Benzofuroxane derivatives as multi-effective agents for the treatment of cardiovascular diabetic complications. Synthesis, functional evaluation, and molecular modeling studies

Article abstract of DOI:10.1021/jm301124s

Diabetes mellitus is the major risk factor for cardiovascular disorders. Aldose reductase, the rate-limiting enzyme of the polyol pathway, plays a key role in the pathogenesis of diabetic complications. Accordingly, inhibition of this enzyme is emerging as a major therapeutic strategy for the treatment of hyperglycemia-induced cardiovascular pathologies. In this study, we describe a series of 5(6)-substituted benzofuroxane derivatives, 5a-k,m, synthesized as aldose reductase inhibitors. Besides inhibiting efficiently the target enzyme, 5a-k,m showed additional NO donor and antioxidant properties, thus emerging as novel multi-effective compounds. The benzyloxy derivative 5a, the most promising of the whole series, showed a well-balanced, multifunctional profile consisting of submicromolar ALR2 inhibitory efficacy (IC₅₀ = 0.99 ± 0.02 μM), significant and spontaneous NO generation properties, and excellent hydroxyl radical scavenging activity. Computational studies of the novel compounds clarified the aldose reductase inhibitory profile observed, thus rationalizing structure-activity relationships of the whole series.

Full text of DOI:10.1021/jm301124s

Article
pubs.acs.org/jmc
Benzofuroxane Derivatives as Multi-Effective Agents for the
Treatment of Cardiovascular Diabetic Complications. Synthesis,
Functional Evaluation, and Molecular Modeling Studies
Stefania Sartini,† Sandro Cosconati,§ Luciana Marinelli,*,‡ Elisabetta Barresi,† Salvatore Di Maro,‡
Francesca Simorini,† Sabrina Taliani,† Silvia Salerno,† Anna Maria Marini,† Federico Da Settimo,†
Ettore Novellino,‡ and Concettina La Motta*,†
†Dipartimento di Farmacia, Universita
̀
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
‡Dipartimento di Chimica Farmaceutica e Tossicologica, Universita
Italy
̀
di Napoli “Federico II”, Via D. Montesano, 49, 80131 Napoli,
§Dipartimento Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Universita
Caserta, Italy
̀
di Napoli, Via Vivaldi 43, 81100
S
* Supporting Information
ABSTRACT: Diabetes mellitus is the major risk factor for
cardiovascular disorders. Aldose reductase, the rate-limiting
enzyme of the polyol pathway, plays a key role in the
pathogenesis of diabetic complications. Accordingly, inhibition
of this enzyme is emerging as a major therapeutic strategy for
the treatment of hyperglycemia-induced cardiovascular path-
ologies. In this study, we describe a series of 5(6)-substituted
benzofuroxane derivatives, 5a−k,m, synthesized as aldose
reductase inhibitors. Besides inhibiting efficiently the target
enzyme, 5a−k,m showed additional NO donor and anti-
oxidant properties, thus emerging as novel multi-effective
compounds. The benzyloxy derivative 5a, the most promising of the whole series, showed a well-balanced, multifunctional profile
consisting of submicromolar ALR2 inhibitory efficacy (IC50 = 0.99 0.02 μM), significant and spontaneous NO generation
properties, and excellent hydroxyl radical scavenging activity. Computational studies of the novel compounds clarified the aldose
reductase inhibitory profile observed, thus rationalizing structure−activity relationships of the whole series.
the major risk factor for cardiovascular disorders, and people
with chronic hyperglycemia are 2−4 times more likely to have a
heart attack, coronary artery diseases, peripheral arterial
diseases, cardiomyopathy, angina, and endothelial disfunction
than people without diabetes.5
INTRODUCTION
■
Diabetes mellitus is a complex and chronic metabolic disorder
now recognized as a public health problem, as it affects a
significant portion of the population worldwide and is
expanding to pandemic dimensions. Actually, according to
epidemiological data, 366 million people were diagnosed with
diabetes in 2011, but this number is expected to rise sharply to
552 million people within the next 20 years.1−3 The economic
cost attributable to the management of this disease markedly
influences countries’ health budgets, being the highest of any
disease category. In 2011, the United States paid $465 billion,
11% of total healthcare expenditures for adults aged 20−79
years, to hold up not only direct costs of diabetes care but also
indirect costs of both short-term or permanent disability and of
premature retirement or death.4 Indeed, although diabetes can
be successfully controlled by the administration of potent
hypoglycemics and/or insulin, it still remains the cause of
significant morbidity and mortality, because of the progressive
development of microvascular and macrovascular alterations. In
particular, according to the Scientific Advisory and Coordinat-
ing Committee of the American Heart Association, diabetes is
Many hypotheses have been proposed to explain the
pathogenic mechanism leading to diabetic complications.
These include nonenzymatic glycation of proteins, alteration
of myoinositol levels, impairment of antioxidant defense,
mitochondrial respiratory chain disruption, and activation of
both the hexosamine pathway and protein kinase C iso-
forms.6−9 However, the prominent theory suggests that they
are causally linked to an increased flux of glucose through the
polyol pathway, ruled by aldose reductase (alditol:NADP+
oxidoreductase, EC 1.1.1.21, ALR2), a monomeric enzyme
belonging to the aldo-keto reductase (AKR) superfamily.10
ALR2 drains away the excess of glucose, catalyzing its reduction
to sorbitol, but the resulting polyol, slowly oxidized to fructose
Received: July 31, 2012
© XXXX American Chemical Society
A
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by the NAD+-dependent sorbitol dehydrogenase, tends to
accumulate inside the cells, thus increasing cellular osmolarity.
In addition to the osmotic imbalance, ALR2 activation causes a
marked impairment of the NADPH/NADP+ free cytosolic
coenzyme ratio, thus inducing a state of pseudohypoxia that
promotes the onset of hyperglycemic oxidative stress through
the accumulation of reactive oxygen species (ROS). Moreover,
as the increased flux of glucose through the polyol pathway is
closely related to myoinositol depletion, protein kinase C
(PKC) activation, and nonenzymatic protein glycation, ALR2
can clearly be identified as the key and critical checkpoint for
the main pathological changes associated with long-term
diabetic complications. Hence, ALR2 represents an excellent
drug target, as its inhibition can significantly minimize the onset
of osmotic and oxidative stress, as well as the activation of other
downstream biochemical pathways, making it possible to
prevent, or at least delay, the progression and severity of
diabetic complications.11,12
tions. Accordingly, a novel and still unexplored scenario is being
elucidated for the use of ARIs, which could be profitably
exploited for the effective treatment of people affected by
hyperglycemia-induced cardiovascular diseases.17−22
Our research group has been involved in the development of
novel ARIs for many years, to identify a drug candidate for the
treatment of long-term diabetic complications.23−31 Through a
virtual screening (VS) campaign, performed by integrating a
receptor-based approach with a ligand-based one, we recently
disclosed a number of viable chemical entities that deserve
further examination in the search for potent ALR2 inhibitors.25
Among the discovered hits, the benzo[c][1,2,5]oxadiazole
derivative KM07100 [1 (Chart 1); IC50 = 35.6
1.8 μM]
emerged as the most intriguing candidate, being characterized
by an unusual core, never exploited before in the ARI field. This
scaffold was predicted to be lodged in the enzyme anion
binding site H-bonding to the Y48, H110, and W111 residues,
which have been demonstrated to be contacted by the
carboxylic and hydantoin groups of the most active ARIs
described so far. However, different from the previously
described ARIs, the benzo[c][1,2,5]oxadiazole scaffold should
not pose, in principle, the risk of failure shown by most ARIs
described in the literature that, although promising during in
vitro studies or in trials with animal models, often fail to
proceed any further, because of either pharmacokinetic
restrictions, typical of inhibitors bearing a carboxylic function,
or unwanted side effects, arising with the use of hydantoinic-like
inhibitors. Prompted by this speculation, we embarked in a hit
to lead optimization campaign and focused our attention on the
benzo[c][1,2,5]oxadiazole synthetic precursor, benzofuroxane,
exploited previously by different authors for its pharmacological
properties.32−34
A huge amount of experimental data, gathered in the decades
since the discovery of ALR2, clearly proved the effectiveness of
ALR2 inhibitors (ARIs) in treating hyperglycemia-induced
pathologies like neuropathy, nephropathy, retinopathy, and
cataract. The main examples of active compounds are
represented by either spiro derivatives, like fidarestat13 and
ranirestat14 (Chart 1), presently undergoing clinical trials in
Chart 1. Aldose Reductase Inhibitors
Actually, the well-known NO donor, vasorelaxant, and
platelet anti-aggregating activities of the benzofuroxane core
made this scaffold more appealing than the parent benzox-
adiazole. As experimental data clearly prove, most of the
diabetic complications affecting the cardiovascular system result
from an impaired endothelium-derived NO production and
action.35,36 Accordingly, a NO donor compound should
certainly represent a privileged structure in the search for a
novel drug candidate for the treatment of diabetes-related
cardiovascular diseases. In addition, the hypoglycemic/anti-
hyperglycemic effect of NO might help to control blood
glucose levels, while its anti-aggregating properties could
profitably counterbalance platelet aggregation observed under
hyperglycemic conditions. Moreover, as demonstrated by
Srivastava and co-workers, endogenous NO is able to regulate
ALR2 activity, as it may inactivate enzyme catalytic function by
inducing a reversible glutathiolation of the highly reactive
residue C298, located at the ALR2 active site. Therefore,
increasing the intracellular levels of NO may certainly
contribute to keeping the enzyme in a partially inhibited
state, thus controlling or even preventing pathological changes
triggered by ALR2.37−40
The choice of the benzofuroxane ring was also suggested by
the necessity of enhancing the interactions of the new ARIs
with the enzyme anion binding site. In fact, preliminary
profiling of the electrostatic potential through ab initio
calculations demonstrated that the benzofuroxane scaffold
should be more electron-rich than the parent benzo[c][1,2,5]-
oxadiazole one, thus being more prone to establishing tight
interactions with the aforementioned ALR2 region (Figure 1a).
Moreover, a preliminary docking study of N-oxide derivative 5a
both the United States and Asia, or acetic acid compounds,
such as epalrestat (Chart 1), which is currently marketed for
the treatment of diabetic neuropathy but is also able to prevent
the progression of both retinopathy and nephropathy.15,16
More recently, a growing body of compelling evidence is
demonstrating that ALR2 also plays a pivotal role in the
development of diabetic cardiomyopathy, atherosclerosis,
hypertension, restenosis, angiopathy, and endothelial dysfunc-
B
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Figure 1. ALR2 and ALR1 inhibition data and predicted binding free energies of benzofuroxane derivatives 5a−k,m.
Scheme 1. Synthesis of Benzofuroxane Derivatives 5a−k,m
in the ALR2 active site revealed that the benzofuroxane scaffold
should be able to engage in well-oriented H-bond interactions
with the side chains of W111, H110, and Y48 in the protein
anion binding pocket, while its benzyloxy pendant chain is
lodged in the specificity pocket forming a π−π stacking
interaction with W111 and hydrophobic contacts with F122,
L300, and Y309 (Figures 1b and Figures S1 and S2 of the
Supporting Information). Analogous results were also achieved
when docking was performed explicitly considering water
molecules during docking calculations employing the newly
described method of Forli and Olson (Figure S3 of the
Supporting Information).41 These encouraging theoretical data
led us to move, once and for all, to the benzofuroxane core as a
viable candidate in the search for noncarboxylic, non-
hydantoinic, ALR2 inhibitors.
aromatic and lipophilic groups. These latter groups were
chosen among the ones already borne by well-known ARIs
from the literature, to plainly comply with pharmacophore
requirements of the ALR2 binding site.42 All the synthesized
compounds were extensively evaluated for their functional
effectiveness, testing their ALR2 inhibitory activity and
selectivity, as well as their ability to generate NO and efficacy
in scavenging reactive oxygen species (ROS). Moreover,
simulations of docking of the test compounds into the
human ALR2 binding site were carried out, to rationalize
structure−activity relationships (SARs) observed and to guide,
prospectively, the development of more effective analogues.
CHEMISTRY
■
The target inhibitors, 5a−k,m were obtained as outlined in
Scheme 1, according to synthetic methods previously reported
in the literature. Chemoselective alkylation of the commercially
available 4-amino-3-nitrophenol, 2, performed with the suitable
In this work, we present the synthesis of the novel
benzofuroxane derivatives, 5a−k,m (Chart 1), whose benzo-
fused ring was suitably substituted through the insertion of
C
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alkyl halide in the presence of anhydrous potassium carbonate,
gave the 4-substituted 2-nitrobenzenamine derivatives 3a−l.43
Reaction of 3a,b,d−k with sodium nitrite and sodium azide led
to the corresponding azide intermediates, 4a,b,d−k, respec-
tively, which afforded target inhibitors, 5a,b,d−k, respectively,
through reaction with refluxing acetic acid.44 Compound 3c,
bearing a 4-methoxybenzyloxy substituent, gave inhibitor 5c
through the Green−Rowe oxidative cyclization, performed with
sodium hypochlorite in a basic alcoholic solution.45 Derivative
3l, carrying a 4-cyanomethoxy group, was treated with 2-
aminobenzenethiol, and the resulting 4-(benzo[d]thiazol-2-
ylmethoxy)-2-nitrobenzenamine, 3m, provided the key inter-
mediate 4m through reaction with sodium nitrite and sodium
azide. Cyclization of 4m, performed with refluxing acetic acid,
led to the desired 5m.
of the ring, left essentially unaltered the potency of the
unsubstituted compound, 5a. Similar results were also obtained
in the presence of the bulkier trifluoromethyl group (5g; IC50
=
1.94 0.61 μM) or the concurrent presence of o-fluoro and p-
bromo atoms (5h; IC50 = 1.41 0.05 μM), which turned out
almost equipotent with parent 5a. Finally, the insertion of a
nitro group at the meta position of the phenyl ring, as in 5i
(IC50 = 0.46 0.01 μM), produced a 2-fold enhancement in
potency with respect to that of parent compound 5a,
confirming once more the fruitful contribution of this key
substituent to the interaction with the amino acid residues
surrounding the ALR2 active site.26,46 To explore the effects of
increasing the distance between the pendant benzyl group and
the benzofuroxane core, a methylene spacer was inserted
between these two key fragments, to obtain derivative 5j. This
distance turned out to be a crucial element in determining the
best spatial relationship between the pharmacophoric groups of
the compounds. Actually, the lengthened derivative 5j, with an
IC50 value of 5.90 0.25 μM, displayed a 6-fold reduction in
inhibitory efficacy with respect to that of the shorter 5a. On the
other hand, widening the secondary lipophilic area of the
compounds turned out to be a successful strategy. Indeed,
although the replacement of the pendant benzyl group with the
naphthalen-2-yl core, as in 5k (IC50 = 1.43 0.04 μM), did not
significantly affect the functional activity, the insertion of the
benzothiazol-2-yl group resulted in a remarkable improvement
RESULTS AND DISCUSSION
■
Functional Evaluation. We started our functional
evaluation by testing the synthesized inhibitors, 5a−k,m, for
their efficacy against ALR2. As reported in Table 1, in which
Table 1. ALR2 and ALR1 Inhibition Data and Predicted
Binding Free Energies of Benzofuroxane Derivatives 5a−k,m
in inhibitory efficacy as the resulting compound, 5m (IC50
0.42 0.01 μM), was shown to be the most active of the whole
series.
=
ALR2 IC
ALR1 IC50 (μM)
ΔGAD4
a50
N
R
(μM)
or % inhibition (kcal/mol)
b
1
35.6 1.80
0.99 0.02
0.83 0.03
4.52 0.19
0.82 0.02
0.92 0.04
7.36 0.37
1.94 0.61
1.41 0.05
0.46 0.01
5.90 0.25
1.43 0.04
0.42 0.01
0.17 0.01
20 0.9
b
All the synthesized compounds, 5a−k,m, were assayed for
their ability to inhibit aldehyde reductase (EC 1.1.1.2, ALR1), a
cytosolic enzyme closely related to ALR2. Besides sharing a
high degree of structural homology, with 65% identical amino
acid sequences, both the enzymes play a common detoxification
role, being key components of the complex antioxidant cell
defense system that ensures the efficient removal of toxic
aldehydes arising from pathological conditions connected with
oxidative stress, like diabetes. Accordingly, a clinically effective
and safe ARI should be able to inhibit ALR2 selectively, leaving
unaltered the detoxification role of ALR1. Tested at 100 μM,
none of the synthesized benzofuroxanes showed appreciable
ALR1 inhibitory activity (Table 1), thus proving to be
completely selective inhibitors of ALR2.
The novel inhibitors were then tested for their ancillary NO
donor properties. The amount of NO released was determined
in a rat hepatic homogenate exploiting the Griess reaction, thus
measuring nitrites obtained as oxidative products of NO. With
the limited exception of 5d, 5h, and 5m, which turned out to be
ineffective, and 5j, showing a modest efficacy, the remaining
synthesized compounds all displayed relevant NO donor
properties. As shown in Figure 2, the substitution pattern of
the benzofuroxane core did not affect significantly the
effectiveness of the test compounds, as the percentage of NO
released by 5a−c, 5e−g, and 5i−k was almost the same.
Actually, after incubation at 37 °C for 1 h, the extent of release
covered a narrow range, going from 2 to 4%. Interestingly, all
the active compounds released NO slowly and spontaneously.
Indeed, differently from previously reported benzofuroxane
derivatives, they did not require the assistance of a cysteine
cofactor, generally exploited to induce the earliest bioactivation
of this kind of compound, necessary for NO release.47
Moreover, the contained amount of NO released by the test
compounds, although smaller than the amount obtained with
5a
5b
5c
5d
5e
5f
C6H5
26 1.2
24 0.8
−7.10
−7.47
−7.62
−7.14
−7.50
−6.94
−7.09
−7.85
−7.33
−7.25
−8.20
−7.96
b
C6H4-4-CH3
C6H4-4-OCH3
C6H4-4-F
c
na
c
na
b
b
C6H4-4-Cl
19 0.9
19 0.7
C6H4-4-Br
b
5g
5h
5i
C6H4-4-CF3
C6H4-2-F-4-Br
C6H4-3-NO2
CH2-C6H5
14
c
na
b
5
0.2
b
b
b
5j
11 0.4
35 1.6
38 1.8
5k
5m
naphthalen-2-yl
benzothiazol-2-yl
a
epalrestat
0.94 0.04
a
IC50 values (means
standard deviation) represent the concen-
b
tration required to produce 50% enzyme inhibition. Percent of ALR1
c
inhibition determined at 100 μM. Not active; no inhibition was
observed at test compound concentrations of up to 100 μM.
biological activities expressed as IC50 values are listed, all the
compounds proved to inhibit the target enzyme, exhibiting
potency levels in the submicromolar to micromolar range. The
benzyloxy-substituted compound 5a (IC50 = 0.99 0.02 μM)
displayed a remarkable inhibitory activity, showing a 36-fold
enhancement in potency with respect to that of VS hit 1 (IC50
= 35.6
1.8 μM). Moving from 5a, we then added either
electron-donating or electron-withdrawing groups to the
pendant ring, to explore their contribution to a fruitful
interaction with the binding site of the enzyme. While the
presence of a methoxy substituent, as in 5c (IC50 = 4.52 0.19
μM), or a bromo atom, as in 5f (IC50 = 7.36
0.37 μM),
slightly decreased the inhibitory efficacy of the resulting
compounds, the insertion of a methyl group (5b; IC50 = 0.83
0.03 μM), a fluoro atom (5d; IC50 = 0.82 0.02 μM), or a
chloro atom (5e; IC50 = 0.92 0.04 μM), in the same position
D
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group, was significantly more effective than the reference α-
tocopherol, inhibiting the production of TBARS by almost 50%
when compared to the control. The addition of electron-
donating substituents at the para position of the pendant
phenyl ring, as for 5b,c, slightly reduced the antioxidant
efficacy, although it led to compounds that were still able to
reduce the level of lipoperoxidation damage. Similar results
were obtained through the insertion of electron-withdrawing
groups at the same position of the ring, like in 5e−i, as well as
through the replacement of the phenyl group with the wider
naphthalene, 5k, and benzisothiazole, 5m. On the other hand,
the presence of a 4-fluorobenzyloxy substituent, as in 5d, or a
phenethoxy group, like in 5j, gave rise to excellent scavenger
properties, and the resulting compounds proved to significantly
protect the rat brain homogenate from lipoperoxidation
damage.
Figure 2. NO generation properties of benzofuroxane derivatives 5a−
k,m. Values are means
the standard deviation. Each value is the
mean of at last two different determinations, performed in triplicate.
Interestingly, among the several promising compounds
described here, benzyloxy derivative 5a emerged for the
excellent correlation between its high ALR2 inhibitory activity
and significant NO donor and ROS scavenger properties, thus
plainly proving the soundness of our choice to exploit suitably
substituted benzofuroxanes as novel structures for develop-
ment.
the inorganic reference compound, sodium nitroprusside
(SNP), represents a distinguishing quality. Actually, it may
profitably control, or even prevent, hyperglycemia-induced
vascular complications without posing the risks of toxicity and
tolerance, often arising from NO donor compounds commonly
available on the market.33,48
Molecular Modeling. To rationalize the achieved inhib-
itory activities of the newly identified ARIs, we performed
docking experiments for compounds 5a−k,m in the enzyme X-
ray structure. These simulations were attained employing
AutoDock4.2 (AD4) that, in our previous paper, successfully
suggested 1 as an ALR2 inhibitor.25 In the same study, the AD4
methodology was included in the so-called “in situ cross-
docking” approach, to simultaneously address multiple ALR2
conformations.49 Furthermore, given the well-known tautomer-
ism of the benzofuroxane core, both tautomers of each
compound, 5a−k,m, were docked.
Finally, bearing in mind the crucial role played by oxidative
stress in the pathogenesis of diabetic complications, we also
investigated the newly synthesized compounds, 5a−k,m, for
their antioxidant properties. In particular, we examined their
effects on hydroxyl radical-dependent lipoperoxidation induced
in rat brain homogenate by the oxidant system Fe(III)/ascorbic
acid, which initiates the Fenton reaction. For a proper
comparison, the well-known antioxidant α-tocopherol was
used as the reference standard. As reported in Figure 3, all the
test compounds exhibited significant antioxidant properties,
proving that the compounds reduce or even inhibit the
production of thiobarbituric acid reactive substances (TBARS),
an index of lipid peroxidation, when assayed at a final
concentration of 100 μM. Compound 5a, bearing a benzyloxy
For all these calculations, AD4 predicted a general preference
of the enzyme in recognizing only one of the two tautomers,
namely the one bearing the aryloxy substituent at position 5 of
the heterocyclic core. Moreover, as seen for parent 1, the newly
discovered ARIs were predicted to establish more stable
complexes with the ALR2 conformation induced by the
IDD594 ARI (Protein Data Bank entry 1US0).50 In addition,
for all these AD4 simulations, the lowest-energy binding
conformation also represented the most populated cluster of
binding poses, demonstrating good convergence behavior.
As seen for 5a, in all the predicted lowest-energy ligand−
ALR2 complexes, the benzofuroxane ring is placed in the ALR2
anion site while the pendant planar, lipophilic, and aromatic
groups are lodged in the enzyme specificity pocket. In this cleft,
the ligands are able to establish hydrophobic contacts with
F122, L300, and Y309 and a π−π interaction with W111. In
particular, among the benzyloxy derivatives, 5i displayed the
highest inhibitory potency against ALR2 because of the
presence of the m-nitro substituent, which is able to enhance
the π-stacking interactions with W111 and establish a H-bond
interaction with T113 (Figure 4a). According to docking
calculations, substitution of the benzyloxy moiety with the
phenethoxy one, as in 5j, negatively influences ALR2
recognition, and in the lowest-energy binding AD4 pose, the
benzofuroxane moiety is placed in the specificity pocket while
the terminal phenyl ring is lodged in the anion site (data not
shown). This might explain why 5j is less active than 5a. On the
other hand, substitution of the terminal phenyl ring with
different aryl substituents generally confers better ALR2
Figure 3. Effects of benzofuroxane derivatives, 5a−k,m, and α-
tocopherol on the production of thiobarbituric reactive substances
(TBARS) in rat brain homogenate treated with the generating reactive
oxygen species Fe(III)-ascorbic acid, expressed as nanomoles of
malondialdehyde produced per 10 mg of wet weight rat brain. The
basal value (rat brain homogenate) was 2.3 0.39 nmol of TBARS/10
mg of wet weight rat brain (n = 18). Values are means the standard
deviation. Each value is the mean of at least two different
determinations, performed in triplicate. Absolute values were
calculated by performing for each experiment a reference curve
using 1,1,3,3-tetramethoxypropane. *p < 0.05; **p < 0.01; ***p <
0.001 [significant difference vs control group (rat brain homogenate
with FeCl3/ascorbic acid)]. C for control and T for α-tocopherol.
E
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multifactor approach. Further studies in animal models will
prove its robustness as a prototypical drug candidate that can
be exploited in preventing the onset and progression of
hyperglycemia-induced cardiovascular diseases.
EXPERIMENTAL SECTION
■
̈
Chemistry. Melting points were determined using a Reichert
Kofler hot-stage apparatus and are uncorrected. Routine nuclear
magnetic resonance spectra were recorded in DMSO-d6 solutions on a
Varian Gemini 200 spectrometer operating at 200 MHz. Mass spectra
were obtained on a Hewlett-Packard 5988 A spectrometer using a
direct injection probe and an electron beam energy of 70 eV.
Evaporation was performed in vacuo (rotary evaporator). Analytical
TLC was conducted on Merck 0.2 mm precoated silica gel aluminum
sheets (60 F-254). The purity of the target inhibitors, 5a−k,m, was
determined by HPLC analysis, using a Merck Hitachi D-7000 liquid
chromatograph (UV detection at 242 nm) and a Discovery C18
column (250 mm × 4.6 mm, 5 μm, Supelco), with a gradient of 40%
water and 60% methanol and a flow rate of 1.4 mL/min. All the
compounds showed percent purity values of ≥95%. 4-Amino-3-
nitrophenol, 2-aminothiophenol, 2-(bromoethyl)benzene, 2-
(bromomethyl)naphthalene, and the suitably substituted benzyl halide,
used to obtain the target inhibitors, were from Alfa Aesar, Aldrich, and
Fluka.
General Procedure for the Synthesis of 4-(4-Substituted
benzyloxy)-2-nitrobenzenamines, 3a−l. A suspension of 4-amino-
3-nitrophenol 2 (154.1 mg, 1.00 mmol) and the appropriate alkyl
halide (1.10 mmol) in DMF was allowed to react at 90 °C in the
presence of potassium carbonate (152.0 mg, 1.10 mmol) until the
starting materials disappeared. Once the reaction was complete (TLC
analysis, 7/3 petroleum ether/AcOEt, 60−80 °C), the mixture was
concentrated to dryness and crushed ice was added. The resulting solid
was collected and purified by recrystallization from the appropriate
solvent, to obtain the title compound as red needles (Tables 1 and 2 of
the Supporting Information).
Synthesis of 4-[(Benzo[d]thiazol-2-yl)methoxy]-2-nitroben-
zenamine, 3m. A solution of 2-(4-amino-3-nitrophenoxy)acetonitrile
(193.2 mg, 1.00 mmol), 3l, and o-aminothiophenol (0.12 mL, 1.00
mmol) in 5 mL of anhydrous ethanol was refluxed while being strirred
until the starting materials disappeared (2 h, TLC analysis, 7/3
petroleum ether/AcOEt, 60−80 °C). After the mixture had cooled, the
resulting solid was collected by filtration and purified by recrystalliza-
tion from ethanol (Tables 1 and 2 of the Supporting Information).
General Procedure for the Synthesis of 1-Azido-4-(4-
substituted-benzyloxy)-2-nitrobenzenes and 2-[(4-Azido-3-
nitrophenoxy)methyl]benzo[d]thiazole, 4a,b,d−k,m. A solution
of sodium nitrite (172.5 mg, 2.50 mmol) in 2.0 mL of water was added
dropwise to an ice-cold solution of the appropriate amine, 3a,b,d−k,m
(1.00 mmol), in concentrated hydrochloric acid. The resulting solution
was stirred at 0 °C for 15 min, and then sodium azide (130.0 mg, 2.00
mmol) in 1.0 mL of water was added and the reaction mixture allowed
to react overnight at room temperature. The target compound
separated as a yellow solid, which was collected by filtration and
purified by recrystallization (Tables 3 and 4 of the Supporting
Information).
General Procedure for the Synthesis of 5(6)-Substituted
Benzofuroxane Derivatives, 5a,b,d−k,m. A solution of the
appropriate azide, 4a,b,d−k,m (1.00 mmol), in 0.5 mL of acetic
acid was heated under reflux until the starting material disappeared
(TLC analysis, 7/3 toluene/CH2Cl2). The crude was poured into
crushed ice, and the solid obtained, collected by filtration, was purified
by recrystallization from the suitable solvent to give the target
comounds, 5a,b,d−k,m, as a mixture of tautomers (Tables 5 and 6 of
the Supporting Information).
Figure 4. Docked conformations of 5i (a) and 5m (b) in the ALR2
structure. Hydrogens have been omitted for the sake of clarity. Ligand
carbon atoms are colored yellow, and key binding site residues are
displayed as blue sticks. Hydrogen bonds are represented by yellow
dashed lines.
inhibitory potencies with the benzothiazol-2-yl ring derivative,
5m, being the most active ARI of this series. In this case, the
higher potency should be ascribed to the enhanced stacking
interactions with W111 and to the presence of additional
interactions in the specificity pocket, namely a H-bond with the
side chain of Y309 and the nitrogen atom of the benzothiazol-
2-yl ring (Figure 4b).
CONCLUDING REMARKS
■
Because of their multifactorial etiology, long-term diabetic
complications, including the ones affecting the cardiovascular
system, should be optimally treated with compounds capable of
affecting simultaneously different biochemical pathways. In this
work, we present a novel class of multi-effective compounds
obtained by optimizing, in a suitably substituted benzofuroxane
core, ALR2 inihibitory activity and selectivity with NO donor
and antioxidant properties. Derivative 5a, 4(5)-benzyloxyfur-
oxane, emerged as a new and viable lead, proving to merge
submicromolar ALR2 inhibitory efficacy with both spontaneous
NO releasing activity and significant hydroxyl radical scavenger
ability. It clearly represents an original compound that, first in
its field and different from the current clinical candidates, is
intended to target diabetic complications taking advantage of a
5(6)-(4-Methoxybenzyloxy)benzofuroxane, 5c. A solution of
sodium hypochlorite (6−14%) was added dropwise to an ice-cold
suspension of 4-(4-methoxybenzyloxy)-2-nitrobenzenamine, 3c (274.1
mg, 1.00 mmol), and KOH (67.34 mg, 1.20 mmol) in anhydrous
EtOH. Once the addition was complete, a yellow solid separated,
F
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which was collected by filtration, washed with water, and recrystallized
from the suitable solvent, to give target 5c as a mixture of tautomers
(Tables 5 and 6 of the Supporting Information).
concentration of NO with a Perkin-Elmer Lambda instrument. The
detected nitrites were evaluated as the oxidative products of nitric
oxide by Griess reaction. Results are expressed as the percentage of
NO2− (moles per mole) determined with respect to compound added
(Figure 2).
Biological Materials and Methods. Assays were realized by
exploiting adult Sprague-Dawley albino rats (body weights of 250−
300), supplied by Harlan Nossan. Aldose reductase (ALR2) and
aldehyde reductase (ALR1) were isolated and purified from rat lens
and kidney, respectively, following a previously described protocol.26
Pyridine coenzyme, D,L-glyceraldehyde, and sodium D-glucuronate
were from Sigma-Aldrich. Epalrestat was obtained from Haorui
Pharma-Chem Inc. All other chemicals were of reagent grade.
Enzymatic Assays. The activity of the two test enzymes was
determined spectrophotometrically by monitoring the change in
absorbance at 340 nm, which is due to the oxidation of NADPH
catalyzed by ALR2 and ALR1. The change in pyridine coenzyme
concentration per minute was determined using a Beckman DU-64
kinetics software program (Solf Pack TM Module).
ALR2 activity was assayed at 30 °C in a reaction mixture containing
0.25 mL of 10 mM D,L-glyceraldehyde, 0.25 mL of 0.104 mM
NADPH, 0.25 mL of 0.1 M sodium phosphate buffer (pH 6.2), 0.1 mL
of enzyme extract, and 0.15 mL of deionized water in a total volume of
1 mL. All these reagents, except D,L-glyceraldehyde, were incubated at
30 °C for 10 min; the substrate was then added to start the reaction,
which was monitored for 5 min. Enzyme activity was calibrated by
diluting the enzymatic solution to obtain an average reaction rate of
0.011 0.0010 absorbance unit/min for the sample.
Thiobarbituric Acid Reagent Substance (TBARS) Assay.
Freshly isolated rat brain was homogenized in 10% (w/v) phosphate
buffer (pH 7.4); 20 μM FeCl3 and 100 μM ascorbic acid (final
concentrations) were added to 100 μL of rat brain homogenate, in the
absence or presence of the test compounds or reference scavenger
molecules, and were diluted to a volume of 1 mL with phosphate
buffer (pH 7.4). Samples were incubated for 30 min at 37 °C in a
water bath while being slightly stirred and were then mixed with 0.5
mL of thiobarbituric acid [1% (w/v) in 0.05 N NaOH] and 0.5 mL of
25% (v/v) HCl. The mixture was boiled for 10 min, and after it had
cooled in an ice-cold water bath, extraction was performed with 3 mL
of n-butanol. After liquid phase separation (centrifugation at 2000g for
10 min), the absorbance of the pink organic phase was spectrophoto-
metrically evaluated at 532 nm, and TBARS was expressed as
nanomoles of malondialdehyde (MDA) per 10 mg of rat brain tissue
(wet weight), using a curve with 1,1,3,3-tetramethoxypropane as the
standard reference27 (Figure 3).
Docking Studies. Structures of human ALR2 in complex with
IDD594, sorbinil, and tolrestat were obtained from the Protein Data
Bank (entries 1US0, 2FZD, and 2PDK, respectively). To add
hydrogen to the structures mentioned above, MolProbity, an online
tool for the validation and correction of protein structures, was used.
To prepare the receptor for the docking procedure, proteins were all
superimposed using their Cα atoms, and a subsite was selected having
a radius of 12 Å from ligand. Residues not enclosed in such a selection
were removed. The three subsites (derived from 1US0, 2FZD, and
2PDK) were then saved in a single file in which, while 1US0 was kept
fixed, 2FZD and 2PDK were linearly aligned along the x-axis. In this
step, to avoid docking results across the border of two adjacent
subsites, the translation of 2FZD and 2PDK (−25 and 25 Å,
respectively) was specified so that a partial overlap between subsites
was present. After being removed from the prepared construct water
molecules, all nonpolar hydrogens were merged to the parent carbon
atoms. Then, all atom values were generated automatically through the
GUI called AutoDockTools (ADT).52 Grids were then generated for
all the ligand atom types sufficient to describe all atoms in the selected
database with the help of AutoGrid4,53 using a grid spacing of 0.5 Å.
The grid was centered on the 1US0 active site and had dimensions of
63 Å × 24 Å × 25 Å, which was sufficiently large to include the entire
construct.
ALR1 activity was determined at 37 °C in a reaction mixture
containing 0.25 mL of 20 mM sodium D-glucuronate, 0.25 mL of 0.12
mM NADPH, 0.25 mL of a dialyzed enzymatic solution, and 0.25 mL
of 0.1 M sodium phosphate buffer (pH 7.2) in a total volume of 1 mL.
The enzyme activity was calibrated by diluting the dialyzed enzymatic
solution to obtain an average reaction rate of 0.015
0.0010
absorbance unit/min for the sample.
Enzymatic Inhibition. The inhibitory activity of the newly
synthesized compounds against ALR2 and ALR1 was assayed by
adding 0.1 mL of the inhibitor solution to the reaction mixture
described above. All the inhibitors were solubilized in water, and the
solubility was facilitated by the addition of a small quantity of DMSO.
To correct for the nonenzymatic oxidation of NADPH and for
absorption by the compounds tested, a reference blank containing all
the assay components listed above except the substrate was prepared.
The inhibitory effect of the new derivatives was routinely estimated at
a concentration of 10−4 M. Those compounds found to be active were
tested at additional concentrations between 10−5 and 10−7 M. The
determination of the IC50 values was performed by linear regression
analysis of the log-dose response curve, which was generated using at
least four concentrations of the inhibitor causing between 20 and 80%
inhibition, with three replicates at each concentration. The 95%
confidence limits (95% CL) were calculated from t values for n − 2,
where n is the total number of determinations.
Quantitative Nitrite Detection in Rat Liver Homogenate. The
NO releasing activity of test compounds, 5a−k,m, was evaluated on
the rat liver homogenate, obtained according to a previously reported
procedure described by Kozlov et al. with slight modifications.51 Liver
was quickly removed, cut into small pieces, and washed with ice-cold
sucrose buffer [100 mM K2HPO4, 1 mM EDTA, 1.15% (p/v) KCl,
0.25 M sucrose, and 0.1% EtOH (pH 7.4)] to remove blood. After
being dried with paper, liver was weighted, suspended in the same
buffer, added in a ratio of 1/6 (liver/buffer, w/v), and homogenized
with Glass-Potter. The homogenate so obtained was filtered through
three layers of surgical gauze and centrifuged for 10 min at 600g and 0
°C to pellet the nuclear fraction. The resulting supernatant, containing
the mitochondrial, microsomal, and cytoplasmic fractions, was
exploited to assess the NO donor properties of the test compounds;
200 μL of the filtrate was mixed with 300 μL of water and 500 μL of
Griess reagent, containing 4% sulfanilamide and 0.2% naphthylethy-
lendiamine dihydrochloride in 10% phosphoric acid, in the presence or
absence of test compounds, at a concentration of 10−4 M. After
incubation at 37 °C for 1 h, the amount of NO released was
determined spectrophotometrically by measuring at 543 nm the
Ligand files were constructed employing the Builder tool and
generated with the Ligprep module within the Maestro suite. Partial
ligand charges and ligand electrostatic potentials were calculated by
means of the Jaguar suite54 within the Schrodinger package and
̈
mapped onto its electron density. The isovalue of 0.0004 electron/
Bhor3 was chosen for the definition of the density surface, while the
electrostatic potential was computed using LACVP* and 6-31G* basis
sets with a scale of −0.0551 (red) to 0.0619 hartree (blue). The
LACVP* basis set uses the standard 6-31G* basis set for light
elements and the LAC pseudopotential (effective core potential for
inner electrons) for the Br atom.55
All the ligands were then converted in the AutoDock4 format file
(.pdbqt). For each ligand, 50 separate docking calculations were
performed. Each docking calculation consisted of 10 million energy
evaluations using the Lamarckian genetic algorithm local search
(GALS) method. The GALS method evaluates a population of
possible docking solutions and propagates the most successful
individuals from each generation into the subsequent generation of
possible solutions. A low-frequency local search according to the
method of Solis and Wets is applied to docking trials to ensure that the
final solution represents a local minimum. All dockings described in
this paper were performed with a population size of 150, and 300
rounds of Solis and Wets local search were applied.
Hydrated Docking Procedure. The same parameters were also
used for explicitly considering water molecules during docking
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calculations of 5a. In this procedure,41 the ligand was hydrated using
the wet.py suite, an additional grid map was calculated for the water
molecules using the mapwater.py suite while, docking results were
processed using the dry.py suite. All images of complexes were
rendered employing UCSF Chimera.56
(13) Asano, T.; Saito, Y.; Kawakami, M.; Yamada, N.; Fidarestat
Clinical Pharmacology Study Group. Fidarestat (SNK-860), a potent
aldose reductase inhibitor, normalizes the elevated sorbitol accumu-
lation in erythrocytes of diabetic patients. J. Diabetes Complications
2002, 16, 133−138.
(14) Bril, V.; Hirose, T.; Tomioka, S.; Buchanan, R.; The Ranirestat
Study Group. Ranirestat for the management of diabetic sensorimotor
polyneuropathy. Diabetes Care 2009, 32, 1256−1260.
ASSOCIATED CONTENT
■
S
* Supporting Information
(15) Ramirez, M. A.; Borja, N. L. Epalrestat: An aldose reductase
inhibitor for the treatment of diabetic neuropathy. Pharmacotherapy
2008, 28, 646−655.
(16) The Aldose Reductase Inhibitor-Diabetes Complications Trial
Study Group. Short report: Treatment long-term clinical effects of
epalrestat, an aldose reductase inhibitor, on progression of diabetic
neuropathy and other microvascular complications: Multivariate
epidemiological analysis based on patient background factors and
severity of diabetic neuropathy. Diabetes Medicine 2012, DOI:
10.1111/j.1464-5491.2012.03684.x.
(17) Reddy, A. B.; Ramana, K. V. Aldose reductase inhibition:
Emerging drug target for the treatment of cardiovascular complica-
tions. Recent Pat. Cardiovasc. Drug Discovery 2010, 5, 25−32.
(18) Ramasamy, R.; Goldberg, I. J. Aldose reductase and
cardiovascular diseases, creating human-like diabetic complications in
an experimental model. Circ. Res. 2010, 14, 1449−1458.
(19) Gleissner, C. A.; Sanders, J. M.; Nadler, J.; Ley, K. Upregulation
of aldose reductase during foam cell formation as possible link among
diabetes, hyperlipidemia, and atherosclerosis. Arterioscler., Thromb.,
Vasc. Biol. 2008, 28, 1137−1143.
Physical, spectral, and purity data of compounds described
(Tables 1−7) and additional molecular modeling figures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*C.L.M.: telephone, (+)390502219593; e-mail, concettina.
lamotta@farm.unipi.it. L.M.: telephone, (+)39081679899; e-
mail, luciana.marinelli@unina.it.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
ALR2, aldose reductase; AKR, aldo-keto reductase; NADPH, β-
nicotinamide adenine dinucleotide phosphate, reduced form;
NADP+, β-nicotinamide adenine dinucleotide phosphate; ROS,
reactive oxygen species; PKC, protein kinase C; ARIs, aldose
reductase inhibitors; VS, virtual screening; NO, nitric oxide;
TBARS, thiobarbituric acid reagent substance
(20) Sellers, D. J.; Chess-Williams, R. The effects of streptozotocin-
induced diabetes and aldose reductase inhibition with sorbinil, on left
and right atrial function in the rat. J. Pharm. Pharmacol. 2000, 52, 687−
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(21) Heather, L. C.; Clarke, K. Metabolism, hypoxia and the diabetic
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Srivastava, S.; Ramana, K. V. Aldose reductase inhibition suppresses
oxidative stress-induced inflammatory disorders. Chem.-Biol. Interact.
2011, 191, 330−338.
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