Discovery of novel aldose reductase inhibitors using a protein structure-based approach: 3D-database search followed by design and synthesis

Article abstract of DOI:10.1021/jm000483h

Aldose reductase (AR) has been implicated in the etiology of diabetic complications. Due to the limited number of currently available drugs for the treatment of diabetic complications, we have carried out structure-based drug design and synthesis in an at

Full text of DOI:10.1021/jm000483h

1718
J . Med. Chem. 2001, 44, 1718-1728
Discover y of Novel Ald ose Red u cta se In h ibitor s Usin g a P r otein
Str u ctu r e-Ba sed Ap p r oa ch : 3D-Da ta ba se Sea r ch F ollow ed by Design a n d
Syn th esis
Yoriko Iwata,† Mitsuhiro Arisawa,‡ Ryuji Hamada,‡ Yasuyuki Kita,‡ Miho Y. Mizutani,§ Nobuo Tomioka,§
Akiko Itai,§ and Shuichi Miyamoto*,†
Exploratory Chemistry Research Laboratories, Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, J apan,
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, J apan, and
Institute of Medicinal Molecular Design, 5-24-5 Hongo, Bunkyo-ku, Tokyo 113-0033, J apan
Received November 13, 2000
Aldose reductase (AR) has been implicated in the etiology of diabetic complications. Due to the
limited number of currently available drugs for the treatment of diabetic complications, we
have carried out structure-based drug design and synthesis in an attempt to find new types of
AR inhibitors. With the ADAM&EVE program, a three-dimensional database (ACD3D) was
searched using the ligand binding site of the AR crystal structure. Out of 179 compounds
selected through this search followed by visual inspection, 36 compounds were purchased and
subjected to a biological assay. Ten compounds showed more than 40% inhibition of AR at a
15 µg/mL concentration. In a subsequent lead optimization, a series of analogues of the most
active compound were synthesized based on the docking mode derived by ADAM&EVE. Many
of these congeners exhibited higher activities compared to the mother compound. Indeed, the
most potent, synthesized compound showed a ∼20-fold increase in inhibitory activity (IC50
)
0.21 vs 4.3 µM). Furthermore, a hydrophobic subsite was newly inferred, which would be useful
for the design of inhibitors with improved affinity for AR.
In tr od u ction
yet been determined, the crystal structures suggest the
involvement of Tyr48 and Lys77 as well as the nicotin-
amide ring in the catalytic reaction.10 A point mutation
study11 and a very recent computer simulation study12
showed that Tyr48 acts as the proton donor in the
reduction, and neutral His110 has a role in substrate
binding during the catalysis although another computer
modeling study13 suggested that His110 was the proton
donor. A potential anion binding site delineated by the
nicotinamide ring, Tyr48, and His110 at the bottom of
the cavity is suggested by the crystallographic and
molecular modeling studies.10,14
The polyol pathway has been implicated in the
etiology of secondary complications of diabetes.1-3
Aldose reductase (AR) is the first enzyme of this
pathway and is widely distributed in mammalian tis-
sues.4 In the presence of NADPH, the enzyme converts
glucose to sorbitol, which is only slowly metabolized to
fructose by sorbitol dehydrogenase, the other enzyme
in the pathway. Aldose reductase inhibitors (ARIs) block
the flux of glucose through the pathway and, in animal
models of diabetes, prevent or reverse functional deficits
and structural damage in the lens, retina, kidney, and
peripheral nerves. A variety of structurally diverse
compounds have been observed to inhibit AR. As can
be seen in Chart 1, these compounds can be divided into
two general classes of ARIs, those containing a car-
boxylic acid moiety and those having rigid spirohydan-
toins or related ring systems.5-8 Although several of
these compounds have progressed to the clinical level,
only a few such drugs are currently on the market.
AR consists of a single polypeptide chain consisting
of 315 residues. Several crystal structures of the enzyme
have been solved by X-ray crystallography. It folds into
a â/R barrel with a core of eight parallel â strands.9 The
ligand binding site is a large, deep, elliptical pocket with
the nicotinamide ring of the NADPH cofactor at the
base. Although a definite catalytic mechanism has not
In line with the current pace of X-ray crystallographic
technologies as well as advances in the human genome
project, structural determinations of macromolecules are
increasing. Structure-based drug design (SBDD) has
therefore become more and more important as a rational
approach to the discovery of leads and their optimiza-
tion.15,16 It includes methods such as de novo design,
3D search, and docking studies and has been applied
to the development of inhibitors, such as those of HIV
protease and integrase.17,18 Due to the limited number
of currently available drugs for the treatment of diabetic
complications, we applied the SBDD to ARIs in an
attempt to find new types of leads.
In the pharmaceutical industry an acceptable lead
typically has a dissociation constant of 10 µM or better.19
Some successful results from a 3D search using the
DOCK program have been summarized by Kuntz.20
Typically, of the 100 to 200 best-scoring compounds, 10-
50 were selected for biological testing and between 2
and 20% of the compounds tested showed inhibition in
the micromolar range (5 to 900 µM). Very recently,
* To whom correspondence should be addressed: Exploratory
Chemistry Research Laboratories, Sankyo Co., Ltd., 1-2-58, Hiromachi,
Shinagawa-ku, Tokyo 140-8710, J apan. Tel: +81-3-3492-3131. Fax:
+81-3-5436-8570. E-mail: miya@shina.sankyo.co.jp.
† Sankyo Co., Ltd.
‡ Osaka University.
§ Institute of Medicinal Molecular Design.
10.1021/jm000483h CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/27/2001

Discovery of Novel Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1719
Ch a r t 1. Chemical Structures of Known ARIs
Meth od s
Com p u ta tion a l Ch em istr y. 1. En zym e Str u ctu r e.
Although several X-ray crystal structures of AR have
been registered in the Protein Data Bank (PDB),28 no
complex structures with potent inhibitors and with side
chain atoms were available when our project started.
The crystal structure of the ternary complex which
includes cofactor NADPH and glucose-6-phosphate (G6P)
was used in this study. Its code name in the PDB is
2acq, and the crystallographic resolution is 1.76 Å.10
Since the crystal structure of bovine lens AR (used for
the inhibition assays in this study) is not known,
calculations were performed on human AR; however, the
two sequences have ∼90% homology, and residues
constituting the binding site are highly conserved. The
His, Asn, and Gln side chains were analyzed using the
Guess_NOHQ program.29 Some terminal conformations
were flipped when they were built the wrong way
around, and the suitable protonation forms of the
imidazole ring were determined.
The oxidized form of NADPH (NADP+), which is the
crystal-bound form of the coenzyme,10 was used in all
calculations although several recent reports30-32 indi-
cated that inhibitor binding is equal to both the AR-
NADPH and AR-NADP+ complexes. With respect to the
partial atomic charges of NADP+, those in the AMBER
database33 were used for the adenine and ribose rings.
As for each of the remaining nicotinamide and diphos-
phate moieties, a single-point ab initio calculation was
carried out with a 6-31G* basis set using the Spartan
program34 to obtain the electrostatic potential charges.
In addition to the solvent water molecules, G6P was
also removed from the crystal structure, and the result-
ing binary complex with NADP+ was used for the
calculation. Hydrogen atoms were added to the complex
structure and geometry-optimized with the adopted-
basis Newton Raphson (ABNR) algorithm35 using the
QUANTA/CHARMm system.36
2. 3D-Da ta ba se Sea r ch . The rectangular region of
16 × 16 × 20 Å3, including the catalytic and the G6P
binding sites, was specified as the ligand binding site
where docking of the compounds was evaluated. Using
the in-house program CALGRID, the van der Waals
interaction, electrostatic interaction, and hydrogen bond-
ing energies were calculated at each grid point with a
0.4 Å interval within the region. On the basis of the grid
data, the ADAM&EVE program searched the 3D data-
base of the Available Chemicals Directory37 (ACD3D)
to identify what would fit into the ligand binding site.
ACD3D consists of around 120 000 reagents whose 3D
structures were constructed in-house from the ACD 2D
database. Although only one conformation is registered
for each entry of ACD3D, ADAM&EVE performed the
systematic conformational search of the ligand at the
time of docking.
Zhang et al. carried out a 3D search to look for protein
tyrosine phosphatase inhibitors.21 From the 2000 com-
pounds identified by DOCK, 25 were purchased for
assay. Seven of them exhibited inhibition in the range
between 21 and 510 µM. Pang et al. also reported on a
virtual screening with the EUDOC program, identifying
four farnesyltransferase inhibitors with IC50 values in
the range of 25 to 100 µM.22 Our 3D search results
obtained by the ADAM&EVE program23 are in no way
inferior to these results.
Recently, Lee et al. presented a docking study in
which some of the compounds shown in Chart 1 were
extensively examined.24 On the basis of the binding
modes and inhibitory activities of those compounds,
pharmacophore requirements for ARIs were discussed.
Mura et al. reported on the design and synthesis of
compounds based on a docking study of a known ARI.25
With respect to the discovery by the 3D search, however,
only a disappointing result has been briefly reported by
Petrash et al. so far.26 That is, among the approximately
30 highest scoring compounds derived by DOCK were
several aromatic aldoximes that had inhibition con-
stants in the micromolar range. Unfortunately, these
were similar to known benzaldoximes with comparable
inhibition constants.27 As far as we know, therefore, the
present study is the first successful example of the
discovery of novel potent ARIs through a 3D-database
search followed by design and synthesis.
After initial positioning of the ligand using two or
three of the potential hydrogen bond sites distributed
over the ligand binding pocket, the ADAM&EVE pro-
gram optimizes the ligand conformation using the
simplex method. The minimum number of hydrogen
bonds formed in the optimized structure was set to 2.
In addition, a hydrogen bond formation with one of the
five atoms located in the bottom of the binding pocket
was set as criteria. These atoms comprise the Oη of

1720 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Iwata et al.
Sch em e 1. Synthesis of Substituted 2-Methyl-indole-3-acetic Acids (19-32)a
a
t
Reagents and conditions: (a) (1) NaNO2, cHCl, (2) SnCl2, cHCl, (3) 1 N HCl-EtOH; (b) levulinic acid buthyl ester, NaOAc, AcOH;
(c) for 30, (1) tBuOK, ClC6H4CBrCH2CH2Ph, DMF, (2) CF3COOH; for 31 and 32, (1) tBuOK, ClC6H4CBrR3, DMF, (2) Pd/C, H2, AcOEt-
p
p
AcOH, (3) R1CO2H, DCC, DMAP, CH2Cl2, (4) neat, 180-190 °C or CF3COOH; (d) (1) BuOK, pClC6H4COCl, THF, (2) Pd/C, H2, AcOEt-
t
AcOH; (e) R1H, DEAD, PPh3, THF; (f) for 21, (1) CF3COOH, (2) Pd/C, H2, AcOEt-AcOH; for 22, (1) Pd/C, H2, AcOEt-AcOH, (2) CF3COOH;
(g) CF3COOH; (h) R1CO2H, DCC, DMAP, CH2Cl2; (i) CF3COOH (only for 28, neat, 180-190 °C).
Tyr48, the Nꢀ2 of His110, the Nꢀ1 of Trp, and the Sγ of
Cys298 as well as the nitrogen atom of the nicotinamide
of NADP+. The maximum energy (i.e., unfavorable limit)
of the van der Waals interaction was set to -27 kcal/
mol. Only compounds with a molecular weight of >250
and at least one ring structure were included.
Among the hit compounds identified using the above
search criteria, further selection was performed based
on the total interaction energy values as well as the
visual inspection with computer graphics using the
GREEN program.38 In this examination, overall fit and
how well the compound complemented the shape of the
bottom of the binding site were taken into account,
together with exclusions based upon considerations of
chemical diversity and structural flexibility.
with the enzyme. Finally, the whole ligand structures
were energy-minimized by the ABNR algorithm with
the enzyme structure fixed. A distance-dependent di-
electric constant of 4r was used in the CHARMm energy
calculations.
Syn th etic Ch em istr y. tButyl 5-benzyloxy-2-methyl-
indole-3-acetate (33) was prepared from commercially
available 4-benzyloxyaniline hydrochloride using Fish-
er’s indole synthesis (Scheme 1). N-Alkylated indoles
(30-32) were synthesized via N-alkylation of 33 in the
t
presence of BuOK followed by chemical transforma-
tions. tButyl 5-benzyloxy-1-(p-chlorobenzoyl)-2-methyl-
indole-3-acetate (34) was prepared from 33 and p-chloro-
t
benzoyl chloride in the presence of BuOK. De-esteri-
fication of 34 gave 1-(p-chlorobenzoyl)-5-hydroxy-2-
methyl-indole-3-acetic acid (19). Treatment of 34 with
the corresponding alkyl halide, DEAD, and PPh3 in THF
afforded the O-alkylated compounds, 20 and 35. Treat-
ment of 20 and 35 with CF3COOH led to the hydrolyzed
compounds 21 and 22, respectively. O-Acylated com-
pounds, 36, 37, 38, 39, 27, and 40 were prepared from
34 and the corresponding carboxylic acid in the presence
of DCC and DMAP. Treatment of 36-39, 27, and 40
3. Design Ba sed on th e Active Com p ou n d . With
respect to the most potent compound yielded by the 3D
search, a rational design was performed with the
QUANTA/CHARMm system starting from the docking
mode derived by ADAM&EVE. First, the modified
structures were built taking into account the van der
Waals interactions and hydrogen bond formations.
Second, the conformations of the modified moieties were
manually changed to search for favorable interactions

Discovery of Novel Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1721
Ta ble 1. AR Inhibitory Activities and van der Waals
Interaction Energies of Compounds 9-18
Ch a r t 2. Chemical Structures of ARIs Discovered in
the 3D Search
inhibition (%)a
IC50
vdw energyb
(kcal/mol)
compd
5 (µg/mL)
15 (µg/mL)
(µM)a
9
10
11
12
13
14
15
16
17
18
49 (51)
40
68 (72)
35 (56)
36
66 (72)
47 (38)
43 (42)
38 (39)
70
56 (47)
83
74 (70)
50 (47)
79 (72)
74
71
63
46 (36)
18
-27.9
-31.5
-29.7
-28.9
-31.9
-29.9
-38.1
-32.7
-33.5
-30.4
5.0 (3.6)
20 (14)
6.0 (4.6)
9.8
13
18
a
Values in parentheses are from a second independent experi-
b
ment. van der Waals interaction energy derived by the
ADAM&EVE program.
with acid or heat gave the hydrolyzed compounds, 23-
26, and 28-29, respectively.
Resu lts a n d Discu ssion
3D-Da ta ba se Sea r ch . The 3D database (ACD3D)
was searched by the ADAM&EVE program to yield a
total of 718 hits. Through visual inspection using
computer graphics and total interaction energy values,
179 compounds were selected. From these, 36 com-
pounds were purchased and subjected to AR inhibition
assay. The 36 compounds were initially tested for their
inhibition of AR at a concentration of 15 µg/mL. Ten
out of the 36 compounds were classified as active. Their
inhibition activities and chemical structures are shown
in Table 1 and Chart 2, respectively. Seven of the
compounds have a carboxyl group in their structures.
Compound 16 has the same fused three-ring structure
as alrestatin but lacks a carboxyl group. Interestingly,
11 is indomethacin, an antiinflammatory drug, and a
similar indole-3-acetic acid ring structure was identified
to have inhibitory activities against AR according to
some of the patent literature. IC50 values were deter-
mined for the potent compounds and are listed in Table
1.
The IC50 values of the three most potent compounds,
11, 14, and 15, are in the low micromolar range, that
is, 4.3 (5.0 and 3.6), 5.3 (6.0 and 4.6), and 9.8 µM,
respectively. Specifically, these figures appear to be
superior to the micromolar range of inhibition constants
observed for several aromatic aldoximes that were found
by Petrash et al. through the 3D search of the Cam-
bridge Structure Database against AR as mentioned
above.24 In general, our results presented here are
among the top 3D-search studies currently in the
literature. Although the potencies of our compounds
were weaker than those of available drugs such as
tolrestat (IC50 ) 0.03-0.1 µM6,14), we believe that the
main purpose of a 3D search is not to find compounds
with ultrapotent activity. A realistic goal of a 3D search
should be the discovery of new leads that can be
subsequently used as the basis for further synthetic
modifications as Wang et al. suggested.39 In addition,
the advantage of using the ADAM&EVE program is
that a rational optimization can be pursued based on
the binding mode obtained as shown in the following
section.
correlation between the docking scores and IC50 values
in their 3D-search study.40 Unfortunately, in our case
as well, there appeared to be no correlation between the
interaction energies derived by ADAM&EVE and the
observed inhibitory potencies (Table 1). Scoring or
energy evaluation is an important and challenging issue
in SBDD.41 Improvement of the interaction energy
evaluation of ADAM&EVE is currently in progress, and
preliminary calculations on another protein‚ligand sys-
tem have provided promising results.
The binding modes derived by ADAM&EVE are
shown in Figure 1 for the three most potent compounds,
11, 14, and 15. They fit into the middle of the long
binding pocket, with good steric complementarity be-
tween the ligand and the bottom of the cavity. Hydrogen
bonds with the Oη of Tyr48 and the Nꢀ2 of His110 in
the anion binding site were formed for 11 and 14. The
ureido group of 14 was hydrogen bonded to the Nꢀ1 of
Trp20 located in the mouth of the cavity. Compound 15
formed hydrogen bonds with the side chain of Cys298
It is well-known that docking scores exhibit poor
correlation with real affinities.19 Hol et al. reported little

1722 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Iwata et al.
F igu r e 1. Complex structure models of AR and 11 (top), 14 (middle), and 15 (bottom). Top view of the large active site (left) and
side view (right). The ligand binding site is represented by the Connolly surface in yellow. The inhibitors are rendered in CPK
form, in which carbon and hydrogen are shown in white, oxygen in red, halogen in purple, and nitrogen in blue.
and the main chains of Ala299 and Leu300. It should
be noted that the side chain of His110 was flipped based
on our analysis. J udging from the docking structures,
it appears that 11 and 14 would not have a favorable
interaction with His110 in the original conformation
registered in the PDB.
Interestingly, the carboxyl group of 15 was not
situated at the bottom of the pocket but faced the
solvent. Using the QUANTA/CHARMm system, a model
was built with the docked orientation of the carboxylic
group in the anion binding site. The interaction energy
of the compound in the former orientation was calcu-
lated to be slightly lower than that in the latter, which
was consistent with the orientation selected by
ADAM&EVE. Geometry optimization of 11 using the
CHARMm force field brought slight positional and
torsional changes of the carboxyl moiety. In this complex
structure, the carboxylate was electrostatically attracted
to the positive nicotinamide ring of NADP+. In addition,
it was held in place with the same three hydrogen bonds
to the Tyr48 hydroxyl, the Nꢀ2 of His110, and the Nꢀ1
of Trp111 (Figure 2) as those observed in the complex
crystal structure of tolrestat.42 The indole ring lay
between the side chains of Trp20, Val47, and Phe122,
and it was close to those of Trp79 and Trp219 with
favorable van der Waals interactions. Although the
carboxyl group of 14 was situated in the anion binding
site, it was, if anything, positioned opposite to Trp20
rather than Trp111. Compound 16 also has the same
fused three-ring system as alrestatin but its sulfonyl
groups are substituted in different positions compared
with the carboxyl group of alrestatin. The sulfonyl

Discovery of Novel Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1723
F igu r e 2. Stereoview of the complex structure model of AR (yellow) and 11 (white) in stick form. Connolly surface of the enzyme
is colored by the electrostatic potential (positive, red; neutral, green; negative, blue) calculated using the QUANTA/CHARMm
system. Hydrogen bonds with Tyr48, His110, and Trp111 are drawn as dashed lines. Nonpolar hydrogens are omitted for clarity.
Ta ble 2. AR Inhibitory Activities of Compound 11 and Its Analogues
compd
R1
R2
R3
IC50 (µM) a
11
19
20
21
22
23
24
25
26
27
28
29
30
31
32
OMe
OH
COOH
COOH
COOtBu
COOH
COOH
COOH
COOH
COOH
COOH
COOtBu
COOH
COOH
COOH
COOH
COOH
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
dO
∼4.3
3.2
OCH2CH2CH2OCH2Ph
OCH2CH2CH2OH
OCH2CH2OH
OCO-n-Hex
OCOCH2CH2CH2CH(Me)2
OCOCH2CH2COCH(Me)2
OCOCH2CH2COOCH(Et)2
OCOCH2CH2COOCH2Ph
OCOCH2CH2COOCH2Ph
OCOCH2CH2COOCH(Me)Ph
OCH2Ph
b
4.2
3.4
2.9
1.9
1.4
0.54
b
0.21
0.31
CH2CH2Ph
9% (9.5 µM)
10% (8.0 µM)
17% (7.6 µM)
OCOCH2CH2COOCH2Ph
OCOCH2CH2COOCH2Ph
CH2CH2Ph
CH2CH2CH2OPh
a
b
IC50 or percent inhibition (at a given micromolar concentration). No inhibition was observed at a concentration of 5 µg/mL.
moiety without the o-amino substituent was located in
the anion binding site and the other sulfonyl group, at
the surface of the complex.
against AR. These compounds were initially tested for
their inhibition at a concentration of 5 µg/mL. Com-
pounds 20 and 27 with the protected carboxyl group lost
their inhibitory activities. This seemed to support the
ADAM&EVE-derived docking mode where the carboxyl
group fit into the anion binding site. The significant
decreases in the inhibitory activities of 30-32 indicated
that the structural modifications of R3 were unsuitable.
The substituents of R3 were assumed to approach
Phe122 and Ala299-Leu301. It was revealed that the
loop Phe121-Thr135 and the short segment Cys298-
Cys303 were liable to incur conformational changes
based on the molecular dynamics simulation13 of AR and
the complex crystal structure with zopolrestat,43 whose
coordinates are as yet not available. These substituents
of R3 may cause unfavorable conformational changes in
this part of the enzyme.
Other analogues of 11 in Table 2 exhibited similar or
higher potencies compared to the lead compound 11. No
gain in activity was observed for 21 or 22. The reason
for this might be that a hydrogen bond could not be
effectively formed with the terminal hydroxy group of
R1 or that they lack an ester carbonyl in the stem of R1.
The compounds with a branched alkyl group in R1
Lea d Op tim iza tion . The most potent compound was
selected as the lead, on which we then carried out
structure modifications. The docking mode derived by
ADAM&EVE (Figures 1 and 2) revealed that the
molecule did not fully occupy the long binding cavity.
It was also found that no hydrogen bond was formed
between the ligand and enzyme residues at the mouth
of the pocket. In designing the modified ligands, there-
fore, we took into account the possibility of hydrogen
bonds with the side chain of Trp20 and the main chains
of Lys21, Val47, Tyr48, Ala299, and Leu300. In addition,
the hydrophobic interactions with the side chains of
Lys21, Val47, Pro23, Pro24, Phe122, Trp219, and Leu301
were considered. The designed models were initially
constructed using the QUANTA/CHARMm system.
After geometry optimization of the modeled ligands by
molecular mechanics calculations, those staying at the
binding pocket as expected were selected as synthetic
candidates.
Table 2 includes the chemical structures of the
synthetic analogues of 11 and their inhibitory activities

1724 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Iwata et al.
F igu r e 3. Stereoview of the complex structure model of AR (green-yellow) and 28 (white). Connolly surface of the enzyme is
shown in yellow. Selected residues of AR are represented in stick form. Nonpolar hydrogen atoms of these residues are omitted
for clarity. Compound 28 is rendered in CPK form, in which carbon and hydrogen are shown in white, oxygen in red, chlorine in
purple, and nitrogen in blue.
showed relatively potent inhibitory activities. Those
with a terminal ester group in R1 exhibited rather
potent activities. Indeed, the IC50 value of 28 was 0.21
µM, indicating the achievement of an around 20-fold
increase in the activity compared to the mother com-
pound.
Figure 3 illustrates the complex model of 28 and AR.
In this docked conformation, the ester carbonyl oxygen
of the root of R1 was hydrogen bonded with the Hꢀ1 of
the side chain of Trp20. The terminal phenyl group fit
into the hydrophobic pocket comprising the methylene
group of the side chains of Pro23, Pro24, Gln49, and
Asn50. Alternatively, it could be accommodated in the
neighboring hydrophobic cavity consisting of the side
chains of Lys21 and Pro23. In this complex model, the
terminal ester moiety was not involved in the hydrogen
bonding but was considered to contribute to the van der
Waals interaction for the sake of good steric comple-
mentarity between the ligand carbonyl group and the
enzyme.
The IC50 value of 0.31 µM for 29 was slightly larger
than that of 0.21 µM for 28. Compound 29 was a racemic
mixture, and one of the isomers was calculated to fit
better into the hydrophobic pocket than the other. It
was therefore presumed that the inhibitory activity of
a suitable isomer of 29 would be comparable to that of
28. With respect to the potent compounds, 26, 28, and
29, the terminal hydrophobic part of R1 was considered
to interact with the alkyl moiety of the side chains of
Lys21, Pro23, Pro24, Gln49, and/or Asn50. This hydro-
phobic subsite of AR was not occupied in the binding of
zopolrestat or tolrestat as was observed in their complex
crystal structures. This newly discovered subsite would
be useful for the design of potent ARIs although
experimental verification such as by mutation studies
is necessary to establish that the hydrophobic subsite
is indeed an important pharmacophore.
On the contrary, 28 was discovered by the 3D-database
search followed by the design and synthesis of a novel
substituent, providing new insight into the interaction
between AR and ARI. Further improvement of potency
and the selectivity evaluation with respect to other
related enzymes are subjects for investigation in the
future.
Con clu sion
The present study is the first successful example of
the discovery of novel potent inhibitors of AR through
a 3D-database search followed by design and synthesis.
Using the ADAM&EVE program, the ACD3D database
was searched based on the ligand binding site of
the crystal structure of AR. This search led to the
identification of 10 inhibitors with significant inhibitory
activities at a concentration of 15 µg/mL. The potent
inhibitions exhibited by around 30% of the compounds
subjected to inhibition assay illustrate the power of the
ADAM&EVE approach for the discovery of new types
of leads. Furthermore, a ∼20-fold increase in inhibitory
activity was achieved by subsequent design and syn-
thesis based on the complex structure model derived by
docking. Our results demonstrate the effectiveness of
SBDD for the discovery of leads and the ensuing
optimization. In addition, the hydrophobic subsite in-
ferred from the complex structure models of the most
potent compounds would be useful for the design of new
ARIs with improved potency.
Exp er im en ta l Section
Syn th etic Ch em istr y. All compounds were fully charac-
terized (1H, IR, mass). IR spectra (cm-1) were recorded using
a KBr pellet. 1H NMR spectra were recorded in CDCl3, unless
otherwise mentioned, at 200, 270, 300, or 500 MHz with TMS
as an internal standard. E. Merck silica gel 60 for column
chromatography and E. Merck precoated TLC plates, silica gel
F
254, for preparative thin-layer chromatography were used. The
The potency of the most effective compound 28 (IC50
) 0.21 µM) is superior to that of sorbinil and recently
reported isoxazolopyridazinones (IC50 ) 3 and ∼5 µM,
organic layers were dried over anhydrous MgSO4 or Na2SO4.
tBu tyl 5-Ben zyloxy-2-m eth yl In d ole 3-Aceta te (33). To
a solution of 4-benzyloxy aniline hydrochloride (2.11 g, 8.94
mmol) in concentrated HCl (15.0 mL) was added a solution of
sodium nitrate (617 mg, 8.94 mmol) in H2O (2.25 mL) dropwise
at 0 °C, and the mixture was stirred for 25 min. To the mixture
was added SnCl2 (4.75 g. 25.0 mmol) dropwise at -25 °C. The
mixture was stirred for 4 h and then filtered, and the residue
was washed with concentrated HCl. The obtained solid was
then dissolved in 25% KOH and Et2O, and the organic
compound was extracted with Et2O, dried over Na2SO4, and
filtered. The organic layer was partially concentrated under
respectively44) but inferior to that of epalrestat (IC50
)
0.01 µM8) and tolrestat (IC50 ) 0.03-0.1 µM6,14). The
IC50 value of 0.21 µM for 28 is comparable to that of
the 0.15 µM that was successfully attained by Mura et
al. through the design and synthesis based on the
docking study of a known ARI.25 Their compound is,
however, a composite of the known tricyclic pyridazi-
none skeleton and the benzothiazole ring of zopolrestat.

Discovery of Novel Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1725
reduced pressure and filtered to get crystals. To these crystals
was added 1 N HCl-EtOH (10.0 mL), and the solvent was
removed to give 4-benzyloxyphenylhydrazine hydrochloride
(897 mg, 40%) as dark yellow plates. To a stirring solution of
the obtained compound (897 mg, 3.58 mmol) in acetic acid (10.0
mL) was added sodium acetate (295 mg, 3.58 mmol) at room
temperature. The mixture was refluxed for 2 h and then
neutralized with aqueous NaOH, and the organic compound
was extracted with Et2O, dried over Na2SO4, and concentrated
in a vacuum. The crude residue was subjected to column
chromatography (n-hexane:AcOEt ) 6:1∼4:1) to give 33 (783
mg, 62%) as a dark yellow plate: 1H NMR (300 MHz, CDCl3)
δ (ppm) 7.49 (1H, s), 7.46-7.28 (5H, m), 7.14 (1H, d, J ) 9.0
Hz), 7.10 (1H, d, J ) 2.4 Hz), 6.84 (1H, dd, J ) 9.0 Hz, 2.4
Hz), 5.10 (2H, s), 3.54 (2H, s), 2.38 (3H, s), 1.42 (9H, s); IR-
(KBr) 3403, 1720 cm-1; LRMS (EI) m/z 351 (M+). HRMS (EI)
calcd for C22H25NO3, 351.1834; found, 351.1837.
solution of the obtained compound (90.0 mg, 0.182 mmol) in
AcOEt (15.0 mL) and AcOH (5.00 mL) was added Pd/C (23.0
mg) under H2 (3.8 atm), and the mixture was stirred for 12 h.
The mixture was filtered, and the solvent was removed. The
crude residue was subjected to column chromatography (CH2-
Cl2:MeOH ) 10:1) to give 21 (51.2 mg, 70%) as a colorless
amorphous solid: 1H NMR (270 MHz, CD3OD) δ (ppm) 7.67
(2H, d, J ) 8.6 Hz), 7.53 (2H, d, J ) 8.6 Hz), 7.04 (1H, s), 6.91
(1H, d, J ) 8.9 Hz), 6.64 (1H, d, J ) 8.9 Hz), 4.07 (2H, t, J )
6.1 Hz), 3.73 (2H, t, J ) 6.3 Hz), 3.55 (2H, s), 2.26 (3H, s),
1.98-1.93 (2H, m); IR(KBr) 3450, 1685, 1612 cm-1; LRMS (EI)
m/z 401 (M+). HRMS (EI) calcd for C21H20NO5Cl, 401.1030;
found, 401.1023.
1-(4-Ch lor oben zyl)-5-(2-h yd r oxyeth oxy)-2-m eth yl In -
d ole 3-Acetic Acid (22). To a solution of 34 (223 mg, 0.656
mmol) in THF (9.00 mL) was added dropwise a mixture of
2-benzyloxyethanol (78.0 mg, 0.597 mmol), PPh3 (147 mg,
0.656 mmol), and DEAD (40% solution in toluene, 0.240 mL,
0.656 mmol) at 0 °C, and the mixture was stirred at the same
temperature for 10 min. The mixture was then stirred at room
temperature for 18 h. To the mixture was added H2O, and then
organic compound was extracted with AcOEt, washed with
brine, dried over Na2SO4, filtered, and concentrated. The crude
residue was subjected to column chromatography (n-hexane:
tBu tyl 5-Ben zyloxy-1-(4-ch lor oben zoyl)-2-m eth yl In -
d ole 3-Aceta te (34). To a solution of 33 (600 mg, 1.71 mmol)
t
in THF (28.0 mL) was added BuOK (229 mg, 2.05 mmol), and
the mixture was stirred for 20 min. To the mixture was added
4-chlorobenzoyl chloride (0.260 mL, 2.05 mmol), and it was
then stirred for 11 h. The reaction was quenched with
saturated aqueous NH4Cl. The organic compound was ex-
tracted with AcOEt, washed with brine, dried over Na2SO4,
filtered, and concentrated. The crude residue was subjected
to column chromatography (n-hexane:AcOEt ) 4:1∼2:1) to give
a 5-benzyloxy derivative (844 mg, 100%) as dark orange
crystals. To the stirring solution of the derivative (1.10 g, 2.24
mmol) in AcOEt (22.0 mL) and AcOH (20.0 mL) was added
Pd/C (110 mg) under H2 (3.8 atm). The mixture was stirred
for 3 h and then filtered, and the crude residue was subjected
to column chromatography (n-hexane:AcOEt ) 3:1) to give 34
(774 mg, 87%) as a pale yellow powder: 1H NMR (300 MHz,
CDCl3) δ (ppm) 7.65 (2H, d, J ) 8.6 Hz), 7.46 (2H, d, J ) 8.6
Hz), 6.91 (1H, d, J ) 2.4 Hz), 6.84 (1H, d, J ) 8.8 Hz), 6.52
(1H, dd, J ) 8.8 Hz, 2.4 Hz), 3.54 (2H, s), 2.35 (3H, s), 1.45
t
AcOEt ) 5:1) to give butyl 5-(2-benzyloxyethoxy)-1-(4-chloro-
benzoyl)-2-methyl indole 3-acetate (58.0 mg, 50%) as a yellow
oil. To a solution of the resulting compound (144 mg, 0.270
mmol) in AcOEt (8.00 mL) and AcOH (8.00 mL) was added
Pd/C (36.0 mg) under H2 (3.8 atm), and the mixture was stirred
for 6.5 h. The mixture was filtered, and the solvent was
removed. The crude residue was subjected to column chroma-
tography (n-hexane:AcOEt ) 3:1∼2:1) to give tbutyl 1-(4-
chlorobenzyl)-5-(2-hydroxyethoxy)-2-methyl indole 3-acetate
(68.0 mg, 57%) as a pale yellow oil. This (57.0 mg, 0.128 mmol)
was dissolved in CF3COOH (2.00 mL) and stirred for 4 h. The
solvent was removed, and the crude residue was subjected to
preparative TLC (CH2Cl2:MeOH ) 10:1) to give 22 (7.00 mg,
14%) as a colorless amorphous solid: 1H NMR (300 MHz, CD3-
OD) δ (ppm) 7.67 (2H, d, J ) 8.7 Hz), 7.54 (2H, d, J ) 8.7 Hz),
7.03 (1H, s), 6.90 (1H, d, J ) 9.2 Hz), 6.70 (1H, d, J ) 9.2 Hz),
4.05 (2H, t, J ) 4.4 Hz), 3.86 (2H, t, J ) 4.4 Hz), 3.71 (2H, s),
2.30 (3H, s); IR(KBr) 2920, 1684 cm-1; LRMS (EI) m/z 387
(M+). HRMS (EI) calcd for C20H18NO5Cl, 387.0873; found,
387.0857.
(9H, s); LRMS (EI) m/z 399 (M+). HRMS (EI) calcd for C22H22
-
NO4Cl, 399.1237; found, 399.1229.
1-(4-Ch lor oben zyl)-5-h yd r oxy-2-m eth yl In d ole 3-Acetic
Acid (19). Compound 34 (201 mg, 0.503 mmol) was dissolved
in CF3COOH (7.00 mL), and the mixture was stirred for 1 h.
The solvent was removed, and the crude residue was recrys-
tallized (from CH2Cl2) to give 19 (152 mg, 88%) as white
crystals: 1H NMR (200 MHz, CDCl3) δ (ppm) 7.67 (2H, d, J )
8.7 Hz), 7.47 (2H, d, J ) 8.7 Hz), 6.89 (1H, d, J ) 2.2 Hz),
6.81 (1H, d, J ) 8.8 Hz), 6.79 (1H, dd, J ) 8.8 Hz, 2.2 Hz),
3.62 (2H, s), 2.37 (3H, s); LRMS (EI) m/z 343 (M+). HRMS (EI)
calcd for C18H14NO4Cl, 343.0611; found, 343.0615.
tBu tyl 5-(3-Ben zyloxyp r op oxy)-1-(4-ch lor oben zoyl)-2-
m eth yl In d ole 3-Aceta te (20). To a solution of 34 (84.9 mg,
0.212 mmol) in THF (3.00 mL) was added dropwise a mixture
of 3-benzyloxypropanol (61.0 mg, 0.193 mmol), PPh3 (105 mg,
0.212 mmol), and DEAD (40% solution in toluene, 0.0950 mL,
0.212 mmol) at 0 °C, and the mixture was stirred at the same
temperature for 10 min. The mixture was then stirred at room
temperature for 2 days. To the mixture was added H2O, and
the organic compound was extracted with AcOEt, washed with
brine, dried over Na2SO4, filtered, and concentrated. The crude
residue was subjected to column chromatography (n-hexane:
AcOEt ) 3:1) to give 20 (58.0 mg, 50%) as a colorless
amorphous solid: 1H NMR (200 MHz, CDCl3) δ (ppm) 7.66
(2H, d, J ) 8.4 Hz), 7.46 (2H, d, J ) 8.4 Hz), 7.34-7.29 (5H,
m), 6.97 (1H, d, J ) 2.4 Hz), 6.88 (1H, d, J ) 9.8 Hz), 6.65
(1H, dd, J ) 9.8 Hz, 2.4 Hz), 4.53 (2H, s), 4.11 (2H, t, J ) 6.2
Hz), 3.68 (2H, d, J ) 6.2 Hz), 3.55 (2H, s), 2.37 (3H, s), 1.44
(9H, s).
Gen er a l P r oced u r e for 23-27 a n d 29. To a solution of
34 (0.100 mmol) in CH2Cl2 (1.50 mL) was added DCC (1.50
equiv), DMAP (0.100 equiv), and the corresponding acid (1.10
equiv), and the mixture was stirred for 12 h. The mixture was
filtered, washed with H2O and brine, dried over Na2SO4,
filtered, and concentrated. The crude residue was subjected
to column chromatography (n-hexane:AcOEt) to give O-acyl-
t
t
ated butyl acetate. To a stirring solution of O-acylated butyl
acetate in CH2Cl2 (1.50 mL) was added CF3COOH (0.400 mL),
and the mixture was stirred for 1.5 h. The solvent was
removed, and the crude residue was subjected to preparative
TLC to give O-acylated acetic acid.
1-(4-Ch lor oben zyl)-5-n h ep ta n oyloxy-2-m eth yl In d ole
3-Acetic Acid (23). A pale yellow oil of 29% yield from 34:
1H NMR (300 MHz, CDCl3) δ (ppm) 7.59 (2H, d, J ) 8.4 Hz),
7.40 (2H, d, J ) 8.4 Hz), 7.12 (1H, s), 6.88 (1H, d, J ) 9.0 Hz),
6.68 (1H, d, J ) 9.0 Hz), 3.59 (2H, s), 2.47 (2H, t, J ) 7.5 Hz),
2.29 (3H, s), 1.67 (2H, t, J ) 7.2 Hz), 1.35-1.25 (6H, m), 0.83-
0.81 (3H, m); LRMS (EI) m/z 455 (M+). HRMS (EI) calcd for
C
25H26NO5Cl, 455.1499; found, 455.1494.
1-(4-Ch lor ob en zyl)-5-ih ep t a n oyloxy-2-m et h yl In d ole
3-Acetic Acid (24). A yellow oil of 43% yield from 34: 1H NMR
(300 MHz, CDCl3) δ (ppm) 7.67 (2H, d, J ) 8.3 Hz), 7.48 (2H,
d, J ) 8.3 Hz), 7.21 (1H, d, J ) 2.1 Hz), 6.95 (1H, d, J ) 8.7
Hz), 6.77 (1H, dd, J ) 8.7 Hz, 2.1 Hz), 3.69 (2H, s), 2.54 (2H,
t, J ) 7.7 Hz), 2.39 (3H, s), 1.81-1.26 (5H, m), 0.91 (6H, d, J
) 6.3 Hz); IR(KBr) 2957, 1755, 1713, 1693 cm-1; LRMS (EI)
m/z 455 (M+). HRMS (EI) calcd for C25H26NO5Cl, 455.1499;
found, 455.1502.
1-(4-Ch lor oben zyl)-5-(3-h yd r oxyp r op oxy)-2-m eth yl In -
d ole 3-Acetic Acid (21). Compound 20 (100 mg, 0.182 mmol)
was dissolved in CF3COOH (5.00 mL), and the mixture was
stirred for 1.5 h. The solvent was removed, and the crude
residue was subjected to preparative TLC (AcOEt) to give 5-(3-
benzyloxypropoxy)-1-(4-chlorobenzoyl)-2-methyl indole 3-acetic
acid (90.0 mg, 100%) as a colorless amorphous solid. To a

1726 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Iwata et al.
Su ccin ic Acid 1-Meth yleth yl Ester 3-Ca r boxym eth yl-
1-(4-ch lor oben zoyl)-2-m eth yl In d ol-5-yl Ester (25). A yel-
low oil of 48% yield from 34: 1H NMR (300 MHz, CDCl3) δ
(ppm) 7.66 (2H, d, J ) 8.6 Hz), 7.47 (2H, d, J ) 8.6 Hz), 7.21
(1H, d, J ) 2.1 Hz), 6.94 (1H, d, J ) 9.0 Hz), 6.77 (1H, dd, J
) 9.0 Hz, 2.1 Hz), 3.67 (2H, s), 2.90-2.61 (5H, m), 2.37 (3H,
s), 1.13 (6H, d, J ) 6.9 Hz); LRMS (EI) m/z 492 (M+ + Na).
HRMS (EI) calcd for C25H24NO6ClNa, 492.1190; found, 492.1186.
Su ccin ic Acid 1-Eth ylp r op yl Ester 3-Ca r boxym eth yl-
1-(4-ch lor oben zoyl)-2-m eth yl In d ol-5-yl Ester (26). A yel-
low oil of 37% yield from 34: 1H NMR (300 MHz, CDCl3) δ
(ppm) 7.66 (2H, d, J ) 8.4 Hz), 7.47 (2H, d, J ) 8.4 Hz), 7.21
(1H, d, J ) 1.8 Hz), 6.95 (1H, d, J ) 9.0 Hz), 6.78 (1H, dd, J
) 9.0 Hz, 1.8 Hz), 4.84-4.76 (1H, m), 3.67 (2H, s), 2.88 (2H, t,
J ) 6.5 Hz), 2.74 (2H, t, J ) 6.6 Hz), 2.37 (3H, s), 1.62-1.52
(4H, m), 0.87 (6H, d, J ) 7.5 Hz); IR(KBr) 2971, 1761, 1732,
1693 cm-1; LRMS (FAB) m/z 536 (M+ + Na); HRMS (FAB)
calcd for C27H28NO7ClNa, 536.1452; found, 536.1450.
Su ccin ic Acid Ben zyl Ester 3-tBu toxyca r boxym eth yl-
1-(4-ch lor oben zoyl)-2-m eth yl In d ol-5-yl Ester (27). A light
brown amorphous solid of 99% yield from 34: 1H NMR (300
MHz, CDCl3) δ (ppm) 7.57 (2H, d, J ) 8.4 Hz), 7.38 (2H, d, J
) 8.4 Hz), 7.30-7.21 (5H, m), 7.15 (1H, s), 6.88 (1H, d, J )
8.8 Hz), 6.64 (1H, d, J ) 8.8 Hz), 5.07 (2H, s), 3.47 (2H, s),
2.87-2.65 (4H, m), 2.28 (3H, s), 1.35 (9H, s); LRMS (FAB) m/z
612 (M+ + Na). HRMS (FAB) calcd for C33H32NO7ClNa,
612.1765; found, 612.1778.
Su ccin ic Acid 1-P h en yleth yl Ester 3-Ca r boxym eth yl-
1-(4-ch lor oben zoyl)-2-m eth yl-in d ol-5-yl Ester (29). A col-
orless oil of 40% yield from 34: 1H NMR (300 MHz, CDCl3) δ
(ppm) 7.60 (2H, d, J ) 8.6 Hz), 7.41 (2H, d, J ) 8.6 Hz), 7.27-
7.12 (5H, m), 7.12 (1H, s), 6.85 (1H, d, J ) 9.0 Hz), 6.63 (1H,
d, J ) 9.0 Hz), 5.85 (1H, q, J ) 6.9 Hz), 3.60 (2H, s), 2.83-
2.66 (4H, m), 2.30 (3H, s), 1.47 (3H, d, J ) 6.9 Hz); IR(KBr)
2930, 1750, 1732, 1715, 1694 cm-1; LRMS (FAB) m/z 570 (M+
+ Na). HRMS (FAB) calcd for C30H26NO7ClNa, 570.1296;
found, 570.1324.
mixture was stirred at room temperature for 14 h. To the
mixture was added H2O. The organic compound was extracted
with AcOEt, washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The crude residue was
subjected to column chromatography (n-hexane:AcOEt
)
4:1∼3:1) to give tbutyl 5-benzyloxy-1-[1-(4-chlorophenyl)-3-
phenyl-propyl]-2-methyl indole 3-acetate (337 mg, 51%) as a
yellow oil. To a solution of the obtained compound (337 mg,
0.581 mmol) in acetic acid (2.00 mL) and AcOEt (10.0 mL) was
added Pd-C (170 mg) under H2 (3.80 atm), and the mixture
was stirred at room temperature for 4.5 h. The mixture was
filtered, and solvent was removed. The residue was subjected
to column chromatography (n-hexane:AcOEt ) 4:1∼3:1) to give
tbutyl 1-[1-(4-chloro-phenyl)-3-phenyl-propyl]-5-hydroxy-2-
methyl indole 3-acetate (185 mg, 65%) as a white powder. To
a solution of the obtained compound (183 mg, 0.373 mmol) in
CH2Cl2 (6.00 mL) was added succinic acid monobenzyl ester
(85.0 mg, 0.410 mmol), DCC (115 mg, 0.560 mmol), and DMAP
(4.50 mg, 0.0373 mmol), and the mixture was stirred for 12 h.
The mixture was filtered, and the solvent was removed. The
crude residue was subjected to column chromatography (n-
hexane:AcOEt ) 3:1) to give succinic acid benzyl ester
3-tbutoxycarbonylmethyl-1-[1-(4-chloro-phenyl)-3-phenyl-pro-
pyl]-2-methyl-1H-indole-5-yl ester (249 mg, 98%) as a pale
yellow oil. The compound (249 mg, 0.366 mmol) and Celite 535
(44.0 mg) were stirred at 180-190 °C for 8 h. The crude residue
was subjected to column chromatography (CH2Cl2:MeOH ) 20:
1) to give 31 (150 mg, 66%) as a yellow oil: 1H NMR (270 MHz,
CDCl3) δ (ppm) 7.33-6.96 (16H, m), 6.70 (1H, dd, J ) 6.8 Hz,
2.2 Hz), 5.43 (1H, m), 5.13 (2H, s), 3.67 (2H, s), 2.88-2.76 (6H,
m), 2.42 (2H,m), 2.13 (3H, s); LRMS (EI) m/z 623 (M+). HRMS
(EI) calcd for C37H34NO6Cl, 623.2074; found, 623.2054.
Su ccin ic Acid Ben zyl Ester 3-Ca r boxym eth yl-1-[1-(4-
ch lor op h en yl)-4-p h en yl-bu tyl]-2-m eth yl In d ol-5-yl Ester
(32). To a solution of 33 (155 mg, 0.441 mmol) and tBuOK (59.0
mg, 0.529 mmol) in DMF (2.00 mL) was added a solution of
p-ClC6H4CHBrCH2CH2OPh (180 mg, 0.529 mmol) in DMF
(1.50 mL), and the mixture was stirred for 12 h. To the mixture
was added H2O, and the organic compound was extracted with
AcOEt, washed with aqueous NaCl, dried over Na2SO4,
filtered, and concentrated. The crude residue was subjected
to column chromatography (n-hexane:AcOEt ) 4:1) to give
N-alkylated compound (96.0 mg, 36%) as a yellow oil. To a
solution of N-alkylated compound (96.0 mg, 0.157 mmol) in
AcOEt (3.00 mL) and AcOH (0.600 mL) was added Pd/C (46.0
mg) under H2 (3.00 atm), and the mixture was stirred for 5 h.
The mixture was filtered, and solvent was removed. The crude
residue was subjected to column chromatography (n-hexane:
AcOEt ) 3:1) to give tbutyl-[1-(4-chloro-phenyl)-4-phenoxy-
butyl]-5-hydroxy-2-methyl indole-3-acetate (50.0 mg, 61.1%)
as a pale yellow powder. To a solution of the resulting
compound (50.0 mg, 0.0961 mmol) in CH2Cl2 (1.60 mL) were
added succinic acid monobenzyl ester (23.0 mg, 0.106 mmol),
DCC (31.0 mg, 0.144 mmol), and DMAP (1.20 mg, 0.00961
mmol), and the mixture was stirred for 12 h. The mixture was
filtered, and the organic compound was washed with water
and brine, dried over Na2SO4, filtered, and concentrated. The
crude residue was subjected to column chromatography (n-
hexane:AcOEt ) 3:1) to give succinic acid benzyl ester
3-tbutoxycarbonyl-methyl-1-[1-(4-chloro-phenyl)-4-phenoxy-bu-
tyl]-2-methyl-1H-indole-5-yl ester (62.0 mg, 91%) as a colorless
oil. To a solution of the obtained compound (41.0 mg, 0.0577
mmol) in CH2Cl2 (1.30 mL) was added CF3COOH (0.300 mL),
and the mixture was stirred for 12 h. The solvent was removed,
and the crude residue was subjected to preparative TLC
(AcOEt:MeOH ) 20:1) to give 32 (18.9 mg, 50%) as a yellow
oil: 1H NMR (300 MHz, CDCl3) δ (ppm) 7.28-6.74 (16H, m),
6.62 (1H, dd, J ) 8.9 Hz, 2.3 Hz), 5.49 (1H, dd, J ) 11.1 Hz,
4.8 Hz), 5.08 (2H, s), 3.87-3.73 (2H, m), 3.60 (2H, s), 2.85-
2.45 (6H, m), 2.26 (3H,s), 1.70-1.30 (2H, m); LRMS (FAB) m/z
676 (M+ + Na); HRMS (FAB) calcd for C38H36NO7ClNa,
676.2078; found, 676.2057.
Su ccin ic Acid Ben zyl Ester 3-Ca r boxym eth yl-1-(4-
ch lor oben zoyl)-2-m eth yl-in d ol-5-yl Ester (28). Compound
27 (284 mg, 0.481 mmol) and Celite 535 (56.0 mg) were stirred
at 190 °C for 30 min. The crude residue was subjected to
column chromatography (CH2Cl2:MeOH ) 15:1) to give 28 (101
mg, 39%) as a brown amorphous solid: 1H NMR (300 MHz,
CDCl3) δ (ppm) 7.55 (2H, d, J ) 8.4 Hz), 7.37 (2H, d, J ) 8.4
Hz), 7.29-7.18 (5H, m), 7.11 (1H, d, J ) 1.5 Hz), 6.84 (1H, d,
J ) 8.8 Hz), 6.64 (1H, dd, J ) 8.8 Hz, 1.5 Hz), 5.05 (2H, s),
3.52 (2H, s), 2.77-2.65 (4H, m), 2.23 (3H, s).
5-Ben zyloxy-1-[1-(4-ch lor op h en yl)-3-p h en yl-p r op yl]-2-
m eth yl In d ole 3-Acetic Acid (30). To a solution of 33 (400
t
mg, 1.14 mmol) and BuOK (154 mg, 1.37 mmol) in DMF (7.00
mL) was added a solution of p-ClC6H4CHBrCH2CH2Ph in DMF
(1.00 mL), and the mixture was stirred at room temperature
for 14 h. To the mixture was added H2O. The organic
compound was extracted with AcOEt, washed with brine, dried
over Na2SO4, and concentrated under reduced pressure. The
crude residue was subjected to column chromatography (n-
t
hexane:AcOEt ) 4:1∼3:1) to give butyl 5-benzyloxy-1-[1-(4-
chlorophenyl)-3-phenyl-propyl]-2-methyl indole 3-acetate (337
mg, 51%) as a yellow oil. To a solution of the obtained
compound (44.0 mg, 0.0758 mmol) in CH2Cl2 (1.50 mL) was
added CH3COOH (0.400 mL) dropwise, and the mixture was
stirred for 3 h. The solvent was removed, and the solid was
subjected to column chromatography (CH2Cl2:MeOH ) 20:1)
to give 30 (15.0 mg, 38%) as a dark yellow oil: 1H NMR (300
MHz, CDCl3) δ (ppm) 7.40-6.84 (16H, m), 6.68 (1H, dd, J )
8.9 Hz, 2.6 Hz), 5.34 (1H, d, J ) 8.0 Hz), 5.00 (2H, s), 3.64
(2H, s), 2.69-2.64 (2H, m), 2.34 (2H, t, J ) 7.5 Hz), 2.07 (3H,
s); LRMS (EI) m/z 523 (M+). HRMS (EI) calcd for C33H30NO3-
Cl, 523.1914; found, 523.1900.
Su ccin ic Acid Ben zyl Ester 3-Ca r boxym eth yl-1-[1-(4-
ch lor op h en yl)-3-p h en yl-p r op yl]-2-m eth yl In d ol-5-yl Es-
t
ter (31). To a solution of 33 (400 mg, 1.14 mmol) and BuOK
(154 mg, 1.37 mmol) in DMF (7.00 mL) was added a solution
of p-ClC6H4CHBrCH2CH2Ph in DMF (1.00 mL), and the
En zym e In h ibition . AR was partially purified from bovine
lenses according to the method of Kinoshita et al.45 The in vitro

Discovery of Novel Aldose Reductase Inhibitors
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