Structure-activity relations on [1-(3,5-difluoro-4-hydroxyphenyl)-1H- pyrrol-3-yl]phenylmethanone. the effect of methoxy substitution on aldose reductase inhibitory activity and selectivity

Article abstract of DOI:10.1016/j.bmc.2011.01.009

Based on our previous work, we studied the effect of methoxy-substitution as well as the regioposition of the benzoyl-moiety of 4a [(1-(3,5-difluoro-4- hydroxyphenyl)-1H-pyrrol-3-yl)(phenyl)methanone]. On this basis, compounds 4b-c and 5a-c were synthesiz

Full text of DOI:10.1016/j.bmc.2011.01.009

Bioorganic & Medicinal Chemistry 19 (2011) 1426–1433
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relations on [1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-
3-yl]phenylmethanone. The effect of methoxy substitution on aldose
reductase inhibitory activity and selectivity
Maria Chatzopoulou a, Eduard Mamadou a, Maria Juskova b, Cathrine Koukoulitsa c, Ioannis Nicolaou a,
,
⇑
Milan Stefek b, Vassilis J. Demopoulos a,
⇑
a Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
b Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dubravska cesta 9, 841 04 Bratislava, Slovakia
c Department of Chemistry, University of Athens, 15784 Zographou, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on our previous work, we studied the effect of methoxy-substitution as well as the regioposition of
the benzoyl-moiety of 4a [(1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(phenyl)methanone]. On
this basis, compounds 4b–c and 5a–c were synthesized and assayed for aldose and aldehyde reductase
inhibitory activity. Furthermore, a 4,6-difluoro-5-hydroxyphenyl pattern (9) was studied, in order to ver-
ify the optimum position of the phenol-moiety. Compound 5b emerged as the most potent and selective
inhibitor. Moreover, further assays proved 5b as a potent antioxidant and an inhibitor of sorbitol accumu-
lation in isolated rat lenses. Combining the above attributes, 5b could serve as a lead compound targeted
at long-term diabetes complications.
Received 21 October 2010
Revised 29 December 2010
Accepted 5 January 2011
Available online 11 January 2011
Keywords:
Aldose and aldehyde reductase
ARI
Long-term diabetic complications
Sorbitol assay
Ó 2011 Elsevier Ltd. All rights reserved.
Docking
1. Introduction
recent statistical analyses in the US, 116 million dollars were spent
on medical expenditures for diabetes during 2007, with 50% of this
Diabetes mellitus is a complex metabolic disorder affecting 285
million adults in 2010 and this number is estimated to increase to
439 million by 2030.1 The increase in numbers is considered as a
result of the dominant lifestyle that encourages low physical activ-
ity and the emergence of obesity, leading this disease to epidemic
proportions. The high morbidity and mortality of diabetes (3.96
million in 20102) is attributed to diabetes’ chronic complications
that develop over the years due to poor glycemic control. Macroan-
giopathies such as coronary artery disease, peripheral vascular and
cerebrovascular disease are the main causes behind the high
lethality of diabetes, accompanied by microangiopathies such as
retinopathy, neuropathy and nephropathy that significantly lower
the quality of the diabetic’s life. Thus, it becomes clear that diabe-
tes consists one of the major health problems worldwide, with the
addition of a significantly high economic burden. According to
amount spent on the treatment of diabetes complications. More-
over, if the indirect costs (reduced productivity, disability, early
mortality) are included the above amount is raised to 174 million
dollars.3
Hyperglycemia, as a result of the instability in glucose regula-
tion in the diabetic individual, is considered as the causative link
on the onset and progression of diabetes chronic complications.
Under hyperglycemic conditions, and especially in cells where
the intake of glucose is non insulin-dependent, there is observed
activation of the polyol pathway as a means to metabolize the glu-
cose excess. This increased flux through the polyol pathway
reaches up to 30% of the total glucose turnover.4 As a consequence,
osmotic, oxidative, reductive, glycative and protein kinase C (PKC)
stress are induced5 with devastating manifestations for the cells.
The polyol pathway was first linked to secondary complications
of diabetes in the mid ’60s.6 Aldose reductase (ALR2, AR, E.C.
1.1.1.21, AKR1B1) is the first and rate-limiting enzyme of the
polyol pathway, reducing glucose to sorbitol using NADPH as
cofactor. Sorbitol is further metabolized to fructose by sorbitol
dehydrogenase (SDH). In the investigations on the significance of
these two enzymes and their implication on diabetes long-term
complications, inhibition of ALR2 emerged as a novel pharmaco-
therapeutic target and the development of inhibitors (ARIs) has
Abbreviations: AKR, aldoketoreductase; ALR1, aldehyde reductase; ALR2, aldose
reductase; ARI, aldose reductase inhibitor; PKC, protein kinase C; rmsd, root-mean
squared deviation; SDH, sorbitol dehydrogenase.
⇑
Corresponding authors. Tel.: +30 2310 998670; fax: +30 2310 997852 (I.N.);
tel.: +30 2310 997626; fax: +30 2310 997852 (V.J.D.).
E-mail addresses: inikolao@pharm.auth.gr (I. Nicolaou), vdem@pharm.auth.gr
(V.J. Demopoulos).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.009

M. Chatzopoulou et al. / Bioorg. Med. Chem. 19 (2011) 1426–1433
1427
Table 1
been gaining attention since then. The most studied classes of
Friedel–Crafts aroylation yields with AlCl3 and BF3ꢀEt2O
inhibitors though, namely carboxylic acids and hydantoins, proved
to have poor bioavailability or manifest adverse side effects.7 As
such, novel and selective chemotypes are sought in order to im-
prove the therapeutic potential of ARIs and explore their clinical
usage.
Compound
Yields
AlCl3 (%)
BF3ꢀEt2O (%)
4a
5a
4b
5b
4c
5c
52
36
26
36
73
10
24
50
22
49
52
44
We have previously reported that in a series of benzoylpyrrol-1-
ylacetic acids the introduction of the methoxy group in the benzoyl
side group generally increases the activity.8 Also, based on bioisos-
tericreplacementoftheacetic acidmoietywith a 2,6-difluorophenol
ring, compound 4a, previously synthesized, proved to be a potent
and selective ARI.9,10 Thus, in order to investigate the effect of phenol
positiononactivityandselectivity, compound9(Scheme2)wassyn-
thesized. Since 4a proved to be a more effective and selective inhib-
itor of ALR2, in contrast to 9, we further processed on 4a in order to
delineate the effect of methoxy-substitution. On this basis, com-
pounds 4b–c and 5a–c (Scheme 1) were synthesized and evaluated
for ALR2 inhibitory activity. Furthermore, the selectivity index of
the newly synthesized pyrrolyldifluorophenol derivatives towards
the closely related enzyme aldehyde reductase (ALR1, E.C. 1.1.1.2,
AKR1A1) was determined. Docking simulations were performed in
order to examine the differences in activity and selectivity among
the most intriguing derivatives. Finally, the antioxidant potential
of the most active and selective compound 5b, as well as its inhibi-
tion of sorbitol accumulation in isolated rat lenses was assessed.
of 70%, whereas aroylation with BF3ꢀEt2O led to the production of
both isomers in a ratio close to 1:1 (52% and 44%).
Compound 9 was synthesized, as depicted in Scheme 2. The first
step involved the reaction of 1,3-difluoro-4-iodo-2-methoxyben-
zene12 (6) in a modified Ullman type coupling reaction with 3-ben-
zoylpyrrole (7). In this modification, previously used for coupling
reactions on a number of substituted pyrroles,13 CuI and trans-
N,N0-dimethylcyclohexane-1,2-diamine were used as catalysts
and K3PO4 as the base. The desired product was derived upon
deprotection of the methoxy-group with HBr cleavage.12
2.2. In vitro results
2.2.1. ALR2 inhibitory activity
ALR2 inhibitory activity was evaluated on partially purified rat
lens ALR2. Human ALR2 exhibits 85% sequence homology to rat
ALR2,14 while the catalytic active sites of both enzymes are consid-
ered identical.15 ALR2 inhibitory activity is expressed as IC50 values
(Table 2). All the compounds exhibited inhibitory activities in the
micromolar or submicromolar range. Changing the position of the
phenol group (compound 9) resulted in the maintenance of the
inhibitory activity, although with a slight decrease in its magnitude.
Furthermore, from the results obtained, it is deduced that the meth-
oxy-substitution generally conserves the inhibitory activity, with
the exception of compound 4b in which activity decreases but is
not totally eliminated. On the role of the position of the pyrrol-sub-
stitution no definite results could be drawn, since the most active
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds 4a–c and 5a–c was
achieved as described in Scheme 1. Friedel–Crafts aroylation of
the respective aryl substituted 2,6-difluoro-4-pyrrol-1-yl-phenyl
esters 3a–c, was followed by alkaline hydrolysis of the crude
formed intermediates. Based on a previous work on the regioselec-
tive synthesis of aroyl-pyrroles,11 two methodologies were applied,
varying on the Lewis acid catalysts. AlCl3 and BF3ꢀEt2O were uti-
lized, producing various ratios among the isomeric products (see
Table 1 for details). The results obtained are in accordance with
those of Kakushima et al.,11 though it should be underlined that
the reactions in their work were highly regiospecific. Generally in
our work, AlCl3 as a catalyst directed mostly to the 3-isomer,
whereas BF3ꢀEt2O to the 2-isomer. It should also be mentioned, that
aroylation of 3c with both catalysts derived mostly the 3-isomer
probably due to stereochemical reasons. Aroylation with AlCl3
led to the massive production of the 3-isomer up to a percentage
compounds, 4a and 5b, are a b-substituted and a
a-substituted pyr-
role derivative, respectively. However, the -substituted pyrrolyl
a
methoxy derivatives (5b, 5c) were more active compared to the
respective b-substituted (4b, 4c). In this respect, it is interesting to
mention that in a lowest energy conformation16 of 4b and 4c the
intramolecular distance between the phenolic oxygen and methoxy
oxygen is close to 14 Å, whereas in 5b and 5c is close to 11 Å.
O
O
R2
R1
O
R2
Cl
Cl
R1
+
R2
N
O
N
N
N
R2
R1
2a-c
O
2a-c
R1
1. b
2. c
a
F
F
F
F
F
F
F
F
O
OH
OH
OH
1
4a-c
5a-c
R1
a: R1, R2 = H
b: R1 = H, R2 = OCH3
c:
R2
3a-c
R1 = OCH3, R2 = H
Scheme 1. Synthesis of compounds 4a–c and 5a–c. Reagents: (a) (CH3CH2CH2CH2)4N(HSO4), NaOH, 1,4-dioxane; (b) AlCl3 or BF3ꢀEt2O, CH2Cl2; (c)H2O/NaOH, 1,4-dioxane.

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M. Chatzopoulou et al. / Bioorg. Med. Chem. 19 (2011) 1426–1433
O
O
I
O
F
b
a
N
F
N
F
+
F
F
OCH3
N
H
F
OCH3
OH
7
8
9
6
Scheme 2. Synthesis of compound 9. Reagents: (a) trans-N,N0-dimethylcyclohexane-1,2-diamine, CuI, K3PO4, toluene; (b) aqueous 48% HBr/CH3COOH.
in the heterogenous system of unilamellar L-a-phosphatidylcho-
Table 2
Aldose and aldehyde reductase inhibitory activity
line dioleoyl (DOPC) liposomes. DOPC were used as model
membranes oxidatively stressed by peroxy radicals generated in
the aqueous phase by thermal decomposition of the hydrophilic
azo-initiator 2,20-azobis(2-amidinopropane)hydro-chloride (AAPH).
In this system factors such as membrane permeability as well as
location of the antioxidant and radical rigidly affect the antioxidant
reactivity. Compound 5b was found to inhibit peroxidation of
DOPC liposomes more efficiently than the known antioxidant trol-
Compound
IC50 SEMa
(l
V)
Selectivity indexb
Rat lens ALR2
Rat kidney ALR1
4a
4b
4c
5a
5b
5c
9
0.40 0.02
24.9 0.08
9.93 0.49
1.06 0.16
0.39 0.02
1.18 0.04
1.26 0.02
0.25 0.01c
13.4 0.58
>100
33.8
>4
5.2
51.8 1.00
4.6 0.17
27.8 5.10
23.5 3.80
1.61 0.01
4.3
71.6
19.9
1.3
ox (IC50 = 35.77 and 93.518,19
lV for 5b and trolox, respectively).
Sorbinil
Valproic acid
56.1 2.7d
2.2.4. Inhibitory activity on rat lens sorbitol accumulation
Increased ALR2 activity as well as elevated sorbitol levels have
been recently reported in type II diabetes patients with diabetic
retinopathy.25 Of the stress-hypotheses mentioned earlier, osmotic
stress caused by sorbitol accumulation has been shown to be of ut-
most importance in the diabetic’s lens, leading eventually to cata-
ract formation.26 On this basis, we studied the effect of the most
active compound 5b on glucose-induced sorbitol accumulation at
an organ level in the rat lens cultivated in the presence of high glu-
cose in vitro. As shown in Table 3, significantly increased sorbitol
levels were recorded in the lenses incubated with glucose in com-
parison with control incubations without glucose, reflecting in-
creased flux through ALR2. Similarly, other authors27 observed
more than 10-fold increase of sorbitol level in the eye lenses incu-
bated with glucose under comparable conditions (50 mM glucose,
4 h incubation). Sorbitol production was significantly inhibited by
compound 5b with efficacy comparable to that of equimolar tetra-
methylene glutaric acid (TMGA). The latter is a specific inhibitor of
ALR2, used also by other authors at the cellular level of isolated red
blood cells.28–30
a
b
c
n = 3.
Defined as IC50 [ALR1]:IC50 [ALR2].
Reported by Alexiou et al.17
d
Reported by Pegklidou et al.18 and Stefek et al.19
2.2.2. ALR1 inhibitory activity
ALR2 belongs to the aldoketoreductase (AKR) superfamily of en-
zymes. Among its many members, ALR2 shares the highest degree
of similarity with ALR1. The two enzymes exhibit 65% sequence
homology, as well as structural homology.20 Since the closely
related AKRs are related with the detoxification of toxic alde-
hydes,21 parallel inhibition could cause unwanted effects. ALR1
inhibitory activity was evaluated on partially purified rat kidney
enzyme and is expressed as IC50 values presented together with
the calculated selectivity index (Table 2). From the obtained results
it becomes clear that the methoxy derivatives are less active on
ALR1 in comparison to their activity on ALR2. Furthermore, the
b-substituted pyrrole derivatives (4a–c) are less active from their
respective
a-substituted analogues (5a–c) on ALR1. Regarding
3. Computational methods
the selectivity index, no selectivity was observed when the phenol
group was relocated (compound 9). By far the most selective inhib-
itor was compound 5b, which is also the most active ARL2
inhibitor.
The structure of ALR2 active site has been extensively analyzed
by both crystallographic and modeling studies. It has proved to be
highly hydrophobic in nature and is formed by aromatic residues
(Trp20, Tyr48, Trp79, Trp111, Phe121, Phe122 and Trp219), apolar
residues (Val47, Pro218, Leu300 and Leu301), and polar residues
(Gln49, Cys298 and His110). The nicotinamide ring of NADP+ is
2.2.3. Antioxidant capacity
Oxidative stress induced by hyperglycemia in diabetes mellitus
has been highly interrelated in the emergence and progression of
long-term diabetes complications.19 The formation of reactive
aldehydes, as well as oxidized glutathione (GSSG) are known prod-
ucts of such conditions that are also substrates for ALR2.22 Further-
more, GSSG has been blamed for oxidation of ALR2 on key cysteine
residues (Cys298) resulting in an oxidized form of the enzyme that
has lower sensitivity to ARIs along with an increase in the affinity
for aldehyde substrates.23,24 Thus, the putative antioxidant activity
of an ARI could account synergistically on the overall effectiveness
of a pharmacotherapeutic agent targeted at diabetic chronic
complications, both by preventing the enzyme’s oxidation and by
reacting with the highly reactive oxidants produced during hyper-
glycemia. The antioxidant inhibitory efficiency of 5b was evaluated
Table 3
Effect of compound 5b on sorbitol accumulation in isolated rat lenses cultivated with
high glucose in comparison with standard TMGA
Incubation
Sorbitol (nmol/g)
n
ꢁGlucosea
39.6 10.3
643.5 133.0
203.5 42.9b
205.8 30.5b
3
+Glucose
11
6
+Glucose + 5b (100 lM)
+Glucose + TMGAc (100
l
M)
3
Results are mean values SD from n independent incubations.
a
Glucose, 50 mM; time of incubation, 3 h; 37 °C.
p <0.001 versus +Glucose.
TMGA = tetramethylene glutaric acid.
b
c

M. Chatzopoulou et al. / Bioorg. Med. Chem. 19 (2011) 1426–1433
1429
centered in the cavity and the three possible proton donors respon-
sible for catalysis are Tyr48, His110 and Trp111 all composing the
anionic binding pocket.5,31,32 Inhibitor selectivity remains an issue
to consider, since the corresponding residues in the ALR1 active
site are highly conserved (Tyr50, His113 and Trp114).33 The differ-
ence in inhibitor potency for the two enzymes has been attributed
to non-conserved residues located in the C-terminal loop lining a
hydrophobic region of the active site called the ‘specificity
pocket’.33 This pocket binds inhibitors that are more effective
against ALR2 than against ALR1.34
Docking simulations of the most intriguing compounds were
performed in order to shed light on their interactions in the active
site of ALR2. The human ALR2 holoenzyme cocrystallized with IDD
594 (PDB entry 1US0) was selected, as it was determined at the
highest resolution (0.66 Å) among all the available structures.
The X-ray crystallography study revealed that the inhibitor IDD
594 induces a conformational change upon binding to ALR2, creat-
ing a specificity pocket localized between Phe122, Trp111, Leu300,
and Ala299. The carboxylate group is firmly anchored in the active
site with hydrogen bonds to His110, Tyr48, and Trp111 and with a
strong electrostatic interaction with NADP+.
The docking simulations were performed by the docking GLUE
program, which has been shown to successfully reproduce experi-
mentally observed binding modes in terms of rmsd (root-mean
squared deviation). The crystallographic structure of IDD 594 was
docked into ALR2 with GLUE provided excellent results as shown
in our previous work.35
Docking calculations showed that the most active compound
(5b) exhibits the lower binding energy (ꢁ28.32 kcal/mol). As illus-
trated in Figure 1a, the compound was placed in the same location
as IDD 594 in the crystal structure. In particular, compound 5b is an-
chored in the anionic binding site forming a polar interaction
between the carbonyl oxygen and Trp111, and electrostatic interac-
tions with the positively charged nicotinamide ring of NADP+. A
hydrogen bond is also formed between the hydrogen of the hydroxyl
group of the 3,5-difluorophenol ring and the oxygen of Thr113.
Further stabilization of the molecule is attributed to the aromatic
interactions of the phenyl rings with Trp20, Trp79, Trp111, and
Van der Waals interactions between phenyl rings and residues
Phe122, and Leu300. It should be noticed that the phenyl ring en-
tered in the specificity pocket localized between Phe122, Trp111,
Leu300, and Ala299, as IDD 594, suggesting a selectivity for ALR2
versus ALR1 (Fig. 1b). This is in accordance with the experimental re-
sult regarding the selectivity index (Table 2), which indicates that
compound 5b is by far the most selective inhibitor. Docking calcula-
tions in the active site of ALR1 were performed in order to compare
the interactions of inhibitor 5b in the two enzymes. The porcine
ALR1 holoenzyme cocrystallized with the potent aldose reductase
inhibitor Fidarestat (PDB entry 3H4G) was selected.33 In both en-
zymes Fidarestat is anchored through its cyclic imide ring to the ac-
tive site and the hydrogen-bonding network with the active-site
residues Tyr50, His, 113, and Trp114 is conserved. Docking simula-
tions of compound 5b (ꢁ11.77 kcal/mol) in the active site of ALR1,
showed that it cannot establish strong interactions with the key
aminoacids of the active site and NADP+. In contrast, compound 9,
for which no selectivity was observed, exhibited lower binding en-
ergy (ꢁ14.43 kcal/mol) in comparison to inhibitor 5b and showed
polar interactions with the key aminoacids of ALR1 (Fig. 1d).
Compound 4a possess similar binding energy (ꢁ27.55 kcal/
mol), and as is depicted in Figure 2a is placed in the same location
as IDD 594 and 5b. The compound is oriented in the active site so
that the fluorine atom at position 3 develops a hydrogen bond with
His110 and polar interactions with Tyr48 and Trp111. The mole-
cule is also stabilized forming polar interactions between the fluo-
rine atom at position 5 and Trp20. A hydrogen bond is also formed
between the hydrogen of the hydroxyl group and the nitrogen of
Trp20. Compound 4b entered the anionic binding site with the 4-
methoxy phenyl ring forming a polar interaction between the car-
bonyl oxygen and the nitrogen of Trp111 (Fig. 2b). It exhibits the
higher binding energy (ꢁ22.78 kcal/mol) among the studied com-
pounds and this result is consistent with the observed reduced
inhibitory activity of compound 4b against ALR2.
Figure 1. Docked orientations of (a) compound 5b with additional depiction of the key aminoacids of ALR2 active site. Polar interactions and hydrogen bonds are shown as
dotted lines; (b) compound 5b in the specificity pocket; (c) compound 5b with additional depiction of the key aminoacids of ALR1 active site; (d) compound 9 with additional
depiction of the key aminoacids of ALR1 active site. Polar interactions and hydrogen bonds are shown as dotted lines.

1430
M. Chatzopoulou et al. / Bioorg. Med. Chem. 19 (2011) 1426–1433
Figure 2. (a) Compound 4a, and (b) compound 4b, with additional depiction of the key aminoacids of ALR2 active site. Polar interactions and hydrogen bonds are shown as
dotted lines; (c) superimposition of the docked compounds 5b, 4a, and 4b and IDD594 in the active site of ALR2.
In Figure 2c is presented the superimposition of the docked
compounds and IDD594 in the active site of ALR2.
and NaOH powder (0.5 g) in 1,4-dioxane (12.5 mL) under a nitrogen
atmosphere, a solution of the appropriate aroyl chloride (2a–c)
(7 mmol) in 1,4-dioxane (5 mL) was added dropwise for 30 min.
The mixture was filtered, washed with 1,4-dioxane (3 ꢂ 10 mL)
and evaporated under reduced pressure. The residue was flashed
chromatographed with a suitable mixture of petroleum ether/
EtOAc. Analytical samples were obtained by recrystallization from
CH2Cl2/petroleum ether.
4. Conclusions
To the previously synthesized 4a we studied the effect of the
phenol position and since 4a proved to be more active and selec-
tive than 9, we further proceeded to examine the effect of meth-
oxy-substitution on the benzoyl-moiety on 4a, as well as the
position of the aroyl-substitution on the pyrrolyl-ring. The newly
synthesized methoxy derivatives (4b–c, 5b–c) retained micromo-
5.2.1.1. 2,6-Difluoro-4-(1H-pyrrol-1-yl)phenyl benzoate (3a). Pre-
pared as described by Nicolaou et al.9
lar inhibitory activities for ALR2 enzyme and furthermore a-substi-
5.2.1.2. 2,6-Difluoro-4-(1H-pyrrol-1-yl)phenyl 4-methoxybenzo-
ate (3b). Prepared from 4-methoxybenzoyl chloride according to
the general procedure A (1.251 g, 77%). Mp 152–153 °C; IR (nujol)
tuted pyrroles (5b–c) shown greater selectivity towards the
homologous enzyme ALR1. The most active compound 5b exhib-
ited submicromolar inhibitory activity towards ALR2 and was
highly selective [selectivity index = 72]. These results are in accor-
dance with docking simulations performed on the most intriguing
compounds and ALR2 and ALR1 enzymes. 5b was further assessed
for its antioxidant capacity on the DOPC liposomes model and
found to be a more potent antioxidant than trolox. Moreover, on
a model of isolated rat lenses, 5b inhibited sorbitol accumulation
to an extend similar of the known ARI TMGA. Considering the
above attributes, 5b could be considered as a lead compound for
further development in the field of novel inhibitors that combine
ALR2 inhibitory activity and selectivity, potent antioxidant capac-
ity and furthermore inhibition of sorbitol accumulation, attributes
that are of key importance in the pathology of diabetes chronic
complications.
1740 cmꢁ1 1H NMR (CDCl3) d 4.05 (s, 3H), 6.30–6.50 (m, 2H),
;
6.95–7.30 (m, 6H), 8.10–8.30 (m, 2H). Anal. Calcd for
C
18H13F2NO3ꢀ0.05CH2Cl2: C, 65.00; H, 3.96; N, 4.20. Found: C,
64.82; H, 3.59; N, 4.16.
5.2.1.3. 2,6-Difluoro-4-(1H-pyrrol-1-yl)phenyl 3-methoxybenzo-
ate (3c). Prepared from 3-methoxybenzoyl chloride according to
the general procedure A (1.307 g, 80%). Mp 147–148 °C; IR (nujol)
1748 cmꢁ1; 1H NMR (CDCl3) d 3.90 (s, 3H), 6.30–6.50 (m, 2H), 7.00–
7.60 (m, 6H), 7.65–8.00 (m, 2H). Anal. Calcd for C18H13F2NO3: C,
65.65; H, 3.98; N, 4.25. Found: C, 65.45; H, 4.05; N, 4.24.
5.2.2. General procedure B. Friedel–Crafts aroylation with AlCl3
as Lewis acid. Preparation of(1-(3,5-difluoro-4-hydroxyphenyl)-
1H-pyrrol-yl)(aroyl)methanones (4a–c, 5a–c)
5. Experimental section
5.1. General notes
In a mixture of anhydrous AlCl3 (6 mmol) in CH2Cl2 (10 mL) the
appropriate aroyl chloride (5.6 mmol) was added slowly and stir-
red at room temperature and under a nitrogen atmosphere. After
10 min, a solution of the substituted ester of an aromatic acid with
2,6-difluoro-4-pyrrol-1-yl-phenol (5 mmol) (3a–c) in CH2Cl2
(5 mL) was added and the resulting mixture was stirred for 3 h
under a nitrogen atmosphere. The reaction was quenched with
ice and water and the product was extracted with CH2Cl2
(2 ꢂ 20 mL). The combined organic extracts were washed with
saturated NaCl solution, dried over anhydrous Na2SO4, and evapo-
rated under reduced pressure. The residue was dissolved in
1,4-dioxane (20 mL) and to this a solution of aqueous 5% NaOH
solution (20 mL) was added. The mixture was stirred at room tem-
perature and monitored for the hydrolysis of the ester group by
TLC. When the reaction ended, the mixture was concentrated to
half of its volume, H2O (50 mL) was added, and it was cooled (ice
bath) and acidified with concentrated HCl. Afterwards, the mixture
was extracted with EtOAc (2 ꢂ 50 mL) and the combined organic
extracts were washed with saturated NaCl solution, dried over
Na2SO4, and the solvents were evaporated under reduced pressure.
All reagents were purchased from Sigma–Aldrich Co. and used
without further purification. IR spectra were taken with a double
beam spectrometer Hitachi U-2001. 1H NMR spectra were recorded
on a Bruker AW80 80 MHz with internal TMS standard. Melting
points are uncorrected and were determined in open glass capillar-
ies using a Mel-Temp II apparatus. Flash column chromatography
was carried out with Merck Silica Gel 60 (230–400 Mesh ASTM).
TLC was run with Fluka Silica gel/TLC-cards. Elemental analyses
were performed by Perkin–Elmer 2400 CHN analyzer and the
results are within 0.4% of the theoretical values.
5.2. Chemistry
5.2.1. General procedure A. Preparation of the 2,6-difluoro-4-
pyrrol-1-yl-phenol esters (3a–c)
To a mixture of 2,6-difluoro-4-pyrrol-1-yl-phenol9 (1) (0.975 g,
5 mmol), tetrabutylammonium hydrogen sulfate (0.1 g, 0.3 mmol)

M. Chatzopoulou et al. / Bioorg. Med. Chem. 19 (2011) 1426–1433
1431
In the residue, H2O (50 mL) was added and it was alkalized with
Et3N. Afterwards, the mixture was extracted with AcOEt (2 ꢂ
50 mL) and dried over anhydrous Na2SO4. The solvents were evap-
orated under reduced pressure and the residue was flash chro-
matographed with petroleum ether/EtOAc (10:1–10:2) in order
tion, dried over anhydrous Na2SO4, and evaporated under reduced
pressure. Hydrolysis and purification proceeded as described above
in general procedure B.
5.2.3.1. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(phe-
nyl)methanone (4a). Prepared from 3a according to general pro-
cedure C (395 mg, 24%).
to purify both
a and b products. Analytical samples were obtained
by recrystallization from toluene/petroleum ether to provide the
desired products.
5.2.3.2. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl)(phe-
nyl)methanone (5a). Prepared from 3a according to the general
procedure C (823 mg, 50%).
5.2.2.1. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(phe-
nyl)methanone (4a). Prepared from 3a according to general pro-
cedure B (778 mg, 52%). Analytical data can be found by Nicolaou
et al.9
5.2.3.3. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(4-
methoxyphenyl)methanone (4b). Prepared from 3b according
to the general procedure C (398 mg, 22%).
5.2.2.2. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl)(phe-
nyl)methanone (5a). Prepared from 3a according to the general
procedure B (593 mg, 36%). Mp 168–169 °C; IR (nujol) 3100,
5.2.3.4. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl)(4-
methoxyphenyl)methanone (5b). Prepared from 3b according
to the general procedure C (887 mg, 49%).
1602 cmꢁ1 1H NMR (CDCl3) d 6.20–6.40 (m, 1H), 6.80–7.10 (m,
;
4H), 7.40–7.60 (m, 4H), 7.75–7.95 (m, 2H). Anal. Calcd for
17H11F2NO2: C, 68.23; H, 3.70; N, 4.68. Found: C, 68.03; H, 3.79;
C
N, 4.59.
5.2.3.5. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(3-
methoxyphenyl)methanone (4c). Prepared from 3c according
to the general procedure C (942 mg, 52%).
5.2.2.3. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(4-
methoxyphenyl)methanone (4b). Prepared from 3b according
to the general procedure B (428 mg, 26%). Mp 214–215 °C; IR
(nujol) 3111, 1611 cmꢁ1 1H NMR (CDCl3/DMSO-d6) d 4.00 (s,
;
5.2.3.6. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl)(3-
methoxyphenyl)methanone (5c). Prepared from 3c according
to the general procedure C (616 mg, 34%).
3H), 6.65–6.80 (m, 1H), 6.90–7.35 (m, 6H), 7.60–7.75 (br s,
1H), 7.80–8.05 (m, 2H). Anal. Calcd for C18H13F2NO3: C, 65.65;
H, 3.98; N, 4.25. Found: C, 65.74; H, 3.99; N, 4.09.
5.2.4. [1-(2,4-Difluoro-3-methoxyphenyl)-1H-pyrrol-3-yl]-
phenylmethanone (8)
5.2.2.4. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl)(4-
methoxyphenyl)methanone (5b). Prepared from 3b according
to the general procedure B (593 mg, 36%). Mp 185–186 °C; IR
To a solution of 612 (1 g, 3.7 mmol) in dry toluene (4 mL), 736
(528 mg 3,08 mmol), trans-tetramethylcyclohexane-1,2-diamine
(88 mg, 0.62 mmol), K3PO4 (1.373 g, 6.47 mmol) and CuI (29 mg,
0.15 mmol) were added and the mixture was refluxed under a
nitrogen atmosphere for 24 h. The reaction mixture was cooled
to room temperature; AcOEt (5 mL) was added, filtered through a
plug of silica, eluted with additional EtOAc (30–50 mL) and the sol-
vents were removed under reduced pressure. The resulting residue
was purified by silica gel flash column chromatography (petroleum
ether/AcOEt 10:1) to provide the title compound (833 mg, 86%) as
(nujol) 3100, 1611–1586 cmꢁ1
;
1H NMR (CDCl3/DMSO-d6) d
3.85 (s, 3H), 6.20–6.40 (m, 1H), 6.70–7.15 (m, 7H), 7.75–8.00
(m, 2H). Anal. Calcd for C18H13F2NO3: C, 65.65; H, 3.98; N, 4.25.
Found: C, 65.29; H, 3.70; N, 4.18.
5.2.2.5. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl)(3-
methoxyphenyl)methanone (4c). Prepared from 3c according
to the general procedure B (1202 mg, 73%). Mp 119–120 °C; IR
(nujol) 3100, 1625 cmꢁ1 1H NMR (CDCl3/DMSO-d6) d 3.80 (s,
;
a yellowish oil. IR (nujol) 1978 cmꢁ1 1H NMR (CDCl3) d 4.05 (s,
;
3H), 6.65–6.80 (m, 1H), 6.80–7.20 (m, 5H), 7.25–7.55 (m, 4H).
Anal. Calcd for C18H13F2NO3: C, 65.65; H, 3.98; N, 4.25. Found:
C, 65.36; H, 3.72; N, 4.36.
3H), 6.79–8.00 (m, 10H). Anal. Calcd for C18H13F2NO2ꢀ0.02CH2Cl2:
C, 68.71; H, 4.17; N, 4.45. Found: C, 68.75; H, 4.00; N, 4.37.
5.2.5. [1-(2,4-Difluoro-3-hydroxyphenyl)-1H-pyrrol-3-yl]-
phenylmethanone (9)
5.2.2.6. (1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl)(3-
methoxyphenyl)methanone (5c). Prepared from 3c according
to the general procedure B (165 mg, 10%). Mp 129–130 °C; IR
To a solution of 8 (200 g, 0.64 mmol) in glacial acetic acid
(3.5 mL), an aqueous 48% HBr solution (3.5 mL) was added and
the mixture was refluxed under a nitrogen atmosphere for 12 h.
The reaction mixture was cooled to room temperature and the
solvents were removed under reduced pressure. The resulting
residue was flash chromatographed with petroleum ether/AcOEt
(4:1) followed by recrystallization from CH2Cl2/petroleum ether
to provide the title compound (89 mg, 46%). Mp: 139–142 °C; IR
(nujol) 3260, 1620 cmꢁ1 1H NMR (CDCl3/DMSO-d6) d 3.80 (s,
;
3H), 6.20–6.40 (m, 1H), 6.80–7.60 (m, 9H). Anal. Calcd for
18H13F2NO3: C, 65.65; H, 3.98; N, 4.25. Found: C, 65.46; H,
C
4.01; N, 4.19.
5.2.3. General procedure C. Friedel–Crafts aroylation with BF3ꢀ
Et2O as Lewis acid. Preparation of (1-(3,5-difluoro-4-hydroxy-
phenyl)-1H-pyrrolyl)(aroyl)methanones (4a–c, 5a–c)
;
(nujol) 3113, 1612 cmꢁ1 1H NMR (CDCl3/DMSO-d6) d 6.79–7.08
(m, 4H), 7.38–7.64 (m, 4H), 7.76–7.98 (m, 2H). Anal. Calcd for
In a mixture of BF3ꢀEt2O (28 mmol) in CH2Cl2 (100 mL), the
appropriate aroyl chloride (28 mmol) was added slowly and stirred
at room temperature and under a nitrogen atmosphere. After
10 min, a solution of the substituted ester of an aromatic acid with
2,6-difluoro-4-pyrrol-1-yl-phenol (5,5 mmol) (3a–c) in CH2Cl2
(5 mL) was added and the resulting mixture was stirred for 72 h un-
der a nitrogen atmosphere. The reaction was quenched with ice and
water and the product was extracted with CH2Cl2 (2 ꢂ 20 mL). The
combined organic extracts were washed with saturated NaCl solu-
C
17H11F2NO2ꢀ0.15CH2Cl2: C, 66.02; H, 3.65; N, 4.49. Found: C,
66.00; H, 3.50; N, 4.42.
5.3. Biological evaluation
5.3.1. ALR2 preparation
The target compounds 4b–c, 5a–c and 9 were dissolved in 10%
aqueous solution of NaHCO3. Lenses were quickly removed from

1432
M. Chatzopoulou et al. / Bioorg. Med. Chem. 19 (2011) 1426–1433
Fischer-344 rats of both sexes following euthanasia. The experi-
ments conform to the law for the protection of experimental ani-
mals (Republic of Greece) and are registered at the Veterinary
Administration of the Republic of Greece. ALR2 from rat lens was
partially purified according to the reported procedure17–19 as fol-
lows: lenses were quickly removed from rats following euthanasia
and stored at ꢁ20 °C until used. The lenses were homogenized in 5
vol of cold distilled water. The homogenate was centrifuged at
10,000g at 0–4 °C for 15 min. The supernatant was precipitated
with saturated ammonium sulfate at 40% salt saturation and this
solution was centrifuged at 10,000g at 0–4 °C for 15 min. The latter
supernatant was either used directly or stored for maximum 24 h
at ꢁ80 °C.
determined by the thiocyanate method by sequentially adding
the CHCl3/MeOH (2:1, v/v) mixture (1.4 mL) and the thiocyanate
reagent (0.1 mL).40 The thiocyanate reagent was prepared by mix-
ing equivalent volumes of a methanolic solution of KSCN (3%) and a
ferrous ammonium sulfate solution (45 mM in 0.2 mM HCl). After
the mixture had been left at ambient temperature for at least
5 min, the absorbance at 500 nm was recorded. The lipid peroxide
value was determined using a calibration curve prepared with
standard cumene hydroperoxide. The value of IC50 was obtained
by least-square analysis of the linear part of the semi logarithmic
plot of I (%, percentage of inhibition) versus antioxidant concentra-
tion (0.936 < r2 < 0.978). The experiment was performed in
triplicate.
5.3.2. ALR1 preparation
5.3.5. Eye lenses sorbitol assay
The tested compounds 4a–c, 5a–c and 9 were dissolved in 10%
aqueous solution of NaHCO3. Kidneys were quickly removed from
Fischer-344 rats of both sexes following euthanasia. The experi-
ments conform to the law for the protection of experimental animals
(Republic of Greece) and are registered at the Veterinary Adminis-
tration of the Republic of Greece. ALR1 from rat kidney was partially
purified according to the reported procedure18,37 as follows: kidneys
were homogenized in a knife homogenizerfollowed by processing in
a glass homogenizer with a teflon pestle in 3 vol of 10 mM sodium
phosphate buffer, pH 7.2, containing 0.25 M sucrose, 2.0 mM EDTA
dipotassium salt, and 2.5 mM b-mercaptoethanol. The homogenate
was centrifuged at 10,000g at 0–4 °C for 30 min and the supernatant
was subjected to ammonium sulfate fractional precipitation at 40%,
50%, and 75% salt saturation. The pellet obtained from the last step,
possessing ALR1 activity, was redissolved in 10 mM sodium phos-
phate buffer, pH 7.2, containing 2.0 mM EDTA dipotassium salt
and 2.0 mM b-mercaptoethanol to achieve total protein concentra-
tion of approx. 20 mg/mL. DEAE DE 52 resin was added to the solu-
tion (33 mg/mL) and after gentle mixing for 15 min removed by
centrifugation. The supernatant containing ALR1 was then stored
in smaller aliquots at ꢁ80 °C. No appreciable contamination by
ALR2 in ALR1 preparations was detected since no activity in terms
of NADPH consumption was observed in the presence of glucose
substrate up to 150 mM.
Male Wistar rats, 8–9 weeks old, weighing 200–250 g, were
used. The animals came from the Breeding Facility of the Institute
of Experimental Pharmacology and Toxicology, Dobra Voda (Slovak
Republic). The study was approved by the Ethics Committee of the
Institute and performed in accordance with the Principles of Labo-
ratory Animal Care (NIH publication 83–25, revised 1985) and the
Slovak law regulating animal experiments (Decree 289, Part 139,
July 9th 2003). The animals in light ether anesthesia were killed
by exsanguinations of the carotid artery and the eye globes were
excised. The lenses were quickly dissected and rinsed with ice-cold
saline.
The compounds studied were added into the tubes containing
freshly dissected eye lenses (1 lens per tube) in 10 mmol/l iso-
tonic phosphate buffered saline (PBS, pH 7.4, 1.9 mM NaH2PO4,
8.1 mM Na2HPO4 and 150 mM NaCl)41 bubbled at 37 °C with
pneumoxid (5% CO2, 95% O2) to the final concentration of
100 lmol/L, 30 min before adding glucose. The incubation was
initiated by adding glucose to the final concentration of
50 mmol/L and then continued at 37 °C with occasional (in about
30-min intervals) bubbling the mixture for approximately 30-s
periods with pneumoxid. The incubations were terminated after
a 3-h period by cooling the mixtures in an ice bath, followed by
washing the lenses three times with ice-cold PBS (1 mL). The
short term cultivations were preferred to avoid substantial per-
meability changes of the eye lenses. The washed lenses were kept
deep-frozen for sorbitol determination.
5.3.3. Enzyme assays
ALR2 and ALR1 activities were assayed spectrophotometrically
by determining NADPH consumption at 340 nm. In order to deter-
mine ALR2 inhibitory activity D,L-glyceraldehyde was used as a
substrate and the measurements took place in 30 °C, whereas
ALR1 inhibitory activity was determined with D-glycuronate as a
substrate and the measurements took place in 37 °C. Compounds
To determine sorbitol, the frozen lenses were let to melt at the
ambient temperature. Then distilled water (0.2 mL/1 lens) was
added. The lenses were disrupted by a glass rod. The rod was
washed twice with distilled water (0.1 mL) and the suspension
was ultra-sounded for 5 min. Thereafter, ice-cold HClO4 (9%,
0.4 mL) was added and mixed thoroughly. The mixture was ul-
tra-sounded for another 5 min and then kept on ice for 30 min to
let proteins precipitate. The precipitated protein was spun off
(15 min at 3000 rpm) at 4 °C. The supernatant was neutralized
with concentrated K2CO3 (4 mol/L).
4a–c, 5a–c and
9 were tested at five concentrations, the
log (dose)–response curves were then constructed from the inhib-
itory data, and the IC50 values were calculated by least-square
analysis of the linear portion of the log (dose) versus response
curves. The experiments were performed in triplicate.
The neutralized supernatant was used for determination of sor-
bitol concentration by modified enzymatic analysis according to
Mylari et al.42 Briefly, sorbitol was oxidized to fructose by SDH
with concomitant reduction of resazurin by diaphorase to the
highly fluorescent resorufin. The final concentrations of the assay
solutions were: diaphorase (11.5 U/25 mL triethanolamine buffer),
5.3.4. DOPC liposome preparation, incubation and LOOH
determination
The experimental protocol is based on a previously described
methodology.17,19,38,39 A suspension of unilamellar L-a-phosphati-
dylcholine dioleoyl (C18:1,[cis]-9; DOPC; 99% grade) liposomes
(1 mM) in phosphate buffer (20 mL, 20 mM, pH 7.4) was prepared.
The liposomes (final concentration 0.8 mM DOPC) were incubated
in the presence of different concentrations of compound 5b
NAD+ (25 mg/25 mL triethanolamine buffer), resazurin (25
lL of
2 mmol/L resazurin solution in 25 mL of triethanolamine buffer),
SDH (15.025 U/L mL triethanolamine buffer). Reaction mixtures
were incubated for 60 min at room temperature with an opaque
cover. The sample fluorescence was determined at 544 nm excita-
tion and 590 nm emission. After the appropriate blanks were sub-
tracted from each sample, the amount of sorbitol in nmol/g of lens
wet weight in each sample was determined by comparison with a
linear regression of sorbitol standards.
(10–300 lM) with the water-soluble initiator AAPH (final concen-
tration 10 mM) at 50 °C for 80 min. Aliquots (1 mL) of the incuba-
tion mixtures were extracted with 2 mL portions of an ice-cold
mixture of CHCl3/MeOH (2:1, v/v) containing 2,6-di-tert-butyl-
p-cresol (BHT) (0.05%). The lipid hydroperoxide content was

M. Chatzopoulou et al. / Bioorg. Med. Chem. 19 (2011) 1426–1433
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Acknowledgements
This work was financially supported by the Greek State Scholar-
ship Foundation and Aristotle University’s Research Comittee. Also,
financial support by grants COST B35 and VEGA 2/0001/08 are
gratefully acknowledged. Furthermore, the authors would like to
thank Professor Gabriele Cruciani (Laboratory for Chemometrics,
School of Chemistry, University of Perugia, Italy) for kindly provid-
ing us the GRID package. At last, many thanks to Dr. Polyxeni Alex-
iou for her help and advice on the experimental protocols.
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