1900 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Costantino et al.
For 7g: yield 53%; mp > 300 °C; 1H NMR (CDCl3) δ 2.2 (s,
3H), 3.3 (br s, 1H, exch with D2O), 4.9 (s, 2H), 7.6-7.7 (m,
6H), 8.3-8.4 (m, 2H). Anal. (C23H15F3N4O3S) C, H, N, S.
Gen er a l P r oced u r e for th e Syn th esis of th e 4-(Su b-
st it u t ed )a m in o-6-p h en yl-p yr id a zin -3-(2H )-on e-2 Acet ic
Acid s (8a -d ). A mixture of the known 1311 (0.13 g; 0.57 mmol)
and the required amine (2.5 mmol) in ethanol (5 mL) was
refluxed overnight. The residue was then purified by flash
chromatography (cyclohexane/ethyl acetate 95/5) to give 14,
which was refluxed with acetic acid (1 mL). After cooling and
alkalinization to pH 8 by diluted sodium hydroxide, the
aminopyridazinones 15 were extracted by CH2Cl2. After drying
over sodium sulfate and evaporation of the solvent, the crude
residue was used as such for the next step. The esters 16 and
their corresponding acids 8 were easily obtained as described
above for 5. (See Table 4 for data.)
NADP+ and with the nearby residues, as indicated by the
binding of other carboxylic acid inhibitors6,7 and previous
modeling results.5,12 Three thousand steps of conjugate gradi-
ent minimization were performed. All protein residues within
10 Å from the inhibitor were allowed to move during minimi-
zation. A distance-dependent dielectric constant with a 4r
dependence and 10 Å nonbonded cutoff were adopted.
Refer en ces
(1) Porte, D., J r.; Schwartz, M. W. Diabetic complications: Why is
glucose potentially toxic? Science 1996, 272, 699-700.
(2) Tomlinson, D. R.; Stevens, E. J .; Diemel, L. Aldose reductase
inhibitors and their potential for the treatment of diabetic
complications. Trends Pharmacol. Sci. 1994, 15, 293-297.
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Synthesis, activity and molecular modeling of a new series of
tryciclic pyridazinones as selective aldose reductase inhibitors.
J . Med. Chem. 1996, 39, 4396-4405.
(6) Wilson, D. K.; Tarle, I.; Petrash, J . M.; Quiocho, F. A. Refined
1.8 Å structure of human aldose reductase complexed with the
protein inhibitor zopolrestast. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 9847-9851.
(7) Urzhumtsev, A.; Tete-Favier, F.; Mitschler, A.; Barbanton, J .;
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En zym e Section . (+)-(S)-6-Fluoro-2,3-dihydrospiro-(4H-1-
benzopyran-4,4′-imidazolidine)-2′,5′-dione (Sorbinil) was a gift
from Pfizer (Groton, CT). Calf lenses for the purification of
Aldose reductase (ALR2, alditol:NADP+ oxidoreductase, EC
1.1.1.21) were obtained locally from freshly slaughtered ani-
mals. The capsule was incised, and the frozen lens was
suspended in sodium potassium phosphate buffer, pH
7
(standard buffer), containing 5 mM DTT (1 g tissue/3.5 mL)
and stirred in an ice cold bath for 1 h. The suspension was
then centrifuged at 22000g at 4 °C for 40 min, and the
supernatant was subjected to ion exchange chromatography
on DE52, affinity chromatography on Orange MatrexA, and
to Sephadex G75 chromatography, as previously described.5
Enzyme activity for all tested enzymes was measured by mon-
itoring the change in absorbance at 340 nm which accompanies
the oxidation of NADPH catalyzed by ALR2. The assay was
performed at 37 °C as previously described,5 using 4.7 mM
D,L-glyceraldehyde as substrate in 0.25 M sodium phosphate
buffer, pH 6.8, containing 0.38 M ammonium sulfate and 0.11
mM NADPH. The sensitivity of the enzymes to inhibition by
different ARIs and newly synthesized compounds was tested
in the above assay conditions by including the inhibitor
dissolved in DMSO at the desired concentration in the reaction
mixture. DMSO in the assay mixture was kept at constant
concentration of 1%. A reference blank containing all the above
reagents except the substrate was used to correct for the
nonenzymatic oxidation of NADPH. IC50 values (the concen-
tration of the inhibitor required to produce 50% inhibition of
the enzyme catalyzed reaction) were determined from least
squares analyses of the linear portion of the log dose-
inhibition curves. Each curve was generated using at least
three concentrations of inhibitor causing an inhibition between
20% and 80% with two replicates at each concentration. The
95% confidence limits (95% CL) were calculated from T values
for n - 2, where n is the total number of determinations.13
Com p u ta tion a l P r oced u r e. Molecular mechanics simula-
tions were performed using the AMBER4.114 program with the
Cornell et al.15 force field on SGI O2 computers. Graphic
display was performed with MIDAS.16 The geometry of inhibi-
tor 5g was completely optimized using the AM1 Hamiltonian.
Parameters for 5g were set consistently to the Cornell et al.15
force field. Missing bond and angle parameters were assigned
on the basis of analogy with known parameters in the database
and calibrated to reproduce the AM1 optimized geometry. The
partial charges on atoms of 5g were calculated from an
electrostatic potential fit to a 6-31G* ab initio wave function
using Gaussian94, followed by RESP analysis.17,18 Parameters
for NADP+ were taken from our previous simulations.5,12 The
crystal structure coordinates of the human ALR2 holoenzyme6
were used. The carboxylate group of the inhibitor 5g was
initially positioned to interact with the nicotinamide ring of
(8) Dal Piaz, V.; Ciciani, G.; Turco, G.; Giovannoni, M. P.; Miceli,
M.; Pirisino, R.; Perretti, M. 5-Acyl-6-aryl-4-nitro-3(2H)-pyridazi-
nones and related 4-amino compounds: Synthesis and pharma-
cological evaluation. J . Pharm. Sci. 1991, 80, 341-348.
(9) Mylari, B. L.; Larson, E. R.; Beyer, T. E.; Zembrowski, W. J .;
Aldinger, C. E.; Dee, M. F.; Siegel, T. W.; Singleton, D. H. Novel,
potent aldose reductase inhibitors: 3,4-dihydro-4-oxo-3[(5-(tri-
fluoromethyl)2-benzothiazolylmethyl]-1-phthalazineacetic acid
(Zopolrestat) and congeners. J . Med. Chem. 1991, 34, 108-122.
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M. Benzothiazol-2-yl-carboxylic acids with diverse spacers:
A
novel class of potent, orally active aldose reductase inhibitors.
Bioorg. Med. Chem. Lett. 1997, 7, 1677-1682.
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general procedure. J . Heterocyclic Chem. 1983, 20, 1473-1476.
(12) Rastelli, G.; Vianello, P.; Barlocco, D.; Costantino, L.; Dal Corso,
A.; Mura U. Structure-based design of an inhibitor modeled at
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lag: New York, 1987.
(14) Pearlman, D. A.; Case, D. A.; Caldwell, J . W.; Ross, W. S.;
Cheatman, T. E., III; Ferguson, D. M.; Seibel, G. L.; Singh, U.
C.; Weiner, P. K.; Kollman, P. A. AMBER 4.1; University of
California: San Francisco, 1995.
(15) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K.
M., J r.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell,
J . W.; Kollman, P. A. A second generation force field for the
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Am. Chem. Soc. 1995, 117, 5179-5197.
(16) Ferrin, T. E.; Huang, C. C.; J arvis, L. E.; Langrifge, R. J . The
MIDAS display system. Mol. Graphics 1988, 6, 13-27.
(17) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A well-
behaved electrostatic potential based method using charge
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Phys. Chem. 1993, 97, 10269-10280.
(18) Cieplak, P.; Bayly, C. I.; Cornell, W. D.; Kollman, P. A. Applica-
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J M981107O