules and
models were displayed and manipulated on a Silicon Graphics O2+
machine. The docking poses reported in Figures 1 and 2 were pre-
pared with the graphic system PyMol.32
14. Brühlmann, C.; Ooms, F.; Carrupt, P. A.; Testa, B.; Catto, M.; Leonetti, F.;
Altomare, C.; Carotti, A. J. Med. Chem. 2001, 44, 3195.
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7.4. Docking simulations
18. Hodge, A. S.; Humphrey, D. R.; Rosenberry, T. L. Mol. Pharmacol. 1992, 41, 931.
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Ther. 1989, 249, 194.
20. Camps, P.; Gómez, E.; Muñoz-Torrero, D.; Badia, V.; Clos, M. V.; Curutchet, C.;
Muñoz-Muriedas, V.; Luque, F. G. J. Med. Chem. 2006, 49, 6833.
The target protein was prepared by adding hydrogen atoms,
completing and optimizing missing residues, removing water and
the cocrystallized fasciculin molecule from the hAChE crystallo-