Synthesis, Activity, and Molecular Modeling Studies of Novel Human Aldose Reductase Inhibitors Based on a Marine Natural Product

Article abstract of DOI:10.1021/jm030957n

Aldose reductase (ALR2) has been implicated in the etiology of diabetic complications, including blindness. Because of the limited number of currently available drugs for the prevention of these long-term complications, the discovery of new ALR2 inhibitor

Full text of DOI:10.1021/jm030957n

5208
J . Med. Chem. 2003, 46, 5208-5221
Syn th esis, Activity, a n d Molecu la r Mod elin g Stu d ies of Novel Hu m a n Ald ose
Red u cta se In h ibitor s Ba sed on a Ma r in e Na tu r a l P r od u ct
J esu´s AÄ ngel de la Fuente,*,† Sonia Manzanaro,† Mar´ıa J esu´s Mart´ın,‡ Teresa G. de Quesada,‡ Isabel Reymundo,‡
Santos M. Luengo,§ and Federico Gago§
Instituto Biomar, S.A., Pol´ıgono Industrial, Edificio CEEI, 24231 Onzonilla, Leo´n, Spain; Pharma Mar, S.A.,
Pol. Ind. La Mina-Norte, Avda. de los Reyes 1, 28770 Colmenar Viejo, Madrid, Spain; and Departamento de Farmacolog´ıa,
Universidad de Alcala´, 28871 Alcala´ de Henares, Madrid, Spain
Received J uly 11, 2003
Aldose reductase (ALR2) has been implicated in the etiology of diabetic complications, including
blindness. Because of the limited number of currently available drugs for the prevention of
these long-term complications, the discovery of new ALR2 inhibitors appears highly desirable.
In this study, a polybrominated diphenyl ether (1) naturally occurring in a marine sponge was
found to inhibit recombinant human ALR2 with an IC50 of 6.4 µM. A series of polyhalogenated
analogues that were synthesized and tested in vitro to explore the structure-activity
relationships displayed various degrees of inhibitory activity. The most active compounds were
also capable of preventing sorbitol accumulation inside human retinal cells. In this cell-based
assay, the most potent synthesized analogue (16) showed a 17-fold increase in inhibitory activity
compared to that of sorbinil (IC50 ) 0.24 vs 4 µM). A molecular representation of human ALR2
in complex with the natural product was built using homology modeling, automated docking,
and energy refinement methods. AMBER parameters for the halogen atoms were derived and
calibrated using condensed phase molecular dynamics simulations of fluorobenzene, chloroben-
zene, and bromobenzene. Inhibitor binding is proposed to cause a conformational change similar
to that recently reported for zenarestat. A free energy perturbation thermodynamic cycle allowed
us to assess the importance of a crucial bromine atom that distinguishes the active lead
compound from a much less active close natural analogue. Remarkably, the spatial location of
this bromine atom is equivalent to that occupied by the only bromine atom present in zenarestat.
Sch em e 1. Polyol Pathway
In tr od u ction
The elevated blood glucose levels that are character-
istic of diabetes mellitus are responsible for the develop-
ment of microvascular pathology in the retina, renal
glomerulus, and peripheral nerve that can lead to
blindness, renal failure, neuropathies, and cardiovas-
cular disease. Among the mechanisms responsible for
these complications, three appear to play a prominent
role: (i) increased advanced glycation end-product
formation, (ii) activation of protein kinase C isoforms,
and (iii) increased flux of glucose through both the polyol
and the hexosamine pathways. An integrating paradigm
postulates a single hyperglycaemia-induced process of
overproduction of superoxide by the mitochondrial
electron-transport chain.1
The accumulated evidence supporting the importance
of hyperglycemia-induced polyol pathway hyperactivity
has led to a special focus on aldose reductase (alditol/
NADP+ oxidoreductase, EC1.1.1.21, ALR2) as a suitable
target for pharmacological intervention.2-6 This enzyme
catalyzes the reduction of the aldehyde form of D-glucose
to D-sorbitol with concomitant conversion of NADPH to
NADP+ (Scheme 1).7 ALR2 inhibitors (ARIs) offer the
possibility of preventing or arresting the progression of
these long-term diabetic complications even in the
presence of elevated blood glucose levels. Besides, since
they have no effect on plasma glucose there is no
associated risk of hypoglycemia.8
A large variety of structurally diverse compounds
have been identified to date as potent in vitro ARIs.5,9-11
The most potent and better characterized orally active
ARIs belong to two main chemical classes: (i) carboxylic
acid derivatives, such as tolrestat, zopolrestat, and
zenarestat, and (ii) spiro-hydantoins, such as sorbinil
(Figure 1).
X-ray crystallographic studies on both porcine12,13 and
human14,15 ALR2 (Figure 2) have shown that it belongs
to the (â/R)8-barrel class of enzymes and that the
coenzyme NAD(P) molecule binds at the C-terminal end
of the â barrel. The large active site pocket is heavily
lined with hydrophobic residues, which is consistent
with the steroid dehydrogenase activity that is concomi-
tantly associated with this enzyme16 and also with the
fact that glucose is an extremely poor substrate. In fact,
currently known ARIs generally make use of both polar
and nonpolar interactions to find complementarity with
the extended enzyme binding pocket, which is best
described as comprising two regions: (i) a polar site with
residues Trp-20, Tyr-48 (the proton donor),17 and His-
110 and the positively charged nicotinamide moiety of
NADP+, which accommodates the anionic carboxylate
* To whom correspondence should be addressed. Telephone: +34-
918466060. Fax: +34-918466001. E-mail: jdelafuente@pharmamar.com.
† Instituto Biomar, S.A.
‡ Pharmamar, S.A.
§ Universidad de Alcala´.
10.1021/jm030957n CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/18/2003

Novel Human Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5209
F igu r e 3. Some marine natural polybrominated diphenyl
ethers.
problems, together with the length of the trials, have
resulted in a lack of observed efficacy.22 Thus, there is
still an urgent need for new ARIs. In the following, we
report on the ALR2 inhibitory properties of a marine
natural product and a series of related novel polyhalo-
genated diphenyl ether derivatives. Their structure-
activity relationships (SAR) have been rationalized on
the basis of molecular modeling studies encompassing
automated docking, molecular dynamics simulations,
and free energy perturbation calculations.
F igu r e 1. Some orally active ALR2 inhibitors.
Resu lts a n d Discu ssion
Ch em istr y a n d Hu m a n ALR2 In h ibitor y Activ-
ity. In our search for new such ARIs, about 2000 marine
natural products from our compound library were
systematically screened. Potent inhibitory activity against
human ALR2 (hALR2) was found in the polybrominated
diphenyl ether 1 (Figure 3), a compound previously
isolated from the marine sponge Dysidea herbacea.23 It
was noteworthy that this marine natural product showed
an inhibitory activity (IC50 ) 6.4 µM) similar to that of
the well-known ARI sorbinil (IC50 ) 3.6 µM) despite the
absence of either a carboxylate or a cyclic imide group
in its molecule. Two more natural polybrominated
diphenyl ethers, 2 and 3 (Figure 3), isolated from the
same sponge and closely related to 1, were also assayed.
Compound 3 showed no significant activity against
hALR2 whereas 2 was shown to be only weakly active
(Table 1). To the best of our knowledge, the only
polybrominated diphenyl ether that has been previously
reported as an ARI is 2′,3,4,4′,5-pentabromo-2-hydroxy-
diphenyl ether,24 although some related polybrominated
phenoxyphenols (including 1 and 3) have been shown
to be micromolar inhibitors of 15-lipoxygenase and
inosine monophosphate dehydrogenase.25 This fact high-
lights the need to address in more detail the issue of
selectivity in future studies. For the time being, and in
view of the encouraging preliminary findings, we have
focused on the potential of these compounds as hALR2
inhibitors. A variety of new analogues were synthesized
and tested (Table 1) to expand the SAR. Our results
show the importance for this activity of both the
hydroxyl groups and the bromine atom at the para
position relative to the hydroxyl group.
F igu r e 2. View of superimposed C(R) traces of porcine ALR2
in complex with NADP and tolrestat (blue), human ALR2 in
complex with NADP and zopolrestat (red), and human ALR2
in complex with NADP and zenarestat (green). NADP is shown
as sticks, and the inhibitors are shown as balls and sticks.
or the ionizable hydantoin group of the ARI, and (ii) a
nonpolar site with residues Trp-111, Thr-113, Phe-115,
Phe-122, and Leu-300, which lodges the nonpolar part
of the inhibitor. Although the charge state of the cofactor
(NADPH vs NADP+) has been shown not to be critical
for inhibitor binding to ALR2,18 it has been conclusively
demonstrated that negatively charged ARIs act prima-
rily by binding to the enzyme complexed with the
oxidized NADP+ to form a ternary dead-end complex
that prevents turnover in the steady state.19 Modeling
studies, on the other hand, have suggested that a
phenoxy group can provide a good structural replace-
ment for the carboxylate group,20 which can account for
the ALR2 inhibitory activity of flavonoid compounds
such as quercetin and the structure-based designed
7-hydroxy-2-(4′-hydroxybenzyl)-4H-1-benzopyran-4-
one.21
Some of these analogues were obtained by modifica-
tion of the marine natural products (Scheme 2). Thus,
6 was obtained by bromination of 1. The chemical shift
of the aromatic proton of 6 was at δ 6.69 indicating that
this proton was ortho to the ether group, as pointed out
previously.26 The substitution pattern of all the other
derivatives with halogen atoms at C-3, C-4, and C-5 was
established by chemical shift comparison to the previous
compounds.
Even though numerous clinical trials for the treat-
ment of diabetic neuropathy have been conducted over
the last 18 years with synthetic ARIs, pharmacokinetic

5210 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
de la Fuente et al.
Ta ble 1. Human ALR2 Inhibitory Activities of the Test Compounds and Some Standard Inhibitors
compd
X
R1
R2
R3
R4
R5
R6
R7
R8
R9
IC50 (µM)a
1
2
3
4
5
6
7
8
9
11
13
14
15
16
20
21
24
27
30
32
33
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
Me
Me
Ac
H
Br
Br
H
Br
Br
Br
Br
Br
H
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
Br
Br
Br
Cl
Br
Br
Br
Br
Br
Br
Br
Br
H
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
H
H
H
H
Br
Br
H
H
H
OH
OH
OMe
OMe
OAc
OH
OCH2COOMe
OCH2COOH
OH
OH
OMe
OH
OH
OH
OH
OH
NH2
OH
OH
OMe
OH
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
H
H
H
H
H
Br
H
H
H
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Cl
OH
OH
Br
Br
F
6.4 ( 1.1
25%
0%
0%
0%
5.5 ( 1.4
0%
25 ( 0.1
0%
32%
0%
46%
5.7 ( 1.6
3.2 ( 1.4
0%
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Br
Br
Br
Br
Br
Br
Br
CH2COOMe
CH2COOH
H
H
Me
H
H
H
Me
H
H
H
H
Me
H
H
H
H
H
H
H
Br
Br
Br
Cl
H
Br
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Br
Br
Br
Br
Br
Br
Br
H
38%
O
O
O
NH
NH
Br
OH
Br
Br
Br
8.9 ( 4.2
4.4 ( 2.5
3.2 ( 2.8
0%
H
H
Br
Br
18%
tolrestat
sorbinil
quercetin
0.4 ( 0.3
3.6 ( 1.0
14%
a
IC50 values represent the concentration required to produce 50% enzyme inhibition or percent of inhibition at the highest concentration
used (25µg/mL). Human recombinant ALR2 was used and all values are the mean of at least three experiments.
Sch em e 2a
Sch em e 3a
a
Reagents and conditions: (a) K2CO3, MeI, 50 °C; (b) Ac2O, Pyr,
a
Reagents and conditions: (a) Br2, CHCl3, -35 °C; (b) Br2,
AcOH, rt; (c) BBr3, ClCH2CH2Cl, rt; (d) Br2, AcOH, reflux, (e) AlCl3,
SO2Cl2, 95 °C.
rt; (c) Br2, AcOH, 90 °C; (d) K2CO3, acetone, BrCH2COOMe, 65
°C; (e) KOH, EtOH, 90 °C; (f) BBr3, ClCH2CH2Cl, rt.
Novel polyhalogenated diphenyl ether derivatives
were obtained by total synthesis (Schemes 3 and 4).
Treatment of the known 12 with bromine afforded 13,
which was transformed into 14 by treatment with boron
tribromide. NOESY experiments of 13 showed correla-
tions between the methoxy group and the proton at δ
7.19, indicating that the bromine atoms were meta and
para to the phenolic OH. This assignment was also
confirmed by Heteronuclear Multiple Bond Coherence
experiments of 14. Other analogues were obtained by
use of the Ullmann reaction between guaiacol and
different bromobenzenes in the presence of copper
catalyst27 (Scheme 5). Compound 31 (Scheme 6) was
obtained as previously described28 and was used to
obtain the derivatives 32 and 33.
The effect of hydroxyl group modification can be
assessed by comparing compound 1 with compounds 4,
5, and 7; the resulting complete loss of activity strongly

Novel Human Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5211
Sch em e 4a
and 33, respectively, were modestly active. On the other
hand, incorporation of two methylencarboxylic groups
in place of the hydroxyls, as in 8, led to an active product
even though its inhibitory properties were decreased
with respect to the parent compound. For other phenolic
ARIs related to acetophenones and benzophenones,29 as
well as several hydroxylated chalcones and benzopiran-
4-ones,21 the markedly detrimental effect on biological
activity of methylation of the hydroxyl groups has been
taken as evidence for the involvement of the dissociated
phenoxy forms in binding to the enzyme. However, in
the present series, substitution of an amino group for
one of the hydroxyls (as in going from 15 to 24) had just
a marginal deleterious effect on the inhibitory activity,
suggesting that this particular hydroxyl group partici-
pates in donating a hydrogen bond to the enzyme
binding site. On the other hand, comparison of the
activities of compounds 15 and 33 clearly illustrates that
an amino group can replace the bridging ether group
but only at a great cost in potency.
a
Reagents and conditions: (a) K2CO3, DMF, rt; (b) Na2S2O4,
CHCl3/H2O, rt; (c) BBr3, ClCH2CH2Cl, rt.
To assess the importance of the halogen group in the
meta position relative to the bridging ether oxygen we
pairwise compared the activities of compounds 1 and
9, 1 and 21, and 14 and 15. From these results (Table
1) it can be inferred that replacement of a bromine at
this meta position with either a hydrogen or a hydroxyl
group is detrimental for potency, regardless of the ring
in which this substitution takes place.
suggests that these free hydroxyl groups play a signifi-
cant role in enzyme binding. This could be due to steric
hindrance or to a direct involvement of one or both OH
groups in crucial interactions with the enzyme binding
site; an alternative possibility would be that a confor-
mational change takes place on the inhibitor upon
substitution that is incompatible with binding at the
site. Further data confirm this SAR, as compounds 13,
20, and 32, all of them with methoxy groups in place of
the hydroxyls, showed no activity, whereas the corre-
sponding compounds with free hydroxyl groups 14, 21,
The importance of the halogen group at the para
position with respect to the ether oxygen in any of the
rings was evaluated by comparing the pairs 1 and 6,
Sch em e 5a
a
Reagents and conditions: (a) NaH, CuBr‚SMe2, DMF, 1-bromo-2-nitrobenzene, reflux; (b) 10% Pd/C, H2, MeOH; (c) Br2, CCl4, rt; (d)
NaOMe, CuCl, Pyr, 1-bromo-2,5-dimethoxybenzene, reflux; (e) BBr3, ClCH2CH2Cl, rt; (f) NaOMe, CuCl, Pyr, 2-bromo-4-fluoroanisole,
reflux; (g) Br2, AcOH, rt.
Sch em e 6a
a
Reagents and conditions: (a) Br2, CCl4, rt; (b) BBr3, ClCH2CH2Cl, rt.

5212 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
de la Fuente et al.
Ta ble 2. Inhibitory Activity on Sorbitol Accumulation in
Human Retinal Cells
compd
IC50 (µM)a
1
6
3
7
15
1
16
24
0.24
6
27
8
30
tolrestat
sorbinil
11
0.02
4
a
IC50 values represent compound concentration that inhibited
sorbitol accumulation by 50%. Retinal human cells were used and
all values are the mean of at least three experiments.
and 15 and 27. Given their similar activities, our
conclusion is that the nature of the substituent at this
para position (a bromine, a hydroxyl or a hydrogen) is
not crucial for modulating the inhibitory properties of
these compounds. The same reasoning can be applied
to the ortho position relative to the bridging ether
oxygen since replacement of a bromine, as in 1 and 6,
with a hydrogen (compound 15) had no effect on potency.
Compounds 16 and 30, in which one or some of the
bromine atoms of 1 or 15 have been replaced with
chlorine or fluorine, allowed us to assess the relative
importance of different halogen atoms on activity. The
potency in both cases increased only 2-fold, which
suggests that the type of halogen has little bearing on
the inhibitory activity.
F igu r e 4. AutoDock results for docking zopolrestat into the
modeled hALR2 enzyme.
Ta ble 3. Bonded and Nonbonded Parameters for the Halogen
Atoms Present in the Halobenzenes Studied
a
b
c
d
bond
Kr
req
angle
Kθ
θeq
atom R*a
ꢀb
CA-F 386.0 1.331 CA-CA-F 70.0 119.0
F
1.63 0.171
1.82 0.475
1.94 0.595
CA-Cl 193.0 1.745 CA-CA-Cl 70.0 119.0 Cl
CA-Br 172.0 1.888 CA-CA-Br 70.0 119.0 Br
a
Bond force constant, kcal/(mol Å2). b Equilibrium bond lengths,
Å. c Angle bending force constant, kcal/(mol radian2). Equilibrium
d
bond angles (deg). e van der Waals minimum. f van der Waals well
depth.
Those compounds with an in vitro IC50 e 10 µM were
then advanced to a secondary screen in which com-
pounds were tested for their ability to prevent the
accumulation of sorbitol inside human retinal cells when
they were cultured in a medium supplemented with 50
mM glucose so as to reproduce in vitro the typical
hyperglycemic conditions of diabetes. The results show
that six out of the seven compounds tested in this assay
have an IC50 under 10 µM (Table 2). Interestingly, the
potency of compounds 15 and 16 was 4-fold and 17-fold
greater, respectively, than that of sorbinil. Moreover,
in this assay compound 16 was 12 times more potent,
on a molar basis, than the lead marine natural product
1.
Molecu la r Mod elin g of h ALR2 a n d Dock in g of
th e In h ibitor s. A structure for the hALR2:NADP+:
zopolrestat ternary complex was modeled starting from
the coordinates deposited for the CR trace of the enzyme,
the cofactor NADP+, and the inhibitor (PDB accession
code: 1mar),15 as described in the Experimental Section.
Energy refinement of this complex resulted in an
enzyme structure possessing an enlarged active site in
an “open” conformation that was suitable for inhibitor
binding. Prior to automated docking of the reported
novel inhibitors, zopolrestat itself was docked as a
means of testing program performance. The largest
cluster found also corresponded to the lowest docking
energy and contained the crystallographic solution
(Figure 4).
F igu r e 5. Time evolution of (left) the calculated density (g
cm-3) and (right) enthalpy of vaporization (∆Hvap, kcal/mol)
during the molecular dynamics simulations of the solvent
boxes: fluorobenzene (open triangles), chlorobenzene (open
circles), and bromobenzene (open squares). For comparative
purposes, dotted lines have been drawn at the respective
experimental values.
was achieved using condensed phase molecular dynam-
ics simulations of three relevant organic solvents (see
under Computational Methods). The good agreement
found between the calculated and experimentally mea-
sured densities and enthalpies of vaporization of these
liquids (Figure 5) attests to the validity of these
parameters, which were similar but not equivalent to
previously reported parameters for the same atoms in
haloalkanes.31
The nonbonded parameters were also transformed
into a form suitable for the automated docking program
(see Methodology and Supporting Information). The
docking results for the diphenyl ethers inhibitors were
heavily dependent on the degree of halogenation. Thus,
for compound 1 a large number of solutions, all of them
outside the active site, were found whereas compound
16 was preferably docked within the specificity pocket
Since the reported inhibitors are halogenated and no
parameters were available for these atoms in the
molecular mechanics force field AMBER (parm96),30
consistent parameters for the halogen atoms had to be
derived to describe the bonded and nonbonded interac-
tions (Table 3). Calibration of these crucial parameters

Novel Human Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5213
F igu r e 6. AutoDock results for representative compounds 1 (left) and 16 (right) into the modeled hALR2 enzyme.
of the active site (Figure 6). The calculated binding
energies, however, were very similar in both cases and,
rather strikingly, could not be used to tell apart those
molecules that were bound in the active site from those
bound elsewhere. The preferred orientation found for
16 was chosen as the most plausible one also for 1 and
the remaining inhibitors. The differences observed
between 1 and 16 were thought to arise from the
increased size of bromine relative to chlorine, which can
hamper docking into the rigidly fixed binding site.
To assess the feasibility of the proposed binding
orientation and study the mutual adaptation between
enzyme and inhibitor, the hALR2-NADP+-1 ternary
complex was energy-minimized and simulated for 950
ps of molecular dynamics. The average structure was
then analyzed in terms of intermolecular energy com-
ponents, desolvation effects, and conformational changes
with respect to the holoenzyme (apoenzyme + coen-
zyme).
reported complex of hALR2 with zenarestat,32 an inhibi-
tor that also contains a bihalogenated phenyl ring
(Figures 1 and 2).
The proposed binding mode can rationalize the SAR
reported above. Replacement of the ether bridge with
an amino group, as in going from 15 to 33, will bring
about a detrimental change in the intra- and intermo-
lecular hydrogen bonding patterns resulting in much
lower affinity (Table 1). Incorporation of two methyl-
enecarboxylic groups in place of the hydroxyls, as in
going from 1 to 8, will likewise result in loss of potency
due to the increase in bulk in positions that are in very
close contact with protein atoms and engaged in crucial
hydrogen bonding interactions. The irrelevant nature
of the substituents present at the ortho and para
positions relative to the bridging ether oxygen is now
rationalized because these positions are mostly exposed
to the bulk solvent.
En er gy An a lysis of th e En zym e-In h ibitor Com -
p lexes. The energy analysis of the hALR2-NADP+-1
complex (Figure 8) reveals a predominance of van der
Waals interactions involving (in decreasing order of
magnitude) Trp-111, Leu-300, Trp-20, Cys-298, Cys-303,
NADP+, Phe-122, Tyr-309, His-110, and Ala-299. The
electrostatic interactions, on the other hand, appear to
be dominated by Cys-298, followed by Lys-77, Trp-111,
Leu-300, and NADP+. When compared with zopolrestat
(Figure 1 of the Supporting Information), the major
differences in electrostatic interaction energy between
these two compounds and the enzyme originate from
those residues that interact with the free carboxylate
group of this negatively charged inhibitor, that is, Lys-
77 and NADP+. However, the counterparts of these
favorable intermolecular interactions are a larger repul-
sion with negatively charged residues, such as Asp-43
and Glu-185, and a larger desolvation penalty associated
with the removal of the interacting polar groups from
water. Thus, in the hALR2:NADP+:1 complex, the
electrostatic contributions to the free energy change
associated with desolvation of both the binding site and
the inhibitor are 4.6 and 1.9 kcal mol-1, respectively,
whereas for the hALR2:NADP+:zopolrestat complex the
corresponding values are 7.2 and 13.3 kcal mol-1. Since
the sum of all residue-based electrostatic contributions
to the binding energy is -14.1 kcal mol-1 for zopolrestat
versus -2.2 kcal mol-1 for 1, the net electrostatic
In the native human holoenzyme (PDB code: 1ads)14
the side chain of Leu300 makes a direct contact with
the aromatic ring of Trp111. The binding orientation
we propose for 1 is such that the more heavily halogen-
ated phenyl ring is sandwiched between the indole ring
of Trp-111 on one side and the side chains of Leu-300
and Cys-303 on the other side (Figure 7). This hydro-
phobic interaction, which is similar but not identical to
that described for the larger benzothiazole ring of
zopolrestat,15 is further stabilized by the phenyl ring of
Phe-122, whose edge contacts both halogenated phenyl
rings. The ether oxygen allows a nearly orthogonal
orientation of these two rings with respect to each other
such that the electronegative bromine atom ortho to the
hydroxyl group in the second phenyl ring makes contact
with both Trp-20 and the pyridinium group of NADP+.
The free hydroxyls engage in hydrogen bonding interac-
tions with the NH of Leu-300 (the OH acts as an
acceptor) and the thiol group of Cys-298 (the OH acts
as a donor). The position of this latter sulfhydryl group
is fixed by a buttressing interaction from the carboxylate
of Glu-185. The inhibitor is thus almost completely
sequestered from the solvent in the apolar region of the
pocket whereas it occupies the polar region of the
binding site only partially. Overall, the orientation is
reminiscent of that of bound zopolrestat and differs with
respect to that of tolrestat (Figure 2). However, the
structural similarity is much greater with the recently

5214 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
de la Fuente et al.
it usually is in very good agreement with calculated
differences in solvation free energies, as we have
recently shown for an unrelated series of antiviral
compounds.34
The free energy difference associated with conversion
of 1 into 2 in aqueous solution was -5.83 ( 0.04 kcal
mol-1, in very good agreement with the change of 5.90
( 0.04 kcal mol-1 obtained from the independent
perturbation of 2 into 1. For the perturbations of the
enzyme-bound inhibitors the corresponding free energy
changes were -4.33 ( 0.52 kcal mol-1 and 3.60 ( 0.52
kcal mol-1, respectively. The binding of 2 to hALR2 is
thus calculated to be disfavored by 1.50 ( 0.30 kcal
mol-1 with respect to the binding of 1, which is in
semiquantitative agreement with the decreased potency
shown by 2 in the enzyme inhibition assay relative to 1
(Table 1). It is remarkable that this relatively large
difference stems from a simple Br f H substitution in
an otherwise equally polyhalogenated compound, but
the more favorable binding energy can be related to both
improved interactions with the enzyme and to a greater
hydrophobic effect. This bromine atom is lodged in a
pocket (made up by Trp-79, Trp-111, Thr-113, Phe-115,
and Cys-303) that is enlarged upon minor side-chain
conformational changes. Interestingly, the position of
this deeply buried bromine atom is spatially equivalent
to that occupied by the only bromine atom present in
zenarestat (Figure 1). The fact that introduction of this
halogen atom alone into a weak inhibitor is enough to
turn it into a more potent one can be important for
future ARI design using a minimal molecular frame-
work.
Con clu sion s
A marine natural polybrominated diphenyl ether, 1,
has been identified as a new hALR2 inhibitor having
an IC50 of 6.4 µM, and a series of analogues have been
synthesized and evaluated to establish the structure-
activity relationships. The most notable structural
feature of the active compounds is the lack of either the
carboxylate or the cyclic imide group commonly present
in the principal classes of currently used inhibitors.
Several of the synthetic derivatives were marginally
more active than the lead compound in the in vitro assay
and one of them, 16, was able to prevent sorbitol
accumulation inside human retinal cells at concentra-
tions 17-fold lower than were necessary with the
standard drug sorbinil.
F igu r e 7. Proposed binding site for 1 in hALR2. (bottom)
Schematic representation of the CR trace of the enzyme (R-
helices are shown as red barrels, â-strands as yellow flat
ribbons, and turns as blue arrows), with protein residues
enveloped by a semitransparent solvent-accessible surface.
Carbon atoms of NADP+ and the inhibitor are colored in cyan
and gray, respectively. (top) Enlarged view of the framed area
shown in the top panel. Protein residues relevant to the
discussion have been labeled, and their side chains are shown
as sticks.
The crucial role of one of the bromine atoms in 1 was
realized when a molecular model of the complex was
constructed and simulated using molecular dynamics.
The spatial position of the relevant Br atom in the
specificity pocket of the active site is coincident with
that of the bromine atom present in zenarestat. More-
over, the described inhibitors and zenarestat give rise
to similar conformational changes in the enzyme. Fur-
ther support for the proposed binding mode was inferred
from the good qualitative agreement that was obtained
between the experimentally measured differences in
inhibitory activity and the calculated differences in
binding free energies using a free energy perturbation
approach.
binding free energy difference between the two com-
pounds is only 2 kcal mol-1
.
Finally, a thermodynamic cycle (Scheme 7) was set
up to test whether the proposed binding orientation
could account for the importance of the crucial bromine
atom in the para position that distinguishes the active
1 from the much less active 2.
Although an increase in hydrophobicity is expected
in going from H to Cl or Br, as assessed from the
incremental π constants derived from log Po/w values for
benzene derivatives,33 such a change is not always
directly correlated with an increase in activity although

Novel Human Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5215
F igu r e 8. Calculated van der Waals (top) and continuum electrostatic (bottom) interaction energies between 1 and individual
protein and cofactor residues.
Sch em e 7a
Elemental analyses were performed on a Perkin-Elmer ana-
lyzer (model 2400 CHN). ESI and APCI mass spectra were
recorded on a HP 1100 LC/MS, EI and FAB mass spectra on
a VG Autospec spectrometer. Thin-layer chromatography was
performed on Merck silica gel plates (DC-60 F254). SDS silica
gel (35-70 µm) and Merck RP-18 (40-63 µm) were used for
normal and reverse phase flash chromatography, respectively.
All reagents were used as received unless otherwise stated.
a
Thermodynamic cycle used to estimate the free energy changes
Compounds 1-3 were isolated from the sponge Dysidea
involved when a bromine in 1 is replaced with a hydrogen (as in
herbacea as previously described,23 and were identical in all
2) in the free ligand in solution (∆G3) and in the solvated complex
respects to those reported in the literature. Compounds 4,26
(∆G4), respectively.
10,35 and 1136 were obtained as previously described.
Exp er im en ta l Section
3,3′,4,5,5′,6-Hexa br om o-2,2′-d ia cetoxyd ip h en yl Eth er
(5). To a solution of 1 (5 mg, 8 µmol) in pyridine (0.4 mL) was
added Ac2O (0.4 mL). The mixture was stirred at room
temperature for 24 h and concentrated in vacuo. The residue
was chromatographed on a silica gel column (Hex-CH2Cl2, 1:1)
to afford 5 (5.4 mg, 96%): 1H NMR (CDCl3): δ 7.48 (s, 1H),
7.47 (s, 1H), 2.35 (s, 3H), 2.24 (s, 3H). MS (ESI-negative) m/z:
717 [M - Ac]-, 674 [M - 2Ac]-.
A. Ch em ica l P r oced u r es. Melting points were determined
in open capillaries with a Bu¨chi B-535 melting point apparatus
and are uncorrected. The NMR spectra were recorded on a
Varian 300 spectrometer at 300 MHz for 1H NMR and at 75
MHz for 13C NMR, with TMS as an internal standard.
Chemical shifts (δ) are reported as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br s (broad singlet).

5216 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
de la Fuente et al.
3,3′,4,4′,5,5′,6-Heptabr om o-2,2′-dih ydr oxydiph en yl Eth er
(6). To a solution of 1 (50 mg, 74 µmol) in acetic acid (5 mL)
at room temperature and stirring was added bromine (0.5 mL,
9.76 mmol). The mixture was heated for 3 days at 90-95 °C
and then cooled to room temperature and concentrated in
vacuo. The residue was chromatographed on a silica gel column
(Hex-acetone, 3:2) to afford 6 (32 mg, 57%): mp:188-190 °C;
149.65 (C-1) and 7.23 (H-3) and 7.06 (H-6); MS (APCI-negative)
m/z: 517 [M - H]-. Anal. (C12H6Br4O3) C; H: calcd, 1.17;
found, 1.13.
3,3′,4,4′,5,5′-Hexa br om o-2,2′-d ih yd r oxyd ip h en yl Eth er
(15). To a solution of 1235 (200 mg, 0.99 mmol) in acetic acid
(6 mL) at room temperature was added dropwise bromine (2
mL, 39.6 mmol) while stirring. The reaction mixture was
heated for 15 h at 90-95°C and then cooled to room temper-
ature and concentrated in vacuo. The residue was chromato-
graphed on a silica gel column (Hex-EtOAc, 7:3) to afford 15
(520 mg, 78%): mp 188-190 °C. 1H NMR (CD3OD): δ 7.16 (s,
2H); 13C NMR (CD3OD): δ 148.21, 145.29, 123.78, 122.98,
116.45, 114.43; MS (APCI-negative) m/z: 675 [M - H]-. Anal.
(C12H4Br6O3) C; H: calcd, 0.90; found, 0.96.
1H NMR (CD3OD): δ 6.69 (s, 1H); 13C NMR (CD3OD):
δ
150.49, 146.81, 145.54, 141.11, 126.94, 121.97, 121.30, 118.92,
117.92, 116.40, 115.58, 113.75; MS (APCI-negative) m/z: 753
[M - H]-, 674 [M - Br]-. Anal. (C12H3Br7O3) C; H: calcd, 0.40;
found, 0.36.
3,3′,4,5,5′,6-Hexabr om o-2,2′-dim eth oxycar bon ylm eth oxy-
d ip h en yl Eth er (7). To a solution of 1 (9 mg, 13 µmol) and
BrCH2COOMe (0.4 mL, 4.2 mmol) in acetone (2 mL) was added
K2CO3 (5 mg, 35 µmol). After stirring at 65 °C for 6 h, the
reaction was cooled to room temperature and filtered. The
solvent was removed in vacuo, and the residue was chromato-
graphed on a silica gel column (Hex-EtOAc, 8:2) to afford 7
(9.9 mg, 91%): 1H NMR (CDCl3): δ 7.43 (d, J ) 2.2 Hz, 1H),
6.49 (d, J ) 2.2 Hz, 1H), 4.75 (s, 2H), 4.67 (s, 2H), 3.80 (s,
3H), 3.75 (s, 3H); 13C NMR (CDCl3): δ 168.51, 167.68, 149.77,
148.91, 144.10, 143.78, 130.08, 126.98, 125.12, 122.31, 121.36,
118.92, 117.52, 117.06, 69.73, 69.58, 52.39, 52.21. MS (ESI-
positive) m/z: 843 [M + Na]+. Anal. (C18H12Br6O7) C; H: calcd,
1.48; found, 1.45.
3,3′,4,4′,5,5′-Hexa ch lor o-2,2′-d ih yd r oxyd ip h en yl Eth er
(16). A suspension of 1235 (120 mg, 0.5 mmol), aluminum
trichloride (35 mg, 0.26 mmol) in sulfuryl chloride (2 mL) was
heated at 95 °C for 6 h. The mixture was cooled to room
temperature and concentrated in vacuo. The residue was taken
up in EtOAc, washed with H2O and brine, dried (Na2SO4), and
concentrated in vacuo. The residue was dissolved in 1,2-
dichloroethane (4 mL) and boron tribromide (1.06 g, 4.23
mmol) was added dropwise under an argon atmosphere. The
mixture was stirred at room temperature for 1 h. Diethyl ether
was added dropwise, and after stirring for 15 min, the mixture
was diluted with EtOAc, washed with H2O and brine, dried
(Na2SO4), and concentrated in vacuo. The residue was chro-
matographed on a silica gel (RP-18) column (MeOH-H2O, 85:
15) to give 16 (14 mg, 6%): 1H NMR (acetone-d6): δ 7.28 (s,
2H); 13C NMR (acetone-d6): δ 146.79, 144.43, 127.52, 123.08,
122.81, 119.26; MS (APCI-negative) m/z: 407 [M - 2H]-. Anal.
(C12H4Cl6O3) C; H: calcd, 0.99; found, 1.03.
3,3′,4,5,5′,6-Hexa br om o-2,2′-d ica r boxym eth oxyd ip h en -
yl Eth er (8). A mixture of 7 (10 mg, 12 µmol), 10% KOH (0.05
mL, 0.89 mmol) and ethanol (3 mL) was heated at 90 °C for 2
h. The reaction mixture was cooled to room temperature and
concentrated in vacuo. The residue was poured into H2O,
acidified with 1M HCl to pH ) 5 and extracted with EtOAc.
The organic layers was washed with H2O and brine, dried (Na2-
SO4) and removed in vacuo to give 8 (9.6 mg, 96%): mp: 185-
187 °C; 1H NMR (DMSO-d6): δ 7.58 (d, J ) 2.4 Hz, 1H), 6.96
(d, J ) 2.4 Hz, 1H), 4.65 (s, 2H), 4.51 (s, 2H); 13C NMR (DMSO-
d6): δ 169.43, 168.75, 149.86, 149.26, 143.72, 143.53, 129.10,
126.22, 123.96, 122.04, 121.01, 118.18, 117.09, 116.29, 69.91,
69.56; MS (FAB-positive) m/z: 814 [M + Na]+. Anal. (C16H8-
Br6O7) C; H: calcd, 1.02; found, 1.10.
2-Br om o-6-(2,3,4,5-t et r a b r om o-6-m et h oxy-p h en oxy)-
1,4-ben zoqu in on e (19). A displacement reaction38 of one
bromine atom from 2,6-dibromobenzoquinone was used to
obtain 19. To this end, to a solution of 1839 (300 mg, 0.68 mmol)
in DMF (9 mL) was added K2CO3 (189 mg, 1.36 mmol). The
mixture was stirred at room temperature for 1 h before adding
a solution of 1740 (181 mg, 0.68 mmol) in DMF (6 mL). After
2 and 5 h at room-temperature two more additions of 17 (203
mg, 0.76 mmol) were done. The mixture was stirred at room
temperature for 24 h, and then it was treated with HCl (2 M),
extracted with diethyl ether, washed with brine, dried (Na2-
SO4), and concentrated in vacuo. The residue was chromato-
graphed on a silica gel column (Hex-CH2Cl2, 3:2) to afford 19
(60 mg, 14%): 1H NMR (CDCl3): δ 7.23 (d, J ) 1.8 Hz, 1H),
5.67 (d, J ) 1.8 Hz, 1H), 3.83 (s, 3H); 13C NMR (acetone-d6):
δ 183.98, 173.33, 154.47, 149.65, 143.95, 138.31, 134.56,
127.52, 124.88, 122.45, 119.89, 111.70, 61.90.
3,4′,5,5′,6′-P en ta br om o-2,2′-d ih yd r oxyd ip h en yl Eth er
(9). To a solution of 3 (2 mg, 3.2 µmol) in 1,2-dichloroethane
(2 mL) was added dropwise boron tribromide37 (100 µL, 1
mmol) under an argon atmosphere. The mixture was stirred
at room temperature overnight. Diethyl ether was added
dropwise, and after stirring for 15 min, the mixture was
diluted with EtOAc, washed with H2O, dried (Na2SO4), and
concentrated in vacuo. The residue was chromatographed on
a silica gel column (Hex-EtOAc, 8:2) to afford the known 9
(1.5 mg, 79%), which was identified by comparison of the 1H
NMR and MS data with published values.25
3,3′,4,5,6-P en tabr om o-2-m eth oxy-2′,5′-dih ydr oxydiph en -
yl Eth er (20). To a mixture of 19 (42 mg, 0.07 mmol) in CHCl3
(2 mL) was added a solution of Na2S2O4 (60 mg, 0.34 mmol)
in H2O (2 mL). After being stirred for 5 h at room temperature,
the reaction mixture was washed with brine, dried (Na2SO4),
and concentrated in vacuo. The residue was chromatographed
on a silica gel column (Hex-EtOAc, 9:1) to give 20 (26 mg,
62%): 1H NMR (acetone-d6): δ 8.31 (s, 1H), 8.17 (s, 1H), 6.75
4,4′,5,5′-Tetr abr om o-2,2′-dim eth oxydiph en yl Eth er (13).
To solution of 1235 (200 mg, 0.87 mmol) in acetic acid (5 mL)
was added dropwise bromine (1 mL) while stirring. The
mixture was stirred at room temperature for 1 h and then was
concentrated in vacuo. The residue was treated with 1M NaOH
and extracted with CH2Cl2. The combined organic layers were
washed successively with H2O and brine, dried (Na2SO4), and
concentrated in vacuo to give quantitatively 13 (474 mg): mp
134-135 °C; 1H NMR (CDCl3): δ 7.19 (s, 2H), 7.02 (s, 2H),
3.83 (s, 6H); a correlation was observed in a NOESY spectrum
in this solvent between δ 3.83 and 7.19; 13C NMR (CDCl3): δ
149.99, 144.72, 122.99, 119.13, 117.30, 114.75, 56.37. Anal.
(C14H10Br4O3) C; H: calcd, 1.85; found, 1.86.
(d, J ) 2.7 Hz, 1H), 6.05 (d, J ) 2.7 Hz, 1H), 3.87 (s, 3H); 13
C
NMR (acetone-d6): δ 152.30, 151.30, 146.78, 146.38, 137.82,
126.11, 124.45, 122.76, 122.42, 113.95, 111.22, 102.43, 61.99.
3,3′,4,5,6,-P en tabr om o-2,2′,5′-tr ih ydr oxydiph en yl Eth er
(21). To a solution of 20 (66 mg, 0.1 mmol) in 1,2-dichloroet-
hane (4 mL) was added dropwise boron tribromide (0.03 mL,
0.3 mmol) under an argon atmosphere. The mixture was
stirred at room temperature for 14 h. Diethyl ether was added
dropwise, and after stirring for 15 min, the mixture was
diluted with EtOAc, washed with H2O and brine, dried (Na2-
SO4), and concentrated in vacuo to afford 21 (50 mg, 80%):
4,4′,5,5′-Tetr abr om o-2,2′-dih ydr oxydiph en yl Eth er (14).
Compound 14 was prepared from 13 (54 mg, 0.1 mmol)
according to the method of preparation for 9. The residue was
chromatographed on a silica gel column (Hex-EtOAc, 3:2) to
1
mp 216-218 °C. H NMR (acetone-d6): δ 6.72 (d, J ) 2.7 Hz,
1
1H), 6.01 (d, J ) 2.7 Hz, 1H); 13C NMR (acetone-d6): δ 151.37,
150.06, 145.91, 140.37, 137.91, 126.24, 121.31, 118.51, 115.41,
113.75, 111.1, 101.68; MS (ESI-negative) m/z: 611[M - H]-,
give 14 (49 mg, 95%): mp 195-197 °C. H NMR (CD3OD): δ
7.23 (s, 2H), 7.06 (s, 2H); 13C NMR (CD3OD): δ 149.65, 145.48,
124.28, 122.41, 119.78, 113.76; gHMBC correlations were
observed between δ113.76 (C-4), 119.78 (C-5), 145.48 (C-2),
531[M - 2H-Br]- Anal. (C12H5Br5O4) C, H.
.

Novel Human Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5217
2-Meth oxy-2′-n itr od ip h en yl Eth er (22). To a suspension
of guaiacol (280 mg, 2.3 mmol), NaH (91 mg, 2.3 mmol), and
CuBr‚SMe2 (1.45 g, 7.03 mmol) in DMF (10 mL) was added
1-bromo-2-nitrobenzene (0.56 g, 2.3 mmol) under an argon
atmosphere. The mixture was heated under refluxing condi-
tions for 3 h and then was cooled to room temperature, diluted
with EtOAc, and washed with HCl (1 M). The organic layer
was washed with H2O and brine, dried (Na2SO4), and removed
in vacuo. The residue was chromatographed on a silica gel
column (Hex-EtOAc, 8:2) to give 2241-42 (203 mg, 36%): mp
69-70 °C; 1H NMR (CDCl3): δ 7.92 (dd, J ) 8.2, 1.5 Hz, 1H);
7.41 (td, J ) 7.9, 1.5 Hz, 1H) 7.20 (td, J ) 7.9, 1.5 Hz, 1H),
7.08 (m, 2H), 6.98 (m, 2H), 6.81 (dd, J ) 8.2, 1.2 Hz, 1H), 3.76
(s, 3H); 13C NMR (CDCl3): δ 151.61, 151.21, 143.13, 139.83,
133.96, 126.28, 125.51, 121.92, 121.74, 121.19, 117.96, 113.08,
55.85.
3,3′,4,5,5′-P en tabr om o-2,2′,4′-tr ih ydr oxydiph en yl Eth er
(27). To a solution of 26 (16 mg, 0.07 mmol) in CCl4 (2 mL)
was added dropwise bromine (1 mL, 19.8 mmol) with stirring.
The mixture was stirred for 24 h at room temperature and
then was concentrated in vacuo. The residue was chromato-
graphed on a silica gel column (CH2Cl2-EtOAc, 95:5) to give
27 (36 mg, 80%): mp 207-209 °C. 1H NMR (acetone-d6): δ
7.35 (s, 1H), 7.13 (s, 1H), 13C NMR (acetone-d6): δ 150.00,
147.71, 146.67, 146.44, 136.66, 123.70, 121.39, 119.71, 114.80,
113.84, 101.64, 99.57. MS (ESI-negative) m/z: 612 [M - H]-,
531 [M - 2H - Br]-. Anal. (C12H5Br5O4) C; H: calcd, 0.82;
found, 0.83.
4-F lu or o-2,2′-d im eth oxyd ip h en yl Eth er (28). Compound
28 was prepared from guaiacol (123 mg, 0.86 mmol) and
2-bromo-4-fluoroanisole (540 mg, 2.58 mmol) according to the
method of preparation for 25. The residue was chromato-
graphed on a silica gel column (Hex-CH2Cl2, 1:3) to afford 28
(134 mg, 63%): 1H NMR (CDCl3): δ 7.13 (m, 1H), 6.93 (m,
4H), 6.70 (m, 1H), 6.46 (dd, J ) 9.5, 2.9 Hz, 1H), 3.86 (s, 3H),
3.82 (s, 3H); 13C NMR (CDCl3): δ 156.81 (d, J ) 239 Hz)
150.89, 147.30 (d, J ) 10 Hz), 146.20 (d, J ) 3 Hz), 144.37,
125.01, 120.93, 120.35, 112.77, 112.60, 108.48 (d, J ) 23.2 Hz),
105.21 (d, J ) 27.2 Hz), 56.50, 55.76.
5-F lu or o-3,3′,4,4′,5′-p en ta br om o-2,2′-d im eth oxyd ip h en -
yl Eth er (29). To a solution of 28 (72 mg, 0.29 mmol) in acetic
acid (2 mL) was added dropwise bromine (0.6 mL) with
stirring. The mixture was stirred at room temperature for 5
days and then was concentrated in vacuo. The residue was
treated with NaOH (1 M) and extracted with CH2Cl2. The
organic layer was successively washed with H2O and brine,
dried (Na2SO4), and removed in vacuo. The residue was
chromatographed (Hex-CH2Cl2, 85:15) to give 29 (118 mg,
64%): 1H NMR (CDCl3): δ 7.21 (s, 1H), 6.71 (d, J ) 8.8 Hz,
1H), 3.88 (s, 3H), 3.87 (s, 3H); 13C NMR (CDCl3): δ 155.82 (d,
J ) 248 Hz), 149.03 (d, J ) 10 Hz), 148.75, 148.36, 145.67 (d,
J ) 4 Hz), 123.75, 123.36, 123.16, 120.02, 108.18 (d, J ) 24
Hz), 106.35 (d, J ) 28 Hz), 102.16 (d, J ) 29 Hz), 61.33, 61.24.
5-F lu or o-3,3′,4,4′,5′-p en ta br om o-2,2′-d ih yd r oxyd ip h en -
yl Eth er (30) To solution of 29 (120 mg, 0.21 mmol) in 1,2-
dichloroethane (3 mL) was added dropwise boron tribromide
(795 mg, 3.17 mmol) under an argon atmosphere. The mixture
was stirred at room temperature for 30 h. Diethyl ether was
added dropwise, and after stirring for 15 min, the mixture was
diluted with EtOAc, washed with H2O, dried (Na2SO4), and
concentrated in vacuo. The residue was chromatographed on
a silica gel column (Hex-EtOAc, 3:2) to give 30 (105 mg, 82%):
1H NMR (CCl3D/CD3OD): δ 7.12 (s, 1H), 6.69 (d, J ) 8.8 Hz,
1H); 13C NMR (CDCl3/CD3OD): δ 152.7 (d, J ) 243 Hz), 145.87,
142.98, 142.85, 142.76, 123.39, 121.55, 115.57, 115.36 (d, J )
30 Hz), 114.11, 108.0 (d, J ) 24 Hz), 105.34 (d, J ) 28 Hz);
MS (APCI-negative) m/z: 614 [M - H]-, 535 [M - Br]-. Anal.
(C12H4Br5FO3) C; H: calcd, 0.66; found, 0.59.
2-Am in o-2′-m eth oxyd ip h en yl Eth er (23). A suspension
of 22 (850 mg, 3.47 mmol), 10% Pd/C (270 mg) in methanol
(25 mL) was stirred at room temperature for 3 h under a
hydrogen atmosphere. The reaction mixture was filtered
throughout Celite and the Celite washed with CH2Cl2. Evapo-
ration of the solvent in vacuo and purification of the residue
by chromatography on a silica gel column (Hex-EtOAc, 7:3)
afforded 2343 (700 mg, 94%): mp 60-63 °C; 1H NMR (CDCl3):
δ 7.06 (m, 1H), 6.98 (m, 1H), 6.94 (m, 1H), 6.87 (m, 2H), 6.79
(m, 2H), 6.68 (td, J ) 7.3, 1.5 Hz, 1H); 13C NMR (CDCl3): δ
150.32, 145.71, 143.91, 137.98, 124.05, 123.68, 120.86, 118.50
(2), 118.32, 116.09, 112.38, 55.77.
2′-Am in o-3,3′,4,4′,5,5′-h exa b r om o-2-h yd r oxyd ip h en yl
Eth er (24). To a solution of 23 (213 mg., 0.99 mmol) in acetic
acid (6 mL) at room temperature was added dropwise bromine
(2 mL, 39.6 mmol) with stirring. The reaction mixture was
heated at 95 °C for 15 h and then cooled to room temperature
and concentrated in vacuo. The residue was chromatographed
on a silica gel column (Hex-CH2Cl2, 3:2) to give 24 (227 mg,
34%): mp 215-216 °C;1H NMR (acetone-d6): δ 7.36 (s, 1H),
7.25 (s, 1H); 13C NMR (acetone-d6): δ 147.55, 144.44, 142.78,
139.96, 123.05, 122.98, 122.39, 121.90, 115.65, 114.21, 111.76,
109.68; MS (APCI-negative) m/z: 674 [M - H]-, 595 [M - Br]-
.
Anal. (C12H5Br6NO2) C; H: calcd, 0.75; found, 0.79; N: calcd,
2.08; found, 2.09.
2,2′,4′-Tr im eth oxyd ip h en yl Eth er (25). To a suspension
of guaiacol (400 mg, 3.2 mmol) in benzene (3 mL) was added
NaOMe (260 mg, 4.8 mmol) under an argon atmosphere. The
mixture was stirred at room temperature for 45 min. The
solvent was evaporated and CuCl (318 mg, 3.2 mmol), 1-bromo-
2,4-dimethoxybenzene (1.2 g, 5.4 mmol), and pyridine (4 mL)
were added. The mixture was heated under refluxing for 3 h
and then cooled to room temperature, diluted with EtOAc, and
washed with HCl (1 M). The organic layer was successively
washed with H2O and brine, dried (Na2SO4), and concentrated
in vacuo. The residue was chromatographed on a silica gel
column (CH2Cl2-Hex, 4:1) to give 2544 (120 mg, 14%): 1H NMR
(CDCl3): δ 6.96 (m, 2H), 6.88 (d, J ) 9 Hz, 1H), 6.80 (m, 1H),
6.68 (dd, J ) 8, 1.5 Hz, 1H), 6.56 (d, J ) 3 Hz, 1H), 6.40 (dd,
J ) 9, 3 Hz, 1H), 3.89 (s, 3H), 3.78 (s, 3H); 13C NMR (CDCl3):
δ 156.74, 151.80, 149.53, 147.32, 138.59, 122.53, 120.86,
120.53, 116.29, 112.09, 103.69, 100.37, 55.81, 55.73, 55.42.
3,3′,4,4′,5,5′-H exa b r om o-2,2′-d im et h oxyd ip h en yla m -
in e (32). To a solution of 2,2′-dimethoxyphenylamine28 (200
mg, 0.87 mmol) in CCl4 (4 mL) was added dropwise bromine
(2.23 g, 19.95 mmol) while stirring. The mixture was stirred
at room temperature for 14 h and then was concentrated in
vacuo. The residue was chromatographed on a silica gel column
(Hex-EtOAc, 3:1) to afford 32 (541 mg, 88%): mp 192-195
°C; 1H NMR (CDCl3) δ 7.04 (s, 2H), 3.62 (s, 6H); 13C NMR
(CDCl3) δ 149.70, 130.67, 117.33, 117.16, 115.32, 113.52, 55.02;
MS (ES-negative) m/z: 702 [M - H]-. Anal. (C14H9Br6NO2) C;
H: calcd, 1.29; found, 1.31; N: calcd, 1.99; found, 2.07.
2,2′,4′-Tr ih yd r oxyd ip h en yl Eth er (26). To a solution of
25 (270 mg, 1.04 mmol) in 1,2-dichloroethane (3 mL) was
added dropwise boron tribromide (1.56 g, 6.23 mmol) under
an argon atmosphere. The mixture was stirred at room
temperature for 30 h. Diethyl ether was added dropwise, and
after stirring for 15 min, the mixture was diluted with EtOAc,
successively washed with H2O and brine, dried (Na2SO4), and
concentrated in vacuo. The residue was chromatographed on
a silica gel column (CH2Cl2-EtOAc; 4:1) to give 2644 (220 mg,
97%): mp 164-166 °C. 1H NMR (acetone-d6): δ 8.16 (br s,
3H), 6.92 (m, 2H), 6.81 (d, J ) 9 Hz, 1H), 6.74 (m, 2H), 6.54
(d, J ) 2.7 Hz, 1H), 6.35 (dd, J ) 9, 2.7 Hz, 1H); 13C NMR
(acetone-d6): δ 155.54, 150.27, 148.12, 146.91, 137.22, 123.89,
122.03, 120.46, 117.34, 117.02, 107.31, 104.71.
3,3′,4,4′,5,5′-H e xa b r om o-2,2′-d ih yd r oxyd ip h e n yla m -
in e (33). Compound 33 was prepared from 32 (100 mg, 0.14
mmol) according to the method of preparation for 26. The
residue was chromatographed on a silica gel column (Hex-
1
EtOAc, 3:1) to give 33 (33 mg, 34%): mp > 300 °C; H NMR
(CD3OD) δ 7.08 (s, 2H), 5.56 (br s, 1H); 13C NMR (CD3OD) δ
151.78, 133.46, 120.83, 119.39, 117.91, 117,16; MS (FAB-
positive) m/z: 675 [M]+. Anal. (C12H5Br6NO2) C; H: calcd, 0.75;
found, 0.80; N: calcd, 2.08; found, 2.12.
B. Biologica l Meth od s. P r od u ction a n d P u r ifica tion
of Recom bin a n t Hu m a n Ald ose Red u cta se. Recombinant

5218 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
de la Fuente et al.
hALR2 was obtained and purified as previously described.45
Insect cells of Spodoptera frugiperda Sf9 (Pharmingen, 21300C)
were cultured in Grace’s medium (Gibco) at 27 °C without CO2.
Once the stationary growing phase was reached, cultures were
infected with baculovirus Autographa californica AcAr that
has been genetically modified by the insertion of the human
aldose reductase gene in its genome.46 These infected cells were
able to produce hALR2, which was recovered and purified from
the culture medium by affinity chromatography in Matrex Gel
Orange A (Amicon). After its elution with NADPH, the enzyme
was stored at -20 °C in storage buffer (50 mM sodium
phosphate, pH 7.0, 5 mM dithiotreitol, and 50% glycerol).
R ecom b in a n t H u m a n Ald ose R ed u ct a se In h ib it ion .
The hALR2 activity assays were carried out according to a
previously described method45 that is based on the quantifica-
tion of NADPH consumption which takes place when hALR2
catalyses the conversion of glyceraldehyde into glycerol. This
assay was carried out in 96-well microtiter plates at 37 °C in
100 mM sodium phosphate buffer pH 6.2, 400 mM ammonium
sulfate, 5 mU/mL of recombinant hALR2 (1 mU of activity was
defined as a change in absorbance of 0.012 units per minute),
and 0.1 mM NADPH. The final reaction volume was 200 µL
per well.
The compounds to be assayed were dissolved in dimethyl
sulfoxide, and the corresponding solution was added to the well
and preincubated for 5 min at 37 °C prior to addition of the
substrate. The reaction was initiated by addition of 10 mM
glyceraldehyde, and the decrease in optical density at 340 nm
was monitored for 6 min at 37 °C in a microtiter plate reader
(MRX-TCII, Dynex Technologies) in three intervals of 2 min
each. The IC50 for each test compound was determined as the
compound concentration that inhibited hALR2 activity by 50%.
Values are given as the mean of three experiments ( the
standard deviation.
were able to reproduce the density (0.876 g cm-3) and enthalpy
of vaporization (∆Hvap ) 8.11 kcal mol-1) of this liquid at 300
K and 1 atm using a compressibility value of 96.7 × 10-6
51
bar-1
.
The starting geometry was provided by the typical
“herringbone” packing arrangement observed in the crystal-
lographic structure of deuterated benzene.52 Once this box was
equilibrated (density ) 0.91 ( 0.01 g cm-3 and ∆Hvap ) 8.35
( 0.13 kcal mol-1), the simple operation of replacing one of
the hydrogen atoms in each benzene molecule by the appropri-
ate halogen atom yielded the preequilibrated boxes of fluo-
robenzene, chlorobenzene, and bromobenzene. The compress-
ibility values (in 10-6 bar-1) used for these solvents were,
respectively, 87.3, 75.1, and 66.8. Molecular dynamics simula-
tions were carried out at 300 K using the SANDER module in
AMBER and a dielectric constant of unity. Both the temper-
ature and the pressure were coupled to thermal and pressure
baths with relaxation times of 0.2 and 0.6 ps, respectively. In
a 20-ps heating phase, the temperature was gradually raised
under constant volume conditions, and the velocities were
reassigned at each new temperature according to a Maxwell-
Boltzmann distribution. This was followed by an equilibration
phase of 300 ps at 300 K, and by a 400-ps sampling period at
constant pressure during which system coordinates were saved
every 50 ps. All bonds involving hydrogens were constrained
to their equilibrium values by means of the SHAKE algorithm,
which allowed an integration time step of 2 fs to be used. A
nonbonded cutoff of 10 Å was employed and the lists of
nonbonded pairs were updated every 25 steps. Density values
were provided directly by the SANDER module whereas ∆Hvap
values were calculated according to the equation:
∆Hvap ) RT - Einter
where Einter is the interaction energy of the system, which
encompasses both the electrostatic (Eele) and van der Waals
(EvdW) components, divided by the number of molecules in
each box. These energy values were obtained directly from the
SANDER output but were corrected to eliminate contributions
from intramolecular nonbonded interactions beyond the ex-
plicitly calculated 1-4. To this end an analysis of each saved
snapshot was carried out with module ANAL setting the cutoff
radius to zero; because of the residue-based cutoff in AMBER,
the Eele and EvdW values thus obtained contain only the
intramolecular nonbonded interactions, which were subtracted
from the corresponding total values provided by SANDER to
yield the required intermolecular components.53
Con str u ction a n d Refin em en t of th e En zym e-In h ibi-
tor Com p lexes. An atomic model of human ALR2 in complex
with zopolrestat was built using as templates both the CR trace
of the zopolrestat-bound human enzyme and the all-atom
structure of the porcine enzyme in complex with tolrestat, both
available from the Protein Data Bank (PDB entries 1mar and
1ah3, respectively). Superposition of the CR trace of both
proteins showed that they are virtually overlapped (root-mean-
square deviation ) 0.45 Å for residues 2-314). In fact, there
are only minimal differences in dihedral angles between both
CR traces (Figure 2 of the Supporting Information) and the
location of the NADP+ atoms in both complexes is almost
identical. The porcine and human ALR2 enzymes differ in 43
out of 315 amino acids: the side chains of those that are
nonequivalent were replaced in porcine ALR2 with those from
their human counterparts using the built-in library of con-
formers within Insight II.54 For each “mutated” residue, the
ø1 angle was maintained and the rotamer producing the lowest
steric clash was chosen. A short optimization run restraining
zopolrestat, NADP+, and all CR atoms of the protein to their
initial coordinates allowed readjustment of covalent bonds and
van der Waals contacts without changing the overall confor-
mation of the protein.
In tr a cellu la r Sor bitol Accu m u la tion in Hu m a n Reti-
n a l Cells ARP E-19. Intracellular concentrations of sorbitol
in human retinal cells ARPE-19 (ATCC CRL 2302) were
measured following the recommended protocol.47 In brief, 10
× 106 retinal human cells were cultured in 2.5 mL of Minimum
Essential Medium (J RH Biosciences) supplemented with 0.5%
fetal calf serum. 50 mM glucose was added to the culture
medium to reproduce in vitro the typical hyperglycemia
conditions of diabetes mellitus. The compounds to be assayed
were dissolved in dimethyl sulfoxide, and the corresponding
solution was added to the well. After an incubation of 16 h at
37 °C with 5% CO2, the accumulated sorbitol inside the cells
was extracted by lysis with 8% perchloric acid and then
neutralized with KOH.48 Quantification of sorbitol was carried
out using a colorimetric method (D-Sorbitol/xylitol, Boehringer
Mannheim, 670 057). The IC50 for each test compound was
determined as the compound concentration that inhibited
sorbitol accumulation by 50%. Values are given as the mean
of three experiments.
C. Com p u ta tion a l Meth od s. F or ce F ield P a r a m eter s.
The second-generation all-atom AMBER molecular mechanics
force field was used for the enzyme, and consistent parameters
for the NADP+ cofactor (Supporting Information), zopolrestat,
and halogenated inhibitors were derived to describe the bonded
and nonbonded interactions. For each inhibitor, molecular
electrostatic potentials (MEPs) were calculated from the
corresponding ab initio wave functions (RHF 6-31G*//3-21G*)
using Gaussian9449 following full energy minimization. Partial
atomic charges were then obtained by fitting each MEP to a
monopole-monopole expression using the RESP methodol-
ogy.50 Atom types for aromatic carbon atoms in the inhibitors
(CA) were taken from the AMBER database. Equilibrium bond
lengths and angles involving halogen atoms were obtained
from the ab initio 6-31G(d) energy-minimized structures of
fluorobenzene, chlorobenzene, and bromobenzene (Table 3).
Nonbonded parameters for F, Cl, and Br in these molecules
were developed and tested essentially by following our previ-
ously reported procedure.51 In brief, a periodic cubic box (24
× 24 × 24 Å3) containing 75 molecules of benzene was first
constructed to ascertain that the standard AMBER parameters
Au t om a t ed Dock in g St u d ies. The Lamarckian genetic
algorithm55 implemented in AutoDock 3.056 was used to
generate docked conformations of each ligand within the
binding site by randomly changing torsion angles and overall
orientation of the molecule. Default settings were used except

Novel Human Aldose Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5219
for number of runs, population size and maximum number of
energy evaluations, which were fixed to 50, 100, and 250000,
respectively. Rapid intra- and intermolecular energy evalua-
tion of each configuration was achieved by having the recep-
tor’s atomic affinity potentials for carbon, oxygen, hydrogen,
and halogen atoms precalculated in a three-dimensional grid
with a spacing of 0.375 Å (additional maps for nitrogen and
sulfur atoms were needed for zopolrestat). AMBER nonbonded
parameters for halogens were transformed into suitable Au-
toDock 3.0 (Table 1 of the Supporting Information) using the
procedure described in the manual and a linear free energy
regression coefficient of 0.1485.
En er gy Refin em en t of th e Com p lexes. To include the
effects of solvation explicitly, a spherical “cap” of TIP3P water
molecules57 was added to the complex within 28.5 Å of the
center of mass of the bound inhibitor. In the solvation process,
any water molecule closer than 1.0 Å to any atom of the
complex was removed. A total of 1398 water molecules were
thus included and restrained from leaving this spherical
boundary by means of a harmonic radial potential with a force
constant of 0.6 kcal mol-1 Å-2. Energy refinement was ac-
complished in a sequential fashion. First, the positions of all
hydrogen atoms were optimized using 1200 steps of steepest
descent followed by conjugate gradient energy minimization
until the root-mean-square value of the potential energy
gradient was below 0.01 kcal mol-1 Å-1. Next, the water
molecules, NADP+, bound inhibitor and those protein residues
within a 14 Å sphere centered on the inhibitor were allowed
to move while the remaining protein residues were fixed at
their starting locations although they were included in the
determination of the forces. The resulting system was then
optimized by using 500 steps of steepest descent energy
minimization followed by conjugate gradient until the root-
mean-square value of the potential energy gradient was below
0.01 kcal mol-1 Å-1. The temperature was gradually raised
from 100 to 298 K over 30 ps. Following 20 ps of equilibration,
Å radius. The potentials at the grid points delimiting the box
were calculated analytically by treating each charged atom as
a Debye-Hu¨ckel sphere.
For energy decomposition, the binding process was divided
in two stages, the first one consisting in desolvating the
apposing surfaces of both the ligand and the receptor, and a
second one consisting in letting the charges of the two
molecules interact in the presence of the surrounding solvent.
The procedure has been described in detail previously.31,59,60
Briefly, the cost of desolvating both the enzyme and the ligand
was calculated by considering the effects on the respective
electrostatic free energies of replacing the high dielectric
medium of the solvent with the low dielectric medium of the
other molecule in those regions that are occupied by the
binding partner in the complex. To calculate the residue-based
ligand-receptor interaction energies, the solvent-corrected
potential generated by the charges on the ligand was computed
at the positions of each of the uncharged atoms of the receptor.
F r ee En er gy P er tu r ba tion Ca lcu la tion s. The molecular
dynamics-thermodynamic integration method, as imple-
mented in AMBER 6.0, was used to investigate the differences
in binding free energy between compounds 1 and 2 and to
verify the reliability of the proposed binding mode. Nonbonded
intramolecular contributions were included in the evaluation
of the free energy differences, as recommended.61 Compound
1 and 2 were each immersed in a rectangular box containing
∼700 water molecules. Following heating and equilibration for
950 ps, as described previously, the Br f H or H f Br
“mutations” took place over a total of 41 “windows”, each
consisting of 5 ps of equilibration and 5 ps of data collection
for averaging. The same mutations were performed in the cap-
solvated hALR2:NADP+:1 and hALR2:NADP+:2 complexes
under the conditions described above. The relative free energy
differences were calculated as ∆∆Gbinding ) ∆Gbinding(2) -
∆Gbinding(1) ) ∆G4 - ∆G3 (Scheme 7).
Ack n ow led gm en t. We thank the O.N.C.E. Founda-
tion (Spain) for their interest and financial support in
the Aldose Reductase Inhibitors Project. We are very
much obliged to Dr. C. Nishimura (National Children’s
Medical Research Center, Tokyo, J apan), Dr. Ulf Ryde
(Department of Theoretical Chemistry, Lund Univer-
sity, Sweden), and Dr. B. Bowden (J ames Cook Univer-
sity, Townsville, Australia) for kindly providing us,
respectively, with the baculovirus, AMBER parameters
for NADP+, and marine natural compounds 1-3. A
predoctoral fellowship to S. Manzanaro from the Span-
ish Ministerio de Ciencia y Tecnolog´ıa (MIT-F2) is
gratefully acknowledged. The authors thank Irene Pala-
cios for technical assistance and Marta Mart´ınez, Ada
Can˜edo, and Susana Gonza´lez for the spectroscopic
studies.
a
950 ps trajectory at 298 K was then simulated, and
coordinates were saved every ps. All bonds involving hydrogens
were constrained to their equilibrium values using SHAKE, a
time step of 2 fs was employed, and the nonbonded pairs list
was updated every 25 steps. The nonbonded cutoff was 13.0
Å, and a dielectric constant of unity was employed. The ANAL
module of AMBER 6.0 was used for energy decomposition of
the refined complexes.
Estim a tion of th e Electr osta tic Con tr ibu tion s to th e
F r ee E n er gies of Bin d in g: Con t in u u m E lect r ost a t ics
Ca lcu la tion s. The overall electrostatic free energy change
upon binding (∆Gele) was calculated from the total electrostatic
energy of the system by running 3 consecutive calculations
on an identically defined grid: one for all the atoms in the
complex, GEelIe, one for the enzyme atoms alone, GEele, and a
third one for the inhibitor atoms alone, GIele. In these calcula-
tions the cofactor was considered to be part of the protein.
Since the grid definition is the same in the three calculations,
the grid energy artifact cancels out when the electrostatic
contribution to the binding free energy is expressed as the
difference in energy between the bound and the unbound
molecules:
Su p p or tin g In for m a tion Ava ila ble: A table containing
AutoDock parameters for the inhibitors, two figures showing
van der Waals and electrostatic contributions to the binding
energy of zopolrestat (Figure 1) and pairwise differences in
CR dihedral angles along the peptide backbone of the different
complexes studied (Figure 2), and the AMBER PREP file used
for NADP+. This material is available free of charge via the
Internet at http://pubs.acs.org.
∆Gele ) GEI - (GEele + GIele
)
ele
The Poisson-Boltzmann equation was solved using a finite
difference method,58 as implemented in the DelPhi module of
Insight II.54 The atomic coordinates employed were those of
the energy-refined average structure. The interior of the
enzyme, the cofactor, and the inhibitor was considered as a
low dielectric medium (ꢀ ) 4) whereas the surrounding solvent
was treated as a high dielectric medium (ꢀ ) 80) with ionic
strength of 0.145 M. The same cubic grid, which was centered
on the complex and had a resolution of 0.5 Å, was used in the
three cases. A minimum separation of 10 Å was left between
any solute atom in the complex and the borders of the box.
The solute boundaries were defined by solvent-accessible
surfaces, which were calculated with a spherical probe of 1.4
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