Novel, highly potent aldose reductase inhibitors: Cyano(2-oxo-2,3-dihydroindol-3-yl)acetic acid derivatives

Article abstract of DOI:10.1021/jm030762f

Cyano(2-oxo-2,3-dihydroindol-3-yl)acetic acid derivatives were synthesized and tested as a novel class of aldose reductase (ALR2) inhibitors. Each compound was evaluated as a diastereomeric mixture, due to tautomeric equilibria in solution. The parent com

Full text of DOI:10.1021/jm030762f

J . Med. Chem. 2003, 46, 1419-1428
1419
Novel, High ly P oten t Ald ose Red u cta se In h ibitor s:
Cya n o(2-oxo-2,3-d ih yd r oin d ol-3-yl)a cetic Acid Der iva tives
Federico Da Settimo,*,§ Giampaolo Primofiore,§ Antonio Da Settimo,§ Concettina La Motta,§ Francesca Simorini,§
Ettore Novellino,‡ Giovanni Greco,‡ Antonio Lavecchia,‡ and Enrico Boldrini†
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Chimica
Farmaceutica e Tossicologica, Universita` di Napoli “Federico II”, Via Domenico Montesano, 49, 80131 Napoli, Italy, and
Farmigea S.p.A., Via Carmignani 2, 56127, Pisa, Italy
Received J anuary 10, 2003
Cyano(2-oxo-2,3-dihydroindol-3-yl)acetic acid derivatives were synthesized and tested as a novel
class of aldose reductase (ALR2) inhibitors. Each compound was evaluated as a diastereomeric
mixture, due to tautomeric equilibria in solution. The parent compound 39 exhibited a good
inhibitory activity with an IC50 value of 0.85 µM, similar to that of the well-known ARI sorbinil
(IC50 0.50 µM). The concurrent introduction of a halogen and a lipophilic group in the 5- and
in the 1-positions, respectively, of the indole nucleus of 39, gave compound 55, cyano[5-fluoro-
1-(4-methylbenzyl)-2-oxo-2,3-dihydroindol-3-yl]acetic acid, which displayed the highest activity
(IC50 0.075 µM, very close to that of tolrestat IC50 0.046 µM), with a good selectivity toward
ALR2 compared with aldehyde reductase (ALR1) (16.4-fold), and no appreciable inhibitory
properties against sorbitol dehydrogenase (SD), or glutathione reductase (GR). The isopropyl
ester 59, a prodrug of 55, was found to be almost as effective as tolrestat in preventing cataract
development in severely galactosemic rats when administered as an eye drop solution. Docking
simulation of 55 into a three-dimensional model of human ALR2 made it possible to formulate
the hypothesis that the 2-hydroxy tautomer was the active species binding into the catalytic
site of the enzyme. This was fully consistent with the structure-activity relationships within
this series of cyanooxoindolylacetic acid derivatives.
Ch a r t 1
In tr od u ction
Progression of chronic diabetes results in long-term
complications, which include neuropathy, nephropathy,
retinopathy and cataract formation. In the tissues
implicated in these pathologies, the excess of free
glucose increases its flux through the polyol pathway
leading to an excessive sorbitol production, which is
thought to cause cellular damage as a result of osmotic
imbalance. Aldose reductase (alditol:NADP+ oxidoreduc-
tase, EC 1.1.1.21, ALR2) is the first enzyme of the polyol
pathway and catalyzes the reduction of glucose by
NADPH to sorbitol, which can in turn be oxidized by
the enzyme sorbitol dehydrogenase (L-iditol:NAD+ 5-ox-
idoreductase, EC 1.1.1.14, SD) and by NAD+ to fructose.
Experimental studies in diabetic animals and men have
shown that aldose reductase inhibitors (ARIs), which
block the flux of the glucose through the polyol pathway
and prevent the intracellular accumulation of sorbitol,
can prevent, retard, or reverse the complications of
chronic diabetes.1-5
Over the past three decades, several ARIs with
diverse structures have been discovered. The structural
classes exhibiting the most potent activity are the spiro-
hydantoins, whose prototype is sorbinil, and the acetic
acid compounds, such as zopolrestat, tolrestat, alresta-
tin, and epalrestat (Chart 1).3,6 However, many of these
compounds have failed clinically due to inadequate
* To whom all correspondence should be addressed. Tel: 39 50
500209. Fax: 39 50 40517. E-mail: fsettimo@farm.unipi.it.
§ Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
‡ Dipartimento di Chimica Farmaceutica e Tossicologica, Universita`
“Federico II” di Napoli.
efficacy, adverse pharmacokinetic properties or toxic
side-effects.5 For these reasons, there is still a need to
† Farmigea S.p.A.
10.1021/jm030762f CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/13/2003

1420 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Da Settimo et al.
F igu r e 1. Superimposition of indole derivative 60 (blue) and
ALR2-bound conformation of zopolrestat (yellow). Note that
the carboxy groups point, in the two structures, toward
different directions.
F igu r e 2. Overlay of 39 (green) on the experimentally
determined ALR2-bound conformation of zopolrestat (yellow)
and TBI derivative (red).
identify and develop clinically effective and well-toler-
ated ARIs.
We have recently described a new class of selective
ARIs: the acetic acid derivatives of [1,2,4]triazino[4,3-
a]benzimidazole (TBI).7 The 10-benzyl TBI derivative
(Chart 1) displayed a high inhibitory activity (IC50 0.36
µM) and was found to be effective in preventing cataract
development in severely galactosemic rats when admin-
istered as an eye drop solution.
As part of an ongoing effort to identify potent and
selective ARIs, in view of our long experience in indole
chemistry,8-11 we decided to support the acetic acid
residue, essential for the inhibition of the enzyme, with
the 2-oxoindole nucleus. It has been reported in the
literature that potent in vitro inhibition of ALR2 has
been observed with a number of acetic acid12 or spiro-
hydantoin13 derivatives of oxoindole. At first, following
a published procedure,14 we prepared the 2-oxoindol-3-
ylacetic acid 60 (Chart 1) and evaluated its ALR2
inhibitory activity. This compound showed only a low
potency, displaying an IC50 value of 10-4 M.
the ALR2 inhibitory activity was indeed improved by 3
orders of magnitude with respect to that of 60.
In this paper we describe the synthesis and the
biological evaluation of a series of cyano(2-oxo-2,3-
dihydroindol-3-yl)acetic acid derivatives (39-57). Com-
pound 55, the most active ALR2 inhibitor among those
examined, and its isopropyl ester 59 were also investi-
gated in vivo for their ability to prevent cataract
development in galactosemic rats. Finally, docking
simulations of 55 into the human ALR2 binding site
were carried out to rationalize the structure-activity
relationships (SARs) observed and to guide, perspec-
tively, the design of new analogues.
Ch em istr y
The synthesis of the title compounds was performed
as outlined in Scheme 1, following a procedure previ-
ously reported for derivative 39.16,20 The appropriately
substituted isatins 1-19 were condensed with cy-
anoacetic acid in dioxane solution, in the presence of
triethylamine to give the indolidene intermediates 20-
38, which were isolated as an E-Z diastereomeric
mixtures (Table 1). These mixtures were used directly
in the following reaction of hydrogenation, performed
in the presence of 10% Pd/C as a catalyst, to obtain the
target acids 39-57. All compounds thus prepared were
characterized as diastereomeric mixtures (Table 2). The
starting substituted isatins 6-19, with the exception
of the commercially available ones 1-5, were prepared
in accordance with a literature procedure involving the
alkylation of isatin 1, 5-fluoroisatin 2, or 5-bromoisatin
3 with the appropriate benzyl halide in DMF solution,
in the presence of sodium hydride.21
To improve the ocular bioavailability of carboxylic acid
55, the more lipophilic ester 59 was also prepared. The
5-fluoro-1-(4-methylbenzyl)isatin 17 was converted with
a good yield to the diastereomeric mixture of the
indolidene intermediate 58 by reaction with isopropyl
cyanoacetate in refluxing isopropyl alcohol in the pres-
ence of piperidine. Hydrogenation of 58 over 10% Pd/C
in an ethanolic solution provided the desired ester 59
as a diastereomeric mixture (Scheme 2; Experimental
Section).
Actually, molecular modeling studies revealed that
compound 60 does not have an excellent overlap with
the ALR2-bound conformation of zopolrestat15 about the
carboxylic groups (Figure 1).
Accordingly, we designed the conformationally con-
strained 2-oxoindolylcyanoacetic acid 3916,17 character-
ized by a coplanar arrangement of the acetic acid chain
with respect to the indole nucleus. As shown in Figure
2, the superposition of 39 (arbitrarily modeled as its R
enantiomer), TBI, and zopolrestat is quite satisfactory
as regards the acetic acid chains and the indole/TBI/
phthalazine systems. The match of the fused-benzene
rings of 39 and TBI suggests that these moieties might
occupy the same domain into the enzyme active site.
A substructure search in the Cambridge Structural
Database (CSD)18 confirmed the rationale of our de-
sign: the retrieved crystal structure of ethyl Z-1-
carboethoxy-2-hydroxy-3-indolinylidenecyanoacetate 61
(reference code layjie, Chart 1),19 a tautomeric form of
the 2-oxoindol-3-ylacetic system, exhibits a nearly total
coplanar arrangement of the cyanoacetic acid fragment
with the indole ring. The attachment of a cyano group
on the R-carbon of the acetic acid residue helps to
increase the coplanarity of the two moieties owing to
the π-conjugation present along the entire system.
Subsequent preparation and testing of 39 revealed that

Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1421
Sch em e 1
Ta ble 1. Physical Properties of Cyano(2-oxo-2,3-dihydroindol-3-ylidene)acetic Acid Derivatives 20-38
no.
X
R
yield (%)
recrystallization solvent
mp (°C)
formulaa
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
F
H
H
H
H
H
50
98
70
96
42
43
67
81
73
57
94
77
81
41
85
61
83
58
36
ethanol
177-180 decb
167-170
C11H6N2O3
ethanol/H2O
methanol
methanol
methanol
ethanol
C11H5FN2O3
C11H5BrN2O3
C11H5N3O5
Br
NO2
OCH3
H
242-245
287-290
188-192 decc
185-189
C12H8N2O4
CH2C6H5
C18H12N2O3
C18H11FN2O3
C19H11F3N2O3
C19H14N2O3
C19H14N2O4
C12H7FN2O3
C13H8ClFN2O3
C14H11FN2O3
C18H11FN2O3
C18H10F2N2O3
C19H10F4N2O3
C19H13FN2O3
C19H13FN2O4
C19H13BrN2O3
H
CH2C6H4-p-F
CH2C6H4-p-CF3
CH2C6H4-p-CH3
CH2C6H4-p-OCH3
CH3
CH2CH2Cl
CH2CH2CH3
CH2C6H5
CH2C6H4-p-F
CH2C6H4-p-CF3
CH2C6H4-p-CH3
CH2C6H4-p-OCH3
CH2C6H4-p-CH3
ethanol
175-179
H
ethanol
165-168
H
ethanol
172-175
H
ethanol
148-152 dec
126-130 dec
154-157 dec
160-163 dec
146-148
F
methanol
methanol
methanol
ethanol
F
F
F
F
ethanol
155-157
F
ethanol
157-160
F
ethanol
168-171
F
ethanol
167-169
Br
methanol
127-128 dec
a
b
Elemental analyses for C, H, N were within (0.4% of the calculated value. Lit.16 mp: 172 °C. c Lit.40 mp: 185-186.
Ta ble 2. Physical Properties and Inhibition of ALR2 by Cyano(2-oxo-2,3-dihydroindol-3-yl)acetic Acid Derivatives 39-57
b
no.
X
R
yield (%)
recrystallization solvent
EtOAc
EtOAc/petroleum ether 60-80 °C 178-180
EtOAc
CH2Cl2
EtOAc
CH2Cl2
CH2Cl2
mp (°C)
179-182 decc
formulaa
IC50 (µM)
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
H
F
H
H
H
H
H
47
30
74
27
50
33
27
23
38
40
62
74
64
13
32
65
45
25
71
C
C
C
C
C
C
C
11H8N2O3
0.85 (0.59-1.10)
0.28 (0.20-0.38)
0.38 (0.25-0.49)
1.20 (0.94-1.46)
5.40 (3.90-7.06)
3.20 (2.44-4.16)
0.57 (0.40-0.74)
11H7FN2O3
11H7BrN2O3
11H7N3O5
Br
NO2
OCH3
H
150-154
177-180
107-110d
126-130
157-160
12H10N2O4
18H14N2O3
18H13FN2O3
CH2C6H5
H
CH2C6H4-p-F
CH2C6H4-p-CF3
CH2C6H4-p-CH3
CH2C6H4-p-OCH3
CH3
H
CH2Cl2/petroleum ether 60-80 °C 151-154
EtOAc/petroleum ether 60-80 °C 169-172
CH2Cl2/petroleum ether 60-80 °C 149-152
EtOAc/petroleum ether 60-80 °C 170-174
EtOAc/petroleum ether 40-60 °C 87-90
EtOAc/petroleum ether 40-60 °C 97-100
CH2Cl2/petroleum ether 60-80 °C 168-172
CH2Cl2/petroleum ether 60-80 °C 149-152
EtOAc/petroleum ether 60-80 °C 80-81
EtOAc/petroleum ether 60-80 °C 173-176
CH2Cl2/petroleum ether 60-80 °C 166-169
C19H13F3N2O3 0.57 (0.43-0.69)
H
C
19H16N2O3
C19H16N2O4
C12H9FN2O3
0.64 (0.44-0.88)
0.63 (0.44-0.80)
0.39 (0.32-0.47)
H
F
F
CH2CH2Cl
CH2CH2CH3
CH2C6H5
CH2C6H4-p-F
CH2C6H4-p-CF3
CH2C6H4-p-CH3
CH2C6H4-p-OCH3
CH2C6H4-p-CH3
C13H10ClFN2O3 1.34 (0.92-1.74)
F
C
14H13FN2O3
C18H13FN2O3
0.28 (0.22-0.35)
0.18 (0.12-0.22)
F
F
C18H12F2N2O3 0.13 (0.09-0.17)
F
C
C
19H10F4N2O3 0.28 (0.23-0.35)
F
19H15FN2O3
C19H15FN2O4
0.075 (0.059-0.090)
0.11 (0.08-0.13)
F
Br
CH2Cl2
167-171
C
19H15BrN2O3 0.25 (0.19-0.30)
0.50 (0.39-0.62)
sorbinil
tolrestat
quercetin
0.046 (0.036-0.055)
8.32 (6.90-9.91)
a
b
Elemental analyses for C, H, N were within (0.4% of the calculated value. IC50 values represent the concentration required to
produce 50% enzyme inhibition. c Lit.16 mp: 179 °C. Lit.40 mp 164-165 °C.
d
Biology a n d P h a r m a cology
enzymatic extract of rat lenses.22-25 IC50 values were
determined by using linear regression analysis of the
log concentration-response curve. Sorbinil,26 tolr-
estat,27,28 and quercetin29 were used as the reference
standards. Selected compounds (40, 47, 53, 55), which
The diastereomeric mixtures of the target compounds
39-57 were used as such to evaluate their in vitro
inhibitory activity against ALR2. Primary in vitro
screening was performed on a water-soluble crude

1422 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Da Settimo et al.
Sch em e 2
Ta ble 3. Enzyme Inhibition Data of Selected Compounds 40, 47, 53, and 55
aldose reductase
aldehyde reductase
sorbitol dehydrogenase
glutathione reductase
a
a
a
a
no.
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
40
47
53
55
sorbinil
tolrestat
quercetin
0.28 (0.19-0.37)
0.46 (0.32-0.69)
0.058 (0.044-0.071)
0.042 (0.030-0.055)
0.65 (0.49-0.82)
0.05 (0.03-0.06)
7.81 (5.47-10.15)
6.79 (4.75-8.82)
3.57 (2.50-4.64)
0.49 (0.31-0.63)
0.69 (0.48-0.89)
0.029 (0.020-0.038)
n.a.
n.a.b
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
2.32 (2.05-2.78)
35.6 (31.6-40.0)
48.8 (34.7-63.4)
a
b
IC50 values represent the concentration required to produce 50% enzyme inhibition. n.a. ) not active.
Ta ble 4. Effect of Treatment with Ophthalmic Solution of 55,
59, and Tolrestat on Development of Nuclear Cataract in
Severely Galactosemic Rats
demonstrated significant inhibitory activity, were as-
sayed for their ability to inhibit ALR2 purified from rat
lenses, SD,30 and two other enzymes not involved in the
polyol pathway, namely aldehyde reductase (ALR1),31
and glutathione reductase (GR).32
The most active derivative 55 and its isopropyl ester
59 were investigated in vivo as eye drop solutions in
the precorneal region for their ability to prevent cataract
development in severely galactosemic rats.26 Their
effectiveness was evaluated with respect to tolrestat as
a highly potent reversible inhibitor of lenticular ALR2
when topically administered to rats fed with 50%
galactose diet.28
rats with nuclear cataract (%)
day of
treatment
55
(3%)
59
(3%)
tolrestat
(1%)
tolrestat
(3%)
control
11
12
13
14
15
16
17
18
19
20
21
13
25
25
25
25
31
50
50
75
88
100
0
0
0
0
0
0
0
0
0
0
0
8
8
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
18
43
57
57
72
84
96
100
23
32
32
32
43
43
47
47
Resu lts a n d Discu ssion
Biologica l Eva lu a tion . To deduce sound structure-
activity relationships, IC50s should on principle be
determined on pure inhibitors. One drawback of testing
mixtures of stereoisomers, unavoidable in our case, was
that we could not assess how each stereoisomer affected
the enzyme. On the other hand, the IC50 values calcu-
lated can be used as a screening method to select
promising inhibitors and to identify the diastereomeric
mixture with the best ability to inhibit the enzyme. All
the cyanoindolylacetic acids 39-57 exhibited a high
inhibitory activity, with IC50 values in the low/sub-
micromolar range (Table 2). In the series of the 1-un-
substituted derivatives 39-43, the best potency was
shown by the 5-halo compounds 40 and 41. All the 5-H
indoles, substituted with a lipophilic area on the indole
nitrogen (44-48), demonstrated almost the same in-
hibitory potency, 44 being the only exception. The 5-F
and 5-Br derivatives 49-57 displayed the best ability
to inhibit ALR2, with compound 55 being the most
potent inhibitor of the whole series, showing an IC50
value (0.075 µM) similar to that of tolrestat (0.046 µM),
and lower than that of sorbinil (0.50 µM).
solution in the precorneal region to investigate its in
vivo ability to prevent cataract development in severely
galactosemic rats. Topical administration can in prin-
ciple achieve significant drug levels in the lens with
negligible effects on other tissues, thus avoiding bio-
availability and/or metabolism-related problems associ-
ated with systemic administration.28 It has been
reported33-35 that esters of acid drugs display a better
corneal permeability and ocular bioavailability by virtue
of their higher lipophilicity. Considering that isopropyl
esters have shown a good permeability in the corneal
tissue,33 the isopropyl ester 59 was prepared and tested
in vivo as a prodrug of compound 55. The pharmacologi-
cal data are reported in Table 4. After 21 days of 50%
galactose diet, 100% of the animals treated only with
vehicle developed nuclear cataract. No protection was
observed in those treated with 3% ophthalmic solution
of the acid 55, whereas an almost complete protection
was detected in rats treated with 3% solution of the
isopropyl ester 59. No nuclear cataract was developed
by animals administered 3% ophthalmic solution of
tolrestat. The high potency displayed by the prodrug 59
with respect to the acid 55 suggests that its higher
lipophilicity is essential for a good permeability in
corneal tissue and that its inhibitory activity is ensured
by a quick hydrolysis to the active acid by corneal
esterases.
Finally, the selected compounds 40, 47, 53, and 55,
when assayed for their ability to inhibit purified ALR2
and other related enzymes (ALR1, SD, and GR), were
all found to be selective inhibitors of ALR2, inhibiting
ALR1 with much less potency (from 8- to 24-fold), and
showing no appreciable inhibitory properties toward SD,
or GR (Table 3).
P h a r m a cologica l Eva lu a tion of Com p ou n d s 55
a n d 59. Compound 55 was administered as an eyedrop
Molecu la r Mod elin g. Derivatives 39-57 are ca-
pable of tautomeric isomerism, as shown in Figure 3.

Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1423
F igu r e 3. Tautomeric equilibria of cyano(2-oxo-2,3-dihydroindol-3-yl)acetic acid derivatives 39-57.
The 1H NMR spectrum of 55 recorded in dimethyl
sulfoxide, a polar nonprotic solvent, showed the presence
of only the carbonyl form I. Nothing can be said about
the tautomeric form present in water solution, since
with deuterium oxide the two signals relative to the H-3
and the acetic CH disappeared, showing that their
acidity/mobility was certainly due to the equilibrium
among the three tautomeric forms.
However, the most stable tautomer in the gas or
solution phases is not necessarily the one preferred by
the enzyme. We hypothesize that our compounds bind
to the enzyme in the hydroxy form III because this
tautomer is not only better superimposable on the
ALR2-bound conformation of zolpolrestat (Figure 2), but
it also fits nicely into the inhibitory site (see below).
Moreover, the crystal strucuture of the cyanoacetate
derivative 6119 (Chart 1), structurally similar to 55, is
frozen in the hydroxy form III, thus suggesting that this
tautomeric arrangement is energetically feasible. This
tautomer receives further stabilization from an in-
tramolecular hydrogen bond formed between the 2-hy-
droxy group and one of the two carboxylate oxygens.
Even though the IC50 values were from diastereomeric
mixtures, we thought it interesting to rationalize the
results obtained within this series of cyano derivatives,
and to formulate a hypothesis about their binding mode
at the active site of the enzyme. For this purpose, we
docked the most potent inhibitor 55 (frozen in the
hydroxy tautomeric form III) into the previously mod-
eled structure of human ALR2.7
F igu r e 4. Schematic representation of the main interactions
observed in the MD simulation of the ALR2/NADP+/55 com-
plex. The interatomic distances are given as mean values of
those observed during the MD simulation, accompanied by
standard deviation.
simulations in a solvated system at room temperature
in order to relieve steric repulsive conflicts between the
flexible amino acid side chains of the enzyme and the
docked ligand.
General binding features are schematically repre-
sented in Figure 4 together with interatomic distances
for all important polar interactions. Details of the
binding mode of the two enantiomers are shown in
Figure 5, where only the amino acids located within 5
Å of the bound ligand are depicted.
The bioactive conformation of the TBI derivative7 and
the experimentally determined ALR2-bound conforma-
tion of zopolrestat15 served as templates to select a trial
conformation of 55 for docking calculations. By manual
adjustment of the torsional angles τ1, τ2, and τ3 (defined
in Figure 4), we identified a low-energy conformation
of 55 superimposable on TBI and zopolrestat about the
carboxylic acid groups and the p-tolyl/phenyl/benzothia-
zole moieties.
Both R and S enantiomers of the cyanoindolylacetic
derivative 55 were considered for docking, because all
the investigated compounds were assayed as racemic
mixtures. These enantiomers are isoenergetic and differ
only in the position of the 2-hydroxy group, which may
project above or below the plane of the ligand, giving
rise in both cases to a hydrogen bond with one of the
two carboxylate oxygen atoms.
Docking was carried out using the automated DOCK
software package,36-39 following the same protocol
described in a previous paper.7 Interestingly, the best-
scoring docking orientation of both (R)-55 and (S)-55
yielded the most satisfactory superposition on the ALR2-
bound conformation of zopolrestat.
The geometries of the ALR2/NADP+/(R)-55 and ALR2/
NADP+/(S)-55 complexes were refined by extensive
energy minimization and molecular dynamics (MD)
As the degree of stereoelectronic complementarity
between the docked enantiomers and the enzyme,
estimated on the basis of the interaction energies, was
rather similar, we predict that the enantiomers of each
inhibitor should not exhibit significant differences in
potency.
As illustrated in Figure 4, the negatively charged
carboxylate group of the cyano derivative 55 makes a
tight hydrogen-bonding network with Tyr48, His110,
and Trp111 of ALR2. Particularly, the Nꢀ2 hydrogen of
His110 is hydrogen-bonded to both of the carboxylate
oxygen atoms O2 and O3, while the Oη hydrogen of Tyr48
and the Nꢀ1 hydrogen of Trp111 form a hydrogen bond
with each of the two carboxylate oxygens. Coordination
of the inhibitor’s carboxylate group to the anionic
binding site was maintained for the entire simulation
time, suggesting that this group is the primary deter-
minant for binding of 55 and its derivatives. Another
favorable interaction between the cyano nitrogen atom
of the ligand and the Nꢀ1 hydrogen of Trp20 was also
shown to contribute to the complex stabilization, al-
though it was observed to be frequently cleaved in the
simulation, giving an average distance longer than that
for an ideal hydrogen bond.
The p-tolyl moiety of 55 fits into the specificity pocket
made up of four aromatic residues (Trp79, Trp111,

1424 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Da Settimo et al.
F igu r e 5. (R)-55 (top) and (S)-55 (bottom) enantiomers docked into the ALR2 binding site. Only amino acids located within 5 Å
distance from the bound ligand are displayed and labeled. The hydrogen bonds discussed in the text are depicted as black dashed
lines.
Phe115, and Phe122), two nonpolar residues (Val130,
Leu300) and five polar residues (Cys80, Thr113, Cys298,
Ser302, Cys303) (Figure 5).
Exp er im en ta l Section
1. Ch em istr y. Melting points were determined using a
Reichert Ko¨fler hot-stage apparatus and are uncorrected. IR
spectra were recorded with a Pye Unicam Infracord Model PU
9516 in Nujol mulls. Routine 1H NMR spectra were determined
on a Varian CFT 20 Spectrometer operating at 80 MHz, using
tetramethylsilane (TMS) as the internal standard and DMSO-
d6 as the solvent. Mass spectra were obtained on a Hewlett-
Packard 5988 A spectrometer using a direct injection probe
and an electron beam energy of 70 eV. Evaporations were
performed in vacuo (rotary evaporator). Analytical TLC was
carried out on Merck 0.2 mm precoated silica gel (60 F-254)
aluminum sheets, with visualization by irradiation with a UV
lamp. Elemental analyses were performed by our Analytical
Laboratory and agreed with theoretical values to within
(0.4%.
The alkyl halides 1-iodopropane, 1-bromo-2-chloroethane,
benzyl bromide, 4-fluorobenzyl chloride, 4-methylbenzyl bro-
mide, 4-(trifluoromethyl)benzyl bromide, and 4-methoxybenzyl
chloride used to obtain compounds 13, 12, 14, 7, 15, 17, 19, 8,
16, 18, respectively, and isatin 1, 5-fluoroisatin 2, 5-bromoi-
satin 3, 5-nitroisatin 4, were from Sigma-Aldrich.
The following products were prepared in accordance with
literature procedures: 5-methoxyisatin 5, mp 198-201 °C
(lit.40 mp 202 °C); 1-benzylisatin 6, mp 130-131 °C (lit.41 mp
131-131.5 °C); 1-(4-methylbenzyl)isatin 9, mp 142-143 °C
(lit.41 mp 141 °C); 1-(4-methoxybenzyl)isatin 10, mp 168-170
°C (lit.42 mp 171-172 °C); 5-fluoro-1-methylisatin 11, mp 157-
159 °C (lit.43 mp 151 °C); 2-oxoindol-3-ylacetic acid 60, mp
142-144 °C (lit.14 mp 140-142 °C).
Phe122 and Trp219 are involved in hydrophobic
contacts with the indole nucleus of the inhibitor. Par-
ticularly, the benzene ring of Phe122 appears to be
optimally oriented for a favorable π-stacking interaction
with the fused aromatic ring of 55: the planes of the
two rings are fairly parallel and separated by a distance
of 4.7 Å. The Trp219 side chain also interacts with the
fused aromatic ring of the inhibitor via a T-shaped
interaction. Such interactions would be consistent with
the activity trend of these compounds showing that
lipophilic and electron-withdrawing substituents in the
5-position of the indole nucleus increase the potency.
In fact, the electron-deficient halogenated fused ring of
indole would more favorably realize a π-stacking charge-
transfer interaction with the electron-rich ring of Phe122,
while the replacement of fluorine or bromine with a
nitro group (42), which has greater electron-withdraw-
ing nature but less lipophilicity than halogens, de-
creases activity.
Con clu sion s
We have described a novel class of highly potent
ARIs: the cyano(2-oxo-2,3-dihydroindol-3-yl)acetic acid
derivatives. Compound 55, the most potent derivative
of the series, exhibited an ALR2 inhibitory activity (IC50
0.075 µM) very close to that of tolrestat (IC50 0.046 µM),
with a 16.4-fold higher selectivity toward ALR2 com-
pared with ALR1, and no appreciable inhibitory proper-
ties against SD or GR. The isopropyl ester 59, a prodrug
of 55, proved to be almost as effective as tolrestat in
preventing cataract development in severely galac-
tosemic rats when administered as an eye drop solution.
Gen er a l P r oced u r e for th e Syn th esis of 1-Alk ylisa tin s
7, 8 a n d 5-Su bstitu ted -1-a lk ylisa tin s 12-19. Sodium hy-
dride (12 mmol, 50% dispersion in mineral oil) was added
portionwise, under a nitrogen atmosphere, to an ice-cooled
solution of isatin 1, 5-fluoroisatin 2, or 5-bromoisatin 3 (10
mmol) in 5 mL of freshly distilled DMF. Once hydrogen
evolution had ceased, the appropriate alkyl halide (12 mmol)
was added dropwise, and the reaction mixture was maintained
under stirring, at room temperature, until the disappearance
of the starting material (0.5-3 h, TLC analysis). The solution
was then slowly poured onto crushed ice, and the solid

Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1425
precipitate was collected, washed with water, and recrystal-
lized. Yields, recrystallization solvents, melting points, and
spectral data for the newly synthesized compounds are listed
below.
of hydrogen was achieved (in the case of the 5-nitro derivative
23 the reduction reaction was stopped after the absorption of
1 mol of hydrogen). The catalyst was then filtered off, and the
solvent was evaporated to dryness under reduced pressure to
obtain an oily residue which solidified by treatment with ice-
cooled petroleum ether (40-60 °C or 60-80 °C). The crude
solid was collected and recrystallized from the appropriate
solvent. Yields, recrystallization solvents, and melting points
of the products, characterized as diastereomeric mixtures, are
reported in Table 2. The specral data of 53 and 55, which are
representative of the title compounds, are listed below.
Cya n o[5-flu or o-1-(4-flu or oben zyl)-2-oxo-2,3-d ih yd r oin -
d ol-3-yl]a cetic Acid 53. IR ν cm-1: 3220-2250, 2200, 1720,
1640. 1H NMR δ: 4.20-4.46 (m, 1H, 3-H, exch. with D2O);
4.87, 4.92 (2s, 2H, CH2); 4.97-5.07 (m, 1H, CHCOOH, exch.
with D2O); 6.76-7.47 (m, 7H, ArH).
1-(4-F lu or oben zyl)isa tin 7. mp 148-149 °C (ethanol);
1
yield 52%. IR ν cm-1: 1720, 1600. H NMR δ, CDCl3: 4.87 (s,
2H, CH2); 6.69-7.62 (m, 8H, ArH). Anal. (C15H10FNO2) C, H,
N.
1-(4-Tr iflu or om eth ylben zyl)isa tin 8. mp 120-121 °C
1
(toluene); yield 35%. IR ν cm-1: 1740, 1720, 1600. H NMR δ,
CDCl3: 5.06 (s, 2H, CH2); 6.75-7.74 (m, 8H, ArH). Anal.
(C16H10F3NO2) C, H, N.
1-(2-Ch lor oeth yl)-5-flu or oisa tin 12. mp 101-102 °C (pe-
troleum ether 60-80 °C); yield 88%. IR ν cm-1: 1730, 1610.
1H NMR δ: 3.84 (t, 2H, NCH2); 4.05 (t, 2H, CH2Cl); 7.30-
7.60 (m, 3H, ArH). Anal. (C10H7ClFNO2) C, H, N.
5-F lu or o-1-n -p r op ylisa tin 13. mp 80-81 °C (petroleum
Cyan o[5-flu or o-1-(4-m eth ylben zyl)-2-oxo-2,3-dih ydr oin -
d ol-3-yl]a cetic Acid 55. IR ν cm-1: 3200-2400, 2275, 1750,
1
ether 60-80 °C); yield 85%. IR ν cm-1: 1730, 1610. H NMR
1
1640. H NMR δ: 2.25 (s, 3H, CH3); 4.36-4.44 (m, 1H, 3-H,
δ: 0.89 (t, 3H, CH3); 1.54-1.65 (m, 2H, CH2); 3.61 (t, 2H,
NCH2); 7.19-7.56 (m, 3H, ArH). Anal. (C11H10FNO2) C, H, N.
1-Ben zyl-5-flu or oisa tin 14. mp 131-132 °C (ethanol);
yield 51%. IR ν cm-1: 1740, 1700, 1620, 1610. 1H NMR δ,
CDCl3: 4.90 (s, 2H, CH2); 6.73-7.36 (m, 8H, ArH). Anal.
(C15H10FNO2) C, H, N.
exch. with D2O); 4.81, 4.87 (2s, 2H, CH2); 4.94-5.06 (m, 1H,
CHCOOH, exch. with D2O); 6.69-7.35 (m, 7H, ArH).
Cyan o[5-flu or o-1-(4-m eth ylben zyl)-2-oxo-2,3-dih ydr oin -
d ol-3-ylid en e]a cetic Acid Isop r op yl Ester 58. A suspension
of 5-fluoro-1-(4-methylbenzyl)isatin 17 (0.269 g, 1 mmol) and
isopropyl cyanoacetate (0.15 mL, 1.2 mmol) in isopropyl alcohol
(5 mL) containing piperidine (0.12 mL, 1.2 mmol) was heated
under reflux for 15 min. After cooling, a deep purple solid
separated, which was collected and recrystallized from iso-
propyl alcohol to give 0.226 g (yield 60%) of pure 58 as an E-Z
diastereomeric mixture. mp 182-184 °C. IR, ν cm-1: 1710,
5-F lu or o-1-(4-flu or oben zyl)isa tin 15. mp 157-158 °C
(petroleum ether 60-80 °C); yield 44%. IR ν cm-1: 1740, 1720,
1
1620, 1600. H NMR δ. CDCl3: 4.88 (s, 2H, CH2); 6.61-7.37
(m, 7H, ArH). Anal. (C15H9F2NO2) C, H, N.
5-F lu or o-1-(4-tr iflu or om eth ylben zyl)isa tin 16. mp 117-
118 °C (ethyl acetate/petroleum ether 60-80 °C); yield 45%.
IR ν cm-1: 1730, 1710, 1600. 1H NMR δ: 4.96 (s, 2H, CH2);
6.58-7.64 (m, 7H, ArH). Anal. (C16H9F4NO2) C, H, N.
5-F lu or o-1-(4-m eth ylben zyl)isa tin 17. mp 140-141 °C
(petroleum ether 60-80 °C); yield 54%. IR ν cm-1: 1740, 1720,
1
1280, 1260. H NMR, δ: 1.34 (d, 6H, CH(CH3)2); 2.25 (s, 3H,
CH3); 4.88 (s, 2H, CH2); 5.18-5.21 (m, 1H, CH); 6.97-8.05 (m,
7H, ArH). MS: m/e 378 (M)+, 105, base. Anal. (C22H19FN2O3)
C, H, N.
1
Cyan o[5-flu or o-1-(4-m eth ylben zyl)-2-oxo-2,3-dih ydr oin -
d ol-3-yl]a cetic Acid Isop r op yl Ester 59. A solution of the
ester 58 (0.378 g, 1 mmol) in isopropyl alcohol (10 mL) was
hydrogenated with 10% Pd/C as a catalyst at atmospheric
pressure and room temperature until the theoretical uptake
of hydrogen was achieved. The catalyst was then filtered off,
and the solvent was evaporated to dryness under reduced
pressure to an oily product which solidified by treatment with
ice-cooled petroleum ether 40-60 °C.
1600. H NMR δ: 2.31 (s, 3H, CH3); 4.85 (s, 2H, CH2); 6.61-
7.40 (m, 7H, ArH). Anal. (C16H12FNO2) C, H, N.
5-F lu or o-1-(4-m eth oxyben zyl)isa tin 18. mp 134-137 °C
(ethanol); yield 48%. IR ν cm-1
:
1720, 1600. 1H NMR δ,
CDCl3: 3.76 (s, 3H, CH3); 4.83 (s, 2H, CH2); 6.63-7.31 (m, 7H,
ArH). Anal. (C16H12FNO3) C, H, N.
5-Br om o-1-(4-m eth ylben zyl)isa tin 19. mp 156-158 °C
(ethyl acetate); yield 75%. IR ν cm-1: 1780, 1760, 1630. 1H
NMR δ: 2.26 (s, 3H, CH3); 4.84 (s, 2H, CH2); 6.85-7.76 (m,
7H, ArH). Anal. (C16H12BrNO2) C, H, N.
The resulting white solid was collected and recrystallized
from isopropyl alcohol to obtain 0.304 g (yield 80%) of pure 59
as a diastereomeric mixture. mp 110-112 °C. IR, ν cm-1: 1740,
Gen er a l P r oced u r e for th e Syn th esis of Cya n o(2-oxo-
2,3-d ih yd r oin d ol-3-ylid en e)a cetic Acid s 20-38. A solution
of cyanoacetic acid (1.02 g, 12 mmol) and triethylamine (1.7
mL, 12 mmol) in dioxane (10 mL) was added dropwise to an
ice-cooled suspension of the appropriate isatin 1-19 (10 mmol)
in dioxane (10 mL). Once addition was complete, stirring was
continued at room temperature until the disappearance of the
starting material (2-4 h, TLC analysis). Then, concentrated
hydrochloric acid (0.3 mL) was added slowly, under stirring
and with cooling, to the dark red reaction mixture. The
resulting solution was left at room temperature for 5-6 days
until the crude product separated. The precipitate was col-
lected, washed with water, and recrystallized from the ap-
propriate solvent. Yields, recrystallization solvents, and melt-
ing points of the target compounds, characterized as an E-Z
diastereomeric mixture, are reported in Table 1. The spectral
data for 21 and 28, which are representative for the title
compounds, are listed below.
1
1700. H NMR, δ: 1.10-1.22 (m, 6H, CH(CH3)2); 2.24 (s, 3H,
CH3); 4.54-5.24 (m, 5H, CH-CH, OCH, NCH2); 6.88-7.25 (m,
7H, ArH). MS: m/e 380 (M)+, 105, base. Anal. (C22H21FN2O3)
C, H, N.
2. Biology. 2.1. Ma ter ia ls a n d Meth od s. Aldose reductase
(ALR2) and aldehyde reductase (ALR1) were obtained from
Sprague Dawley albino rats, 120-140 g b.w., supplied by
Harlan Nossan, Italy. In ordered to minimize cross-contamina-
tion between ALR2 and ALR1 in the enzyme preparation, rat
lens, in which ALR2 is the predominant enzyme, and kidney,
where ALR1 shows the highest concentration, were used for
isolation of ALR2 and ALR1, respectively.
Glutathione reductase (GR) type IV from bakers’yeast (100-
300 U/mg), sorbitol dehydrogenase (SD) from sheep liver (10
U/mg of protein), pyridine coenzymes, D,L-glyceraldehyde,
glutathione disulfide, sodium D-glucuronate, sorbitol, and
quercetin were from Sigma Chemical Co. Sorbinil was a gift
from Pfizer, Groton CT. Tolrestat was obtained from Lorestat
Recordati, Italy. All other chemicals were of reagent grade.
2.2. En zym e P r ep a r a tion . 2.2.1. Ald ose Red u cta se
(ALR2). A purified rat lens extract was prepared in accordance
with the method of Hayman and Kinoshita44 with slight
modifications. Lens were quickly removed from normal killed
rats and homogenized (Glas-Potter) in 3 volumes of cold
deionized water. The homogenate was centrifuged at 12 000
rpm at 0-4 °C for 30 min. Saturated ammonium sulfate
solution was added to the supernatant fraction to form a 40%
solution, which was stirred for 30 min at 0-4 °C and then
Cya n o(5-flu or o-2-oxo-2,3-d ih yd r oin d ol-3-ylid en e)a ce-
tic Acid 21. IR ν cm-1: 3350, 3200-2400, 2200, 1700, 1620,
1
1560. H NMR δ: 6.75-7.97 (m, 3H, ArH); 10.96 (s, 1H, NH).
Cyan o[5-flu or o-1-(4-m eth ylben zyl)-2-oxo-2,3-dih ydr oin -
d ol-3-ylid en e]a cetic Acid 28. IR ν cm-1: 3250-2600, 2200,
1
1700, 1600, 1560. H NMR δ: 2.25 (s, 3H, CH3); 4.87 (s, 2H,
CH2); 6.74-8.03 (m, 7H, ArH).
Gen er a l P r oced u r e for th e Syn th esis of Cya n o(2-oxo-
2,3-d ih yd r oin d ol-3-yl)a cetic Acid s 39-57. A solution of the
appropriate acid 20-38 (1 mmol) in absolute ethanol (10 mL)
was hydrogenated with 10% Pd/C as a catalyst at atmospheric
pressure and room temperature until the theoretical uptake

1426 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Da Settimo et al.
centrifuged at 12 000 rpm for 15 min. Following this same
procedure, the recovered supernatant was subsequently frac-
tionated with saturated ammonium sulfate solution using first
50% and then 75% salt saturation. The precipitate recovered
from the 75% saturated fraction, containing ALR2 activity, was
redissolved in 0.05 M NaCl and dialyzed overnight in 0.05 M
NaCl. The dialyzed material was used for the enzymatic assay.
2.2.2. Ald eh yd e r ed u cta se (ALR1). Rat kidney ALR1 was
prepared in accordance with a previously reported method.31
Kidneys were quickly removed from normal killed rats and
homogenized (Glas-Potter) in 3 volumes of 10 mM sodium
phosphate buffer, pH ) 7.2, containing 0.25 M sucrose, 2.0
mM EDTA dipotassium salt and 2.5 mM â-mercaptoethanol.
The homogenate was centrifuged at 12 000 rpm at 0-4 °C for
30 min, and the supernatant was subjected to a 40-75%
ammonium sulfate fractionation, following the same procedure
previously described for ALR2. The precipitate obtained from
the 75% ammonium sulfate saturation, containing ALR1
activity, was redissolved in 50 volumes of 10 mM sodium
phosphate buffer, pH ) 7.2, containing 2.0 mM EDTA dipo-
tassium salt and 2.0 mM â-mercaptoethanol and dialyzed
overnight using the same buffer. The dialyzed material was
used in the enzymatic assay.
2.3. En zym a tic Assa ys. The activity of the four test
enzymes was determined spectrophotometrically by monitoring
the change in absorbance at 340 nm, which is due to the
oxidation of NADPH or the reduction of NAD+ catalyzed by
ALR2, ALR1, and GR or SD, respectively. The change in
pyridine coenzyme concentration/min was determined using
a Beckman DU-64 kinetics software program (Solf Pack TM
Module).
ALR2 activity was assayed at 30 °C in a reaction mixture
containing 0.25 mL of 10 mM d,L-glyceraldehyde, 0.25 mL of
0.104 mM NADPH, 0.25 mL of 0.1 M sodium phosphate buffer
(pH ) 6.2), 0.1 mL of enzyme extract and 0.15 mL of deionized
water in a total volume of 1 mL. All the above reagents, except
D,L-glyceraldehyde, were incubated at 30 °C for 10 min; the
substrate was then added to start the reaction, which was
monitored for 5 min. Enzyme activity was calibrated by
diluting the enzymatic solution in order to obtain an average
reaction rate of 0.011 ( 0.0010 absorbance units/min for the
sample.
found to be active were tested at additional concentrations
between 10-5 and 10-9 M. The determination of the IC50 values
was performed by using linear regression analysis of the log-
dose-response curve, which was generated using at least four
concentrations of inhibitors 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.
3. P h a r m a cology. 3.1. Ma ter ia ls a n d Meth od s. Experi-
ments were carried out using Sprague Dawley albino rats, 45-
55 g b.w., supplied by Harlan-Nossan Italy. Animal care and
treatment conformed to the ARVO Resolution on the Use of
Animals in Ophthalmic and Vision Research. The galactose
diet consisted of a pulverized mixture of 50% D-galactose and
50% TRM (Harlan Teckland U.K.), laboratory chow, and the
control diet consisted of normal TRM. Both control and
experimental rats had access to food and water ad libitum.
3.2. P r even tion of Ca ta r a ct Develop m en t. Animals were
randomly divided into groups of equal average body weight
with 15 rats per group. The test compounds 55, 59, and
tolrestat were administered four times daily as eyedrops of
appropriate concentrations. The vehicle in which ARIs were
contained was administered with the same dose regimen to
the control group, which was given access to the galactose diet,
and to the group fed with normal diet, which was included to
record the aspect of normal lenses. Groups treated with the
tested compounds were predosed 1 day before switching their
diet to galactose-containing chow. Lenses were examined using
slit-lamp microscopy, after dilating the pupils with atropina
1% Farmigea, Italy, to establish their status of integrity.
Nuclear cataracts, which appeared as a pronounced central
opacity readily visible as a white spot, were considered. The
number of animals which attained this stage was recorded and
the ability of the test compounds to prevent cataract develop-
ment was assessed on the basis of comparison with galac-
tosemic rats treated only with the vehicle.
4. Com p u ta tion a l P r oced u r es. Molecular modeling and
other graphical manipulations were performed using the
SYBYL 6.845 software package, running on a Silicon Graphics
R10000 workstation. Model building of compound 55 was
accomplished with the TRIPOS force field46 available within
SYBYL. Atomic point charges of this inhibitor were calculated
with the semiempirical AM147 method implemented in the
MOPAC program.48 Energy minimizations and MD calcula-
tions were performed with the AMBER 4.1 program,49,50 using
the all-atom Cornell et al. force field.51
4.1. Liga n d Dock in g a n d MD Sim u la tion . A starting
model of compound 55 was obtained by modifying the crystal
structure retrieved from a search by substructures using the
October 2001 release of the Cambridge Structural Database
(CSD)18 (refcode: LAYJ IE). The carboxylate of the inhibitor
was taken as dissociated. Atom-centered partial charges were
calculated using the AM1 Hamiltonian47 as implemented in
MOPAC 6.048 (CHARGE ) -1; keyword: MMOK). Geometry
optimizations were achieved with the SYBYL/MAXIMIN2
minimizer by applying the BFGS (Broyden, Fletcher, Goldfarb
and Shannon) algorithm52 and setting a root-mean-square
gradient of the forces acting on each atom of 0.05 kcal/mol Å
as gradient convergence criteria.
The pharmacophore-based conformations of R-55 and S-55,
obtained as described in Results and Discussion, were sub-
jected to docking calculations into the ALR2 active site using
the DOCK 3.5 suite of programs.36-39 Interestingly, the best
scoring value for both enantiomers was achieved by the
conformation yielding the best overlay on the TBI and the
ALR2-bound conformation of zopolrestat. Docking of the R-55
enantiomer generated 1054 orientations. Out of these, 83 were
within 5 kcal/mol of the best orientation based on the scoring
function (force field score of -35.9 kcal/mol). For the S-55
enantiomer, out of the 1158 orientations generated by DOCK,
the top-ranking 124 were within 5 kcal/mol from the best
orientation based on the scoring function (force field score of
-34.9 kcal/mol). Inspection of the docked structures revealed
that the best and many of the top-scoring orientations of both
ALR1 activity was determined at 37 °C in a reaction mixture
containing 0.25 mL of 20 mM sodium D-glucuronate, 0.25 mL
of 0.12 mM NADPH, 0.25 mL of dialyzed enzymatic solution,
and 0.25 mL of 0.1 M sodium phosphate buffer (pH ) 7.2) in
a total volume of 1 mL. The enzyme activity was calibrated
by diluting the dialyzed enzymatic solution in order to obtain
an average reaction rate of 0.015 ( 0.0010 absorbance units/
min for the sample.
SD activity30 was determined at 37 °C in a reaction mixture
containing 0.25 mL of 10 mM sorbitol, 0.25 mL of 0.47 mM
NAD+, 0.25 mL of 3.75 mU/mL enzymatic solution, and 0.25
mL of 100 mM TRIS/HCl buffer (pH ) 8) in a total volume of
1 mL. All the reagents were incubated at 37 °C for 1 min, after
which the reaction was monitored for 3 min.
GR activity32 was determined at 37 °C in
a mixture
containing 0.25 mL of 1 mM glutathione disulfide, 0.25 mL of
0.36 mM NADPH, 0.25 mL of 4.5 mU/mL enzymatic solution,
and 0.25 mL of 0.125 sodium phosphate buffer (pH ) 7.4)
supplemented with 6.3 mM EDTA potassium salt, in a total
volume of 1 mL.
2.4. En zym a tic In h ibition . The inhibitory activity of the
newly synthesized compounds against ALR2, ALR1, SD, and
GR was assayed adding 0.1 mL of the inhibitor solution to the
reaction mixture described above. All the inhibitors were
dissolved in water, and the solubility was facilitated by
adjustment to a favorable pH. After complete dissolution, the
pH was readjusted to 7. To correct for the nonenzymatic
oxidation of NADPH or reduction of NAD+ and for absorption
by the compounds tested, a reference blank containing all the
above assay components except the substrate was prepared.
The inhibitory effect of the new derivatives was routinely
estimated at a concentration of 10-5 M. Those compounds

Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1427
(14) Szabo-Pusztay, K.; Szabo, L. A Simple General Method for the
Oxidation of Indoles to Oxindoles. Synthesis 1979, 276-277.
(15) Wilson, D. K.; Tarle, I.; Petrash, J . M.; Quiocho, F. A. Refined
1.8 Å Structure of Human Aldose Reductase Complexed with
the Potent Inhibitor Zopolrestat. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 9847-9851.
(16) Pietra, S. Indole Derivatives. Farmaco 1957, 12, 946-953.
(17) Harley-Mason, J .; Ingleby, R. F. J . Hydroxytryptamines. Part
IV. Synthesis and Reactions of 2-3′-Oxindolylethylamines. J .
Chem. Soc. 1958, 3639-3642.
enantiomers placed the negatively charged carboxylate group
of 55 in the anionic binding site of ALR2 lined by residues
Tyr48, His110, and Trp111 so as to make a network of
hydrogen bonds. From this cluster of structures, we selected
the top-scoring orientation of the two enantiomers in which
the p-tolyl ring was located in the same hydrophobic pocket
occupied by the benzothiazole ring of zopolrestat in the crystal
structure.
The parameters of the ligandwere set consistently to the
Cornell et al. 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 complex was solvated by the addition of 217
TIP3P water molecules53 within 20 Å of the inhibitor. After
the protein, NADP+ and the inhibitor were frozen, and the
water molecules alone were minimized (20 000 cycles or 0.1
kcal/mol rms deviation in energy) and equilibrated for 5 ps in
a constant temperature (300 K) bath. SANDER energy mini-
mization (<0.01 kcal/mol rms deviation) and 200 ps MD
simulation of the entire system followed this. During dynamics,
the positional constraints on the protein backbone were
gradually reduced from 5 to 0.1 kcal/Å2/mol. The SHAKE was
used to constrain bonds involving hydrogen. A 1-fs time step
was used along with a nonbonded cutoff of 8 Å at 1 atm of
constant pressure. The temperature was maintained at 300
K using Berendsen’s algorithm54 with a coupling constant of
0.2 ps. Four snapshots, extracted each 25 ps from the last 100
ps MD simulation, were found to be very similar in terms of
rms deviation. An average structure was calculated from the
last 100 ps trajectory and energy-minimized using the steepest
descent and conjugate gradient methods available within the
SANDER module of AMBER as specified above.
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