Decreasing acidity in a series of aldose reductase inhibitors: 2-Fluoro-4-(1H-pyrrol-1-yl)phenol as a scaffold for improved membrane permeation

Article abstract of DOI:10.1016/j.bmc.2014.02.016

Targeting long-term diabetic complications, as well as inflammatory pathologies, aldose reductase inhibitors (ARIs) have been gaining attention over the years. In the present work, in order to address the poor membrane permeation of previously reported ARIs, derivatives of N-phenylpyrrole, bearing groups with putative pK_a ≥ 7.4, were synthesized and evaluated for aldose reductase inhibitory activity. The 2-fluorophenol group proved the most promising moiety, and further modifications were explored. The most active compound (31), identified as a submicromolar inhibitor (IC₅₀ = 0.443 μM), was also selective against the homologous enzyme aldehyde reductase. Cross-docking revealed that 31 displays a peculiar interaction network that may be responsible for high affinity. Physicochemical profiling of 31 showed a pK_a of 7.64, rendering it less than 50% ionized in the physiological pH range, with potentially favorable membrane permeation. The latter was supported from the successful inhibition of sorbitol formation in rat lenses and the ability to permeate rat jejunum.

Full text of DOI:10.1016/j.bmc.2014.02.016

Bioorganic & Medicinal Chemistry 22 (2014) 2194–2207
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Decreasing acidity in a series of aldose reductase inhibitors:
2-Fluoro-4-(1H-pyrrol-1-yl)phenol as a scaffold for improved
membrane permeation
Maria Chatzopoulou a, Alexandros Patsilinakos b, , Theodosia Vallianatou c, , Marta Soltesova Prnova d,
e
b
d
e
c
ˇ
Simon Zakelj , Rino Ragno , Milan Stefek , Albin Kristl , Anna Tsantili-Kakoulidou ,
Vassilis J. Demopoulos a,
⇑
a Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
b Rome Center for Molecular Design, Dipartimento di Chimica e Tecnologie del Farmaco, University of Rome ‘Sapienza’, 00185 Rome, Italy
c Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimiopolis, Zografou, 157 71 Athens, Greece
d Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dubravska cesta 9, 841 04 Bratislava, Slovakia
e Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
Targeting long-term diabetic complications, as well as inflammatory pathologies, aldose reductase inhib-
itors (ARIs) have been gaining attention over the years. In the present work, in order to address the poor
membrane permeation of previously reported ARIs, derivatives of N-phenylpyrrole, bearing groups with
putative pKa P 7.4, were synthesized and evaluated for aldose reductase inhibitory activity. The 2-fluo-
rophenol group proved the most promising moiety, and further modifications were explored. The most
Received 10 December 2013
Revised 6 February 2014
Accepted 13 February 2014
Available online 26 February 2014
active compound (31), identified as a submicromolar inhibitor (IC50 = 0.443 lM), was also selective
Keywords:
against the homologous enzyme aldehyde reductase. Cross-docking revealed that 31 displays a peculiar
interaction network that may be responsible for high affinity. Physicochemical profiling of 31 showed a
pKa of 7.64, rendering it less than 50% ionized in the physiological pH range, with potentially favorable
membrane permeation. The latter was supported from the successful inhibition of sorbitol formation
in rat lenses and the ability to permeate rat jejunum.
Non-anionic aldose reductase inhibitors
Fluoroacetylation
Cross-docking
Physicochemical profiling
Inhibition of sorbitol accumulation
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
been proposed as the main biochemical changes that are observed
upon the manifestation of long-term diabetic complications.5–7
Discovered by Hers in 1956,1 aldose reductase (AR, ALR2,
AKR1B1, EC 1.1.1.21) has been studied for over half a century
now, though it has been a truly evasive target. The enzyme’s
involvement in the polyol pathway of glucose metabolism initially
connected it to the onset and progression of long-term diabetic
complications (i.e., retinopathy, neuropathy, nephropathy and vas-
cular complications) that are caused due to poor glycemic con-
trol.2–4 In the first and rate-limiting step of this pathway, ALR2
reduces glucose to sorbitol which is then slowly metabolized to
fructose by sorbitol dehydrogenase (SDH). Increased flux through
the polyol pathway occurs during hyperglycemia and can induce
oxidative, reductive, glycative, and PKC stress, all of which have
More recent evidence suggests that ALR2 also plays a central
role in inflammatory pathologies in non-diabetic subjects.8,9 There-
fore, there are growing literature reports on the therapeutic poten-
tial of aldose reductase inhibitors (ARIs) on endotoxin-related
inflammatory diseases10,11 (i.e., asthma, sepsis, and uveitis) and
inflammation-mediated neoplasias12 (i.e., colon, lung, breast, and
prostate cancers). As a result, ALR2 stands as an important pharma-
cotherapeutic target for the treatment of common and devastating
diseases.
Up to now, two inhibitors have reached the market. Tolrestat
was marketed in several countries in Europe during the 1990s,
but was shortly withdrawn due to hepatotoxicity.13,14 On the other
hand, epalrestat is still marketed for the treatment of diabetic neu-
ropathy in a number of countries in Asia, and appears to be well
tolerated, however it has a short half-life, and its efficiency is con-
sidered marginal.3,15 Both these compounds belong to the carbox-
ylic acid class of ARIs (Fig. 1). Carboxylic acid derivatives comprise
⇑
Corresponding author. Tel.: +30 2310997626; fax: +30 2310997852.
E-mail address: vdem@pharm.auth.gr (V.J. Demopoulos).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.02.016
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
2195
Carboxylic acid ARIs
O
O
O
S
N
CF3
OH
OH
OH
O
Cl
N
O
O
Br
N
N
N
N
OH
N
S
H3CO
S
O
S
CF3
F
O
Epalrestat (EPR)
Zenarestat (ZES)
Zopolrestat (ZST)
Tolrestat (TOL)
Spiroimide ARIs
O
O
O
O
HN
HN
NH
NH
HN
HN
O
O
O
N
O
O
O
F
O
Br
Br
F
F
N
N
NH2
O
O
O
F
O
F
Sorbinil (SBI)
Minalrestat (BFI)
Fidarestat (FID)
Ranirestat (NA*)
Figure 1. Representative drug candidates from the two most common classes of ARIs. In brackets are reported the ligand IDs as found in the PDB for those compounds
available as co-crystals. ⁄NA: not available.
the majority of reported ARIs, although they exhibit pharmacoki-
netic shortcomings such as poor permeability (due to their high
degree of ionization at physiological pH), elevated protein binding,
and susceptibility to phase 2 metabolism.16
Previously, we disclosed that the benzoylpyrrole scaffold is fa-
vored in different series of ARIs (Fig. 2). However, in all these stud-
ies, it is linked to an acidic moiety such as the acetic acid,29
tetrazole30 or 2,6-difluorophenol.31,32 Aiming to the reduction of
acidity in the above classes of compounds, a scaffold morphing ap-
proach of the compounds, depicted in Figure 3, was undertaken by
From another perspective, spiroimide derivatives are promising
ARIs. The four drug candidates depicted in Figure 1 are among the
most studied in this group, and along with carboxylic acids repre-
sent the two most common classes of ARIs. From these candidates,
the hydantoin sorbinil, although having a higher pKa (7.817 or
8.718) than carboxylic acid ARIs, exhibited hypersensitivity reac-
tions (common to the hydantoin moiety) and liver toxicity, while
fidarestat was paused on phase II trials.3,5 Poor selectivity of sorbi-
nil against the closely related enzyme aldehyde reductase (ALR1)
was thought to be the main reason behind the side effects.19 Final-
ly, minalrestat is a precursor of ranirestat, and the latter is the only
candidate that proved safe in phase III trials for the treatment of
diabetic neuropathy. Nevertheless, a relative high placebo re-
sponse rendered the results of the trial inconclusive20 and results
from the latest clinical trial, that was completed earlier this year,
are awaited. It should also be noted that the succinimide ranirestat
has a much lower pKa (5.6)21 than sorbinil and should be mostly
ionized at physiological pH.
O
O
O
N
N
N
OH
N
N
N
NH
F
F
O
OH
I
III
II
Figure 2. N-substituted acidic ARIs of the 3-benzoylpyrrole moiety.
OH
As research for ARIs appears to have reached a plateau, novel
chemotypes are sought, that can overcome the adversities posed
from the above classes of compounds. To this end, a novel scaffold
should retain potent ALR2 inhibitory activity, with considerable
selectivity against ALR1. Moreover, in order to reach a cytoplasmic
enzyme such as ALR2, as well as target tissues, the putative thera-
peutic agent needs to be highly permeable through biological bar-
riers. This is in accordance with the studies of Ko22 and Mylari,23
who suggested that ARIs’ in vivo efficacy is significantly optimized
when their ionization potential is low.
7
F
OH
F
6
8
NO2
N
O
OH
29
9
OH
F
N
NO2
28
N
Literature search, with the aid of ChEMBL database (https://
www.ebi.ac.uk/chembldb/), revealed only scarce reports of ARI
structures with percentage of anion formation at physiological
pH less than 50%. Prominent examples are the piplartine analogues
OH
OH
10
OH
reported by Rao et al.24 (IC50 P 4
l
M), the 2,4-thiazolidinediones
reported by Maccari et al.25–27 (IC50 P 1.79
M), and the
pyrido[1,2-a]pyrimidin-4-ones reported by La Motta et al.28
(IC50 P 0.1 M). Thus, it could be argued that the field of non-
N
OH
OH
23
N
OH
l
21
OH
N
l
22
OH
anionic ARIs remains an unexplored area and further studies
should be undertaken.
Figure 3. Potential alternatives for the 2,6-difluorophenol moiety.

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M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
(A)
NH2
N
a
+
O
O
O
F
F
F
F
1
O
(B1)
O
N
N
X3
X2
1) b
2) c
Cl
X3
X2
+
X1
X1
6 X1,X2 = F, X3 = H
1
X1,X2 = F, X3 = H
2 X1,X2 = H, X3 = OH
X1,X3 = H, X2 = OH
7
X1,X2 = H, X3 = OH
8 X1,X3 = H, X2 = OH
3
4
9
X1 = OH, X2,X3 = H
X
1 = OH, X2,X3 = H
O
(B2)
Y1
O
N
Y3
Y2
N
1) b
2) c
Cl
+
10 Y1,Y2,Y3 = H
Y1
Y3
F
11
Y1 = OCH3, Y2,Y3 = H
12 Y1,Y3 = H, Y2 = OCH3
13
Y2
F
OH
OH
Y
1 = OCF3, Y2,Y3 = H
5
14 Y1 = Br, Y2 = H, Y3 = F
15 Y1 = Ph, Y2,Y3 = H
O
Z
(C)
Z
O
N
N
N
1) d
2) c
O
Cl
+
+
Z
F
F
F
OH
OH
OH
5
10 Z = H
11 Z = OCH3
16
Z = H
17 Z = OCH3
Scheme 1. Synthesis of derivatives 6–17. (A) Clauson–Kaas reaction for the synthesis of 1. (B1) regio-Selective Friedel–Crafts aroylation reactions with pyrrolyl compounds 1–
4 and benzoyl chloride. (B2) regio-Selective Friedel–Crafts aroylation reactions with 5 and aroyl chlorides. (C) Friedel–Crafts aroylation reactions for the formation of both
regio-isomeric pyrrolyl derivatives. Reagents and conditions: (a) 4-chloropyridine hydrochloride, 1,4-dioxane, (b) AlCl3 (1:1 ratio of AlCl3/aroyl chloride), 1,2-dichloroethane,
(c) 5% aq NaOH, 1,4-dioxane, (d) AlCl3 (1:2 ratio of AlCl3/aroyl chloride), 1,2-dichloroethane.
evaluating their activity against rat lens ALR2. In all these com-
pounds, the phenyl substructures on the position 1 of the pyrrole
ring share the ability to form hydrogen bonds as donors or accep-
tors, while they have putative pKa values in the range of physiolog-
ical pH or higher (see experimental pKa values of the used phenyl
substructures in Table 1 of Supplementary data (Sd), along with
the predicted values of pKa and the ratios of logD7.4/logP of the
synthesized compounds in Table 2). It has been previously sug-
Of the compounds tested, derivative 10, bearing the 2-fluoro-
phenol structure, attained the highest inhibitory activity and fur-
ther synthetic efforts were undertaken, so that structure activity
relationships could be developed. Initially, our focus was directed
at the benzoyl core, where several substituents were examined,
along with its spatial disposition on the pyrrole ring. Since the a-
position delivered inactive compounds, we focused on the b-regio
isomers. Finally, a second substitution at C-2 of the pyrrole ring
with the fluoroacetyl group was investigated (30 and 31,
Scheme 4), based on previously published encouraging results.34
The respective monosubstituted fluoroacetyl derivatives (32 and
33, Scheme 5) were also synthesized in order to develop further
the structure activity relationships. Compounds that were found
gested that penetration of lens tissue is favored by high logD7.4
/
logratios.23 The synthesized compounds are therefore expected
to achieve better membrane permeation. Phenol derivatives 7–9,
as well as 2-fluorophenol 10 were designed as less acidic isosteres
of the 2,6-difluorophenol III. Salicylaldoximes 21–23 and nitroal-
doximes 28 and 29 were also targeted, since the incorporated sub-
structures have been previously shown to possess ALR2 inhibitory
activity.33 Finally, the 3,4-difluorophenyl derivative 6 was pre-
pared in an attempt to remove the remaining phenolic acidic
hydrogen by replacing the hydroxy group with its classical fluorine
isostere.
significantly active against ALR2 (IC50 <100 lM) were also assessed
for their selectivity against the homologous enzyme ALR1. Com-
pound 31, was the most active derivative of this series, while it
was also found to be the most selective. Cross docking experiments
were conducted on derivatives 10, 31, and 33 in order to delineate
their putative binding mode. An extensive structure based study,

M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
2197
validated on several experimental ALR2 crystal structures, indi-
cates that the newly synthesized derivative 31 shares the most
important features of a generic pharmacophoric model. Addition-
ally, inhibition of sorbitol formation in rat lenses was determined
for compounds 30 and 31, in an attempt to explore the ability of
membrane permeation in combination with ALR2 inhibitory activ-
ity. Further studies were conducted on both compounds in order to
determine their experimental pKa, logD, logP values and their
retention factor logkw on Immobilized Artificial Membrane (IAM)
chromatography. Finally, compound 31 was studied for its ability
to permeate rat jejunum.
The synthetic procedure to obtain the salicylaldoxime deriva-
tives 21–23 is illustrated in Scheme 2. Pyrrolylphenols 7–9 were
formylated in the presence of MgCl2/Et3N, based on a method de-
scribed by Hofslokken and Skattebol.38 The incomplete formyla-
tion, even after prolonged refluxing, can be attributed to the
electron-withdrawing effect of the pyrrolyl substituent that leads
to the reduction of phenol reactivity. In the next step, the reaction
of the derived aldehydes 18–20 with hydroxylamine hydrochlo-
ride,39 led to the formation of the desired salicylaldoximes 21–23.
Nitroaldoximes 28 and 29 were synthesized (Scheme 3) by first
coupling40 of either 2441 or 2542 with 3-benzoylpyrrole to yield nit-
roaldehydes 26 and 27, followed by aldoxime formation.43
All the derived oximes (21–23 and 28 and 29) were identified as
the E-isomers based on their 1H NMR spectra. Hydrogens on the
carbon of E-oximes are deshielded in comparison to the Z-isomers
and present 1H NMR signals with d >8.33 In the synthesized ana-
logues, the respective signals were in the range of 8.42–8.60.
Initial efforts to synthesize the bis-ketosubstituted pyrrole
derivative 30 with conventional heating of 10 with trifluoroacetic
anhydride in the presence or absence of the base EtN(iPr)2 and in
the presence or absence of the nucleophile 4-(pyrrolidin-1-yl)pyr-
idine failed, even after refluxing for 120 h. Finally, the bis-substi-
tuted derivatives 30 and 31 were synthesized from 10 in a
reaction with the appropriate fluoroacetic anhydride under pulsed
microwave irradiation (Scheme 4). The intrinsic difficulty of the
reaction is also depicted in the prolonged period that is needed
for the reaction’s completion even under microwave irradiation,
where the best yield was obtained by exposing the reaction to
ten 30 min sequences with intermittent cooling.
2. Results and discussion
2.1. Chemistry
The difluorophenylpyrrolyl derivative 1 was synthesized from
3,4-difluoroaniline in the presence of 4-chloropyridine hydrochlo-
ride, based on a previously reported method35 (Scheme 1A). In a
similar way, pyrrolylphenols 2–5 were prepared via a modified
Clauson–Kaas reaction from the respective amine hydrochloride
salts in the presence of nicotinamide, and are reported else-
where.36 In order to obtain the desired products 6–17
(Scheme 1B and C), Friedel–Crafts aroylation reactions were car-
ried out, followed by hydrolysis of the postulated intermediate
formed esters. The latter was based on the report37 that a phenol
could be esterified during the course of an AlCl3 catalyzed acylation
reaction. In a previous study,34 we have found that by adjusting the
ratio between AlCl3 and the aroylchloride, selectively b-substituted
The monosubstituted fluoroacetic derivatives 32 and 33 were
synthesized, as previously reported,34 in a pyrrole acylation reac-
tion from 5 and the appropriate fluoroacetic anhydride. In this
case, the reaction on the unsubstistuted pyrrole ring proceeded
with ease and was completed after 4 h of refluxing (Scheme 5).
or a mixture of a- and b-substituted pyrrolyl-derivatives could be
obtained. In the present work, we followed a similar stoichiometry,
and successfully prepared the b-substituted pyrrolyl derivatives
(6–15, Schemes 1B), or both
a
- and b-pyrrolyl-derivatives (10
and 16 or 11 and 17, Scheme 1C).
O
O
O
N
N
N
a
b
X3
X2
Y3
Y2
Z3
Z2
Y1
Z1
X1
21
Z
1 = H, Z2 = CHNOH, Z3 = OH
22 Z1 = CHNOH, Z2 = OH, Z3 = H
23
7 X1,X2 = H, X3 = OH
X1,X3 = H, X2 = OH
9 X1 = OH, X2,X3 = H
18
Y1 = H, Y2 = CHO, Y3 = OH
19 Y1 = CHO, Y2 = OH, Y3 = H
20
8
Z
1 = OH, Z2 = CHNOH, Z3 = H
Y1 = OH, Y2 = CHO, Y3 = H
Scheme 2. Synthesis of salicylaldoxime derivatives 21–23. Reagents and conditions: (a) MgCl2, Et3N, HCHO, CH3CN, (b) NH2OHꢀHCl, EtOH–H2O.
O
O
O
Br
X1
N
N
a
b
+
N
H
X2
X2
Y2
X1
Y1
24
25
26 X1 = CHO, X2 = NO2
28 Y1 = CHNOH, Y2 = NO2
29
Y1 = NO2, Y2 = CHNOH
X1 = CHO, X2 = NO2
X1 = NO2, X2 = CHO
27
X1 = NO2, X2 = CHO
Scheme 3. Synthesis of nitroaldoxime derivatives 28 and 29. Reagents and conditions: (a) Pd(OAc)2, xanthphos, K2CO3, o-xylene, (b) NH2OHꢀHCl, CH3COONa, EtOH.

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M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
relocation of the hydroxy group from the para-position (9) to
R
ortho- or meta-positions (7 and 8) was detrimental for activity,
while replacement of the hydroxy group with fluorine (6) resulted
in total loss of activity.
N
N
O
a
Derivatives of 10 were then synthesized in order to further ex-
plore the structure activity relationships. As shown in Table 1, the
optimal aroyl substitution was on the b-position of the pyrrole
32
R = CF3
33 R = CHF2
F
5
F
OH
OH
Scheme 4. Synthesis of the bis-ketosubstituted derivatives 30 and 31. Reagents and
conditions: (a) (RCO)2O, 1,2-dichloroethane, MW irradiation.
ring. Both the two
a
-regio isomers synthesized (16 and 17) had
M. Further substitution on the benzoyl moi-
IC50 values over 100
l
ety with a methoxy group (11 and 12) appeared well tolerated and
resulted in a slight improvement of activity. In another step, and in
order to avoid its metabolic liability, the methoxy group was re-
placed by the trifluoromethoxy group (13). However, activity was
significantly decreased. The trifluoromethoxy group adopts a
different conformation than the methoxy group,46 and this confor-
mation is probably not favorable for activity. In compound 14, the
4-bromo-2-fluorophenyl moiety was introduced. This aromatic
substituent appears to be a ‘privileged scaffold’ in ARIs and has
been successfully used in the past in clinical candidates such as
ponalrestat, zenarestat, minalrestat, and ranirestat (Fig. 1). Never-
theless, inhibitory activity was decreased in the case of derivative
14. Finally, it appears that there is no tolerance for a bulkier substi-
tuent, since the biphenyl derivative 15 was inactive.
From another point of view, we sought to introduce a second
substituent on the pyrrole ring. The trifluoroacetyl group was con-
sidered as a candidate since it has already been successfully used
as a substituent in a 2,6-difluorophenol analogue.34 Fluorine has
been extensively used in medicinal chemistry and more recently
there is growing literature on fluorophilic protein environ-
ments.47–49 Although such environments have not yet been re-
ported for ALR2 we wanted to explore the possibility for such
interactions. Our results appear to be positive since activity was
significantly improved by tenfold in 30 and twentyfold in 31, com-
pared to compound 10. The monosubstituted derivatives 32 and
33, though less active, retain ALR2 inhibitory activity in the micro-
molar range. At this point, it is interesting to note that in compar-
ing the potency of 32 with its 2,6-difluoro analogue,34 we find a
similar negative trend to that of 10 and its 2,6-difluoro analogue.31
O
O
R
N
N
O
a
30
R = CF3
31 R = CHF2
F
10
F
OH
OH
Scheme 5. Synthesis of b-substituted pyrrole derivatives 32-33. (a) (RCO)2O, 1,2-
dichloroethane, reflux.
2.2. ALR2/ALR1 inhibition
ALR2 inhibitory activity was evaluated on partially purified rat
lens ALR2. It has been shown that human ALR2 exhibits 85% se-
quence homology to rat lens ALR2,44 while the catalytic active sites
of both enzymes are considered identical.45 ALR2 inhibitory activ-
ity is expressed as IC50 values and was determined for the com-
pounds that exhibited more than 50% inhibition at 100 lM.
Sorbinil was used as the reference compound. The results are
shown in Table 1.
From the structural motifs (Fig. 3) tested for inhibitory activity
against ALR2, the aldoximes (21–23, 28 and 29) were marginally
active on ALR2, while phenol 9 and 2-fluorophenol 10 exhibited
significant inhibitory activity (IC50 <100 lM). In the phenol series,
The most active compounds (IC50 <100 lM) were also assayed
Table 1
for their selectivity against ALR1. ALR2 belongs to the aldoketore-
ductase (AKR) superfamily of enzymes. 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.50 Since these closely related AKRs are responsible for
the detoxification of toxic aldehydes,51 parallel inhibition could
Inhibitory activities of the synthesized compounds against rat lens ALR2, rat kidney
ALR1 and the calculated selectivity index
Cmpd
IC50 SEMa
(l
M)
Selectivity indexb
Rat lens ALR2
Rat kidney ALR1
6
7
8
9
10
11
5.81 0.38%c
16.08 0.79%c
21.49 0.43%c
55.42 0.48
10.10 0.11
9.23 0.14
cause unwanted effects. ALR1 inhibitory activity of the most active
½ALR2ꢁ
compounds (IC50 <100 lM) is expressed as IC50 values presented
>100
>1.80
3.79
2.84
4.25
>2.40
1.43
together with the calculated selectivity index (Table 1), along with
the reference compound valproic acid.
From the obtained results it becomes clear that all compounds
tested are less active on ALR1, although in most cases not to a great
38.24 0.67
26.19 2.61
28.69 0.54
>100
12
6.75 0.03
13
14
15
16
17
21
22
23
41.67 0.07
24.84 0.33
14.16 0.07%c
37.23 1.13%c
43.66 0.14%c
23.47 0.53%c
33.68 4.73%c
35.28 0.00%c
60.53 1.29%c
36.32 0.86%c
1.04 0.02
35.50 0.05
extent. Introduction of a second substituent in the
a-position ap-
pears to significantly improve the selectivity indices, and deriva-
tives 30 and, in particular, 31 are the most selective. It should be
noted that compound 31 is also the most active ARI in this series.
Finally, considering the molecular obesity indices of ligand effi-
ciency (LE)52 and ligand efficiency-dependent lipophilicity
(LELP),53 the calculated values were 0.39 and 8.79, respectively,
for compound 31. These data, taken together with the reported
activity and selectivity, render compound 31 a ‘healthy’ lead for
further development.
28
29
30
20.38 0.51
12.10 0.46
>100
19.60
27.31
>1.36
>1.65
31
32
33
0.443 0.03
73.77 1.42
60.76 2.12
0.25 0.003
>100
Sorbinil
Valproic acid
56.1 1.56
2.3. Docking simulations
a
b
c
n = 3.
Defined as IC
% Inhibition observed at 100 lM.
[ALR1]
[ALR2]
The cross-docking protocol assessment as described in Musmuca
et al.54 proved Autodock Vina suitable as molecular docking
/IC
.
50
50

M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
2199
with low RSMD values (see Table 4, Sd) with a docking accuracy
(DA)55 of 75% and 42.9% for re-docking and cross-docking experi-
ments, respectively. It could be argued that the DA values are not
high enough; nevertheless, in trials using other docking softwares
or implementing side chain flexibility (data not shown), no better
DA was obtained.
Based on the above, the most active of the synthesized com-
pounds, 31, was cross-docked to the above 14 selected ALR2 pro-
teins. Supportively, derivative 10, lacking the difluoroacetyl
moiety, and derivative 33, lacking the benzoyl substituent, were
also cross-docked. These three compounds show good agreement
with many of the molecular features found in the 14 co-crystal-
lized ligands. The latter can be summarized in a four point pharma-
cophore comprising three hydrophobic centers (HY-1, HY-2 and
HY-3 in Fig. 4) and an electrostatic one (EL in Fig. 4). Results from
Table 5 in Sd clearly state that the more potent compounds
matched HY-1, HY-2 and EL features, while the less active com-
pounds lack overlapping with HY-1 and EL. HY-3 is the least impor-
tant feature and it is shared among the low endowed pIC50 ARIs.
From the cross-docking of 10, 31 and 33, lowest energy docked
conformations were found in 1US0, 3T42 and 1PWL, respectively
(Table 6, Sd).
At a deeper analysis, the three derivatives (10, 31 and 33) share
similar binding modes (Fig. 1, Sd), but only the most active com-
pound (31) can place optimal interactions by fulfilling both of
the ALR2 binding site sub-pockets, as well as the three most impor-
tant pharmacophoric features (HY-1, HY-2 and EL) in a fashion
similar to the reference compound 3T4 (Fig. 5).
In particular, the difluoromethyl group is involved in positively
contributing van der Waals (vdW) interactions in the ALR2 active
site with the NADP+ nicotinamide moiety and three surrounding
residues’ side chains (Trp20, Tyr48 and His110). The adjacent car-
bonyl oxygen is at a weak hydrogen bonding distance from the
Figure 4. Pharmacophore depicted from co-crystallized ARIs. HY-1, HY-2, and HY-3
(in gray) are hydrophobic zones. In red is the electrostatic feature EL. All the 14
co-crystallized ligands listed in Table 5 (Sd), are shown.
software for ALR2. Briefly, the ligands’ experimental conforma-
tions, extracted from 14 ALR2 co-crystals (Table 4, Sd), were ran-
domized in their transformation in
a dimensionless smiles
structure and a new 3-D conformation was generated. Each ran-
domized ligand was either docked in the native protein (re-dock-
ing) or in the other ALR2 crystal structures (cross-docking). For
each ligand, the lowest docking energy complex was then com-
pared with the experimental to check the ability to reproduce
the bioactive conformation in term of root mean square deviation
(RMSD) value. For the majority of ligands, the procedure proved
Autodock Vina to be able to reproduce the binding conformation
Figure 5. Proposed binding mode for derivative 31 (colored in orchid). (A) Superimposition with reference compound 3T4 (PDB ID 3T42, colored in red) and matching with
the pharmacophore features. (B) Details on the difluoroacetyl moiety binding in the pocket corresponding to EL. (C) Detail on the benzoyl group in the HY-1 pocket. (D) Details
on the 3-fluoro-4-hydroxyphenyl N-pyrrolyl substituent in the HY-2 pocket. In B, C and D the NADP+ structure and surface are reported in yellow; the ALR2 pocket
corresponding to the EL feature in green, the pocket for HY-1 in orange and for HY-2 in gray.

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M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
are expected to be less acidic than the di-substituted 2,6-difluor-
ophenols (Table 2, Sd). Indeed, the software ACD provided calcu-
lated pKa values equal to 7.68 for compound 30, and 7.73 for
compound 31 (Table 3, Sd). Less evident is the effect of fluorine
atoms to lipophilicity. Earlier aspects suggested that upon replace-
ment of hydrogen by fluorine lipophilicity remains the same.56
More recent findings, however, have shown that changes in octa-
nol–water logD values upon one H/F exchange follow a Gaussian
distribution with the maximum roughly at 0.25 log units, while
an increase up to 1.5 as well as a decrease up to ꢂ1 log units
may be observed.46 Thus, it is not easy to predict the lipophilicity
modulation between compounds 30 and 31 which differ by a gem-
inal fluorine atom. Calculations of logP by MedChem Designer and
ClogP were found equal to 3.81 and 4.11, respectively, for the tri-
fluoroacetyl derivative 30. For the difluoroacetyl derivarive 31,
however, MedChem Designer computed a significantly lower logP
equal to 3.43, while ClogP did not recognize any change in lipo-
philicity, providing logP = 4.11. (Table 3, Sd). Experimental deter-
mination of the logD-pH profile of compounds 30 and 31 over a
wide pH range proved the significant decrease in lipophilicity of
the difluoroacetyl derivative. For both compounds similar curves
were obtained, with the profile of compound 31 lying below that
of compound 30 (Graph 1, Sd). Estimation of logP and pKa by non-
linear fitting was performed according to Eq. 1, with high correla-
tion coefficients in both cases (compd 30: r = 0.997, compd 31:
r = 0.996).
logD ¼ logP ꢂ logð1 þ 10pHꢂpK Þ:
ð1Þ
a
The logP values generated for compounds 30 and 31 were 4.01
( 0.18) and 3.43 ( 0.11) respectively ( logP=0.58). The pKa values
D
of compounds 30 and 31 practically did not differ and were found
to be equal to 7.70 ( 0.22) and 7.64 ( 0.16), respectively, in good
agreement with ACD predictions. The experimental results lead
to a high logD7.4/logP ratio, 0.96 and 0.90 for compounds 30 and
31, respectively, favoring lens penetration.23For an overview of
the detailed results along with the logD/pH profiles see Sd (Table 3,
Graph 1).
2.5. Permeability indices
Figure 6. Details of 31 binding mode. (A) ALR2 active site. (B) Aromatic rich pocket.
(C) ALR2 entrance. Compound 31 is depicted with orchid colored carbon atoms and
NADP+ in yellow.
IAM chromatography provides a valuable tool for rapid estima-
tion of cell membrane permeability potential.57 As IAM stationary
phases consist of phospholipid monolayers, covalently bound to
silica, they may be considered to simulate better the cell mem-
brane than isotropic n-octanol. Retention on IAM surface has been
found to involve additional interaction forces due to the presence
of charged groups not existing in the octanol/water partitioning
system.58
e-NH His110 side chain. The benzoyl moiety overlaps with the 3T4
benzyl group, filling the aromatic rich pocket formed by Trp79,
Cys80, Trp111, Thr113, Phe115, Cys303, Tyr309, Pro310, and
Phe311 side chains. The N-pyrrolyl phenyl ring is placed in the ac-
tive site entrance channel, making vdW interactions with Trp20,
Val47, Try48, Phe122 and Trp219 side chains (Fig. 6). Finally, the
calculated Autodock Vina scoring function proved to also be quite
good in correctly predicting the activity trend (Table 6, Sd).
The ionized form of 31 was considered in the docking studies
and almost full superimposed binding mode was observed with
that of the neutral species (Fig. 2, Sd).
Extrapolated IAM retention factors (logkIwAM), corresponding to
100% aqueous phase, (logkw) were derived using three isocratic
logk values, according to Eq. 2:
logk ¼ ꢂSu þ logkw
ð2Þ
u
being the volume fraction of methanol and S the slope of the
regression curve obtained by linear regression analysis with corre-
lation coefficients (r2) 0.999 in both cases (Graph 2 in Sd). A signif-
icant higher retention was observed for the trifluoroacetyl
derivative 30 in comparison to that of the difluoroacetyl derivative
31 with extrapolated logkw values 3.40 and 2.52, respectively
2.4. Physicochemical properties
Given the significance of an ARI’s ability to be highly permeable
through biological barriers, we proceeded to estimate the compu-
tational and experimental values of pKa and logP for compounds
30 and 31.
In the series of 2-fluorophenol derivatives, the presence of a
fluorine atom in the ortho-position to the hydroxy group may af-
fect the ionization constant, increasing acidity in comparison to
the unsubstituted phenol, although 2-fluorophenol derivatives
(Dlogkw = 0.88). The large difference in IAM affinity upon replace-
ment of fluorine by hydrogen may be explained by the strong
electronegativity of the fluorine atom in proximity also to the
keto-oxygen, which may lead to additional electrostatic interactions
with the positively charged choline nitrogen of the IAM surface. In
this aspect, derivative 30 may have the tendency to stay stronger on

M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
2201
the membrane, not favoring permeability; thus the reduced, but
still high, IAM retention of compound 31 may reflect a better
permeability potential.
to bear the best characteristics for potency as well as an optimal
physicochemical profile. Development of structure activity rela-
tionships in this moiety identified compound 31 as a submicrom-
olar inhibitor, which is also selective against the closely related
enzyme ALR1. Binding mode analyses, through cross-docking, re-
vealed that 31 displays a peculiar interaction network that over-
laps with those of the experimentally co-crystallized ALR2
inhibitors. Additional experimental data for compound 31 con-
firmed that it has a pKa of 7.64, thus 64% of the compound would
be neutral at physiological pH. The ability of compound 31 to be
readily permeable through biological barriers was evaluated by
IAM chromatography and in a model of permeability through rat
jejunum. The good membrane permeation is further supported
from experimental data at the tissue level, since 31 efficiently
inhibited sorbitol formation in isolated rat lenses. Molecular obes-
ity indices (LE and LELP) are also in the preferential range, render-
ing 31 as a promising new lead for the development of non-anionic
ARIs.
Following this line of evidence, 31, which is the most active ARI
of the series, was further studied for its ability to permeate rat jeju-
num. The average permeability coefficient measured for 31
through the rat jejunum was (6.4 1.2) ꢃ 10ꢂ6 cm/s (AVG SD;
n = 4) and the permeability coefficient of the internal low-perme-
ability standard fluorescein was (5.2 0.8) ꢃ 10ꢂ6 cm/s. The high-
est permeability of the external low-permeability standard from
the list of the FDA-recommended standards measured in our
laboratory was that of furosemide (5.9 3.0) ꢃ 10ꢂ6 cm/s, and
the lowest permeability of an external high-permeability standard
was measured for propranolol (11.1 2.3) ꢃ 10ꢂ6 cm/s.59 With
these data available it would be rational to expect the in vivo
absorption of 31 in the range of 50–85%.
2.6. Inhibition of sorbitol formation in rat lenses
In diabetic patients, under conditions of hyperglycemia, intracellu-
lar accumulation of sorbitol mediated by ALR2 results in a hyperos-
motic effect, that may initiate a cascade of biochemical steps
resulting in lens opacification. Analogically, as shown in Figure 7
and Table 7 of Sd, increased sorbitol levels were recorded in the iso-
lated lenses incubated with glucose, in comparison with control incu-
bations without glucose, reflecting increased flux of glucose through
lens cytosolic ALR2. Similarly, other authors60 observed more than
10-fold increase of sorbitol levels in the isolated eye lenses incubated
with glucose under comparable conditions (50 mM glucose, 4 h
incubation). Sorbitol accumulation was significantly inhibited by
compounds 30 and 31, present in the incubation medium at a
4. Experimental section
All reagents for syntheses were purchased from Sigma–Aldrich
Co. and used without further purification, except for the solvents
used for flash chromatography and recrystallization. The chlorides
used for the Friedel–Crafts aroylation reaction were commercially
available with the exception of 4-bromo-2-fluorobenzoyl chloride
which was prepared as described before.34 Trifluoroacetic anhy-
dride for pyrrole acylation was commercially available and difluo-
roacetic anhydride was prepared as previously described from
difluoroacetic acid.61 Bromo-nitrobenzaldehydes 24 and 25 were
prepared according to previously reported methods.41,42 Phe-
nyl(1H-pyrrol-3-yl)methanone was prepared as previously de-
scribed from 1-(phenylsulfonyl)-1H-pyrrole.62 IR spectra were
taken with a Perkin–Elmer FT-IR System Spectrum BX. NMR spec-
tra were recorded at 300 MHz (1H NMR) and 75.5 MHz (13C NMR)
on a Bruker AM 300. Chemical shifts are given in d referenced to
the residual solvent peak, with TMS as an internal standard. UV–
vis measurements were taken with a UV/Vis Perkin–Elmer Spec-
trometer Lamda 20. Melting points are uncorrected and were
determined in open glass capillaries using a Mel-Temp II appara-
tus. Elemental analyses were performed at Galbraith Laboratories,
Inc., Knoxville, TN. All compounds possess a purity of at least 95%.
Flash column chromatography was carried out with Merck silica
gel 60 (230–400 mesh ASTM). TLC was run with Fluka Silica gel/
TLC-cards. All solvents used for column chromatography were rou-
tinely distilled prior to use. Petroleum ether refers to the fraction
with bp 40–60 °C, unless stated otherwise.
concentration as low as 10 lM (Fig. 7). Between the two, there is a
slight improvement with compound 31. These results, obtained at
the organ level of isolated eye lenses, point to the ready uptake of
compounds 30 and 31 by the eye lens tissue. It is important to note
at this point, that the interplay of physicochemical properties
(namely lipophilicity and ionization) with inhibitory activity/
efficiency leads to the final response at the tissue level.
3. Conclusions
In this work, an effort to discover potent and non-anionic ARIs
was realized. From the moieties tested, 2-fluorophenol appeared
1000
cmpd 30
cmpd 31
*
800
600
400
200
0
4.1. Chemistry
**
**
4.1.1. 1-(3,4-Difluorophenyl)-1H-pyrrole (1)
**
**
A mixture of 3,4-fluoroaniline (129 mg, 1 mmol), 2,5-dime-
thoxytetrahydrofuran (213 mg, 1.61 mmol) and 4-chloropyridine
hydrochloride (236 mg, 1.57 mmol) in 1,4-dioxane (17 mL) was re-
fluxed under a nitrogen atmosphere for 4 h. The mixture was then
cooled to room temperature and concentrated under reduced pres-
sure. The residue was then diluted in CH2Cl2, sonicated for 15 min,
and filtered. The procedure was repeated one more time and the
organic extracts were concentrated under reduced pressure. The
residue was flash chromatographed with petroleum ether to pro-
vide the title compound (136 mg, 76%). An analytical sample was
obtained by recrystallization from petroleum ether (bp 80–
10 μΜ
50 μΜ
- Glucose
+ Glucose
Figure 7. Effect of compounds 30 and 31 on sorbitol accumulation in isolated rat
lenses cultivated with high glucose. Glucose, 50 mM; time of incubation, 3 h; 37 °C.
Results are mean values SEM from 5 to 22 independent incubations. ⁄p < 0.001
versus (ꢂ)glucose; ⁄⁄p < 0.001 versus (+)glucose.
100 °C). Mp 62–64 °C [lit.63 62–63 °C]; IR (KBr) 1614 (C@C) cmꢂ1
;
1H NMR (CDCl3) d 6.33–6.40 (m, 2H, pyrrolyl H-3, H-4), 6.98–
7.05 (m, 2H, pyrrolyl H-2, H-5), 7.10–7.17 (m, 1H, phenyl H-5),

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M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
7.18–7.27 (m, 2H, phenyl H-2, H-6). Anal. Calcd for C10H7F2N: C,
67.04; H, 3.94; N, 7.82. Found: C, 66.80; H, 3.97; N, 7.74.
1H NMR (CDCl3, DMSO-d6) d 6.66–6.70 (m, 1H, pyrrolyl H-4),
6.81 (d, J = 8.8 Hz, 2H, phenyl H-3, H-5), 6.86–6.91 (m, 1H, pyrrolyl
H-5), 7.12 (d, J = 8.8 Hz, 2H, phenyl H-2, H-6), 7.31-7.46 (m, 4H,
benzoyl H-3, H-4, H-5 and pyrrolyl H-2), 7.74 (d, J = 6.8 Hz, 2H,
benzoyl H-2, H-6), 8.95 (s, 1H, OH); Anal. Calcd for C17H13
NO2⁄⁄0.01CH2Cl2: C, 77.35; H, 4.97; N: 5.30. Found: C, 76.99; H,
4.96; N, 5.07.
4.1.2. General procedure 1. Friedel–Crafts aroylation reaction.
Selective formation of b-pyrrolyl derivatives.
In a suspension of anhydrous AlCl3 (187 mg, 1.40 mmol) in
1,2-dichloroethane (7.5 mL) the appropriate aroyl chloride
(1.31 mmol) was added slowly and stirred at room temperature
and under a nitrogen atmosphere. After 15 min, a solution of the
appropriate pyrrolyl derivative 1–5 (0.57 mmol) in 1,2-dichloro-
ethane (1.5 mL) was added and the reaction was stirred for 24–
72 h. The reaction was then quenched with ice and water and
the product was extracted with CHCl3 (2 ꢃ 20 mL). The combined
organic extracts were washed with saturated NaCl solution, dried
over anhydrous Na2SO4 and the solvents were removed under re-
duced pressure. The residue was dissolved in 1,4-dioxane
(14 mL) and a solution of 5% NaOH (14 mL) was added. The mix-
ture was stirred at room temperature and under a nitrogen atmo-
sphere overnight. The mixture was then concentrated to half its
volume, H2O (14 mL) was added, and it was cooled and acidified
with concentrated HCl. Afterwards, the mixture was extracted with
CH2Cl2 (2 ꢃ 25 mL) and the organic extracts were washed with 1%
NaHCO3, saturated NaCl solution and dried over Na2SO4. The sol-
vent was evaporated under reduced pressure and the residue
was flash chromatographed with petroleum ether/EtOAc (18:1–
2:1). Analytical samples were obtained by recrystallization from
CH2Cl2 (6–9, 12–17) or CH2Cl2/petroleum ether (10 and 11).
4.1.2.5. [1-(3-Fluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]phenyl-
methanone (10).
Prepared from 536 and benzoyl chloride
according to general procedure l with a reaction time of 48 h
(115 mg, 72%). Mp 172–173 °C; IR (KBr) 3118 (OAH), 1593 (C@O)
cmꢂ1 1H NMR (CDCl3, DMSO-d6) d 6.74–6.79 (m, 1H, pyrrolyl
;
H-4), 6.92–7.11 (m, 4H, phenyl H-2, H-5, H-6, and pyrrolyl H-5),
7.38–7.53 (m, 4H, benzoyl H-3, H-4, H-5, and pyrrolyl H-2), 7.80
(d, J = 6.8 Hz, 2H, benzoyl H-2, H-6); Anal. Calcd for C17H12FNO2:
C, 72.59; H, 4.30; N, 4.98. Found: C, 72.22; H, 4.29; N, 4.67.
4.1.2.6.
[1-(3-Fluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]
Prepared from 536 and
(4-methoxyphenyl)methanone (11).
4-methoxybenzoyl chloride according to general procedure l with
a reaction time of 4.5 h (91 mg, 58%). Mp 188–191 °C; IR (KBr)
3141 (OAH), 1599 (C@O) cmꢂ1 1H NMR (CDCl3) d 3.85 (s, 3H,
;
CH3), 6.72–6.79 (m, 1H, pyrrolyl H-4), 6.88–7.14 (m, 6H, phenyl
H-2, H-5, H-6, benzoyl H-3, H-5, and pyrrolyl H-5), 7.42–7.51 (m,
1H, pyrrolyl H-2), 7.87 (d, J = 9.0 Hz, 2H, benzoyl H-2, H-6), 8.91
(br s, 1H, OH); Anal. Calcd for C18H14FNO3: C, 69.45; H, 4.53; N,
4.50. Found: C, 69.17; H, 4.54; N, 4.33.
4.1.2.1. [1-(3,4-Difluorophenyl)-1H-pyrrol-3-yl]phenylmetha-
none (6).
Prepared from 1 and benzoylchloride according to
4.1.2.7.
[1-(3-Fluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]
Prepared from 536 and
general procedure 1 with a reaction time of 48 h, but the hydrolysis
(3-methoxyphenyl)methanone (12).
step was omitted (73 mg, 45%). Mp 97–97.5 °C; IR (KBr) 1632
3-methoxybenzoyl chloride according to general procedure l with
(C@O) cmꢂ1
;
1H NMR (CDCl3) d 6.87–6.99 (m, 1H, pyrrolyl H-4),
a reaction time of 48 h (142 mg, 80%). Mp 131 °C; IR (KBr) 3125
7.03–7.14 (m, 1H, pyrrolyl H-5), 7.16–7.25 (m, 1H, phenyl H-5),
7.27–7.40 (m, 2H, phenyl H-2, H-6), 7.47–7.71 (m, 4H, benzoyl
H-3, H-4, H-5 and pyrrolyl H-2), 7.89 (d, J = 6.9 Hz, 2H, benzoyl
H-2, H-6). Anal. Calcd for C17H11F2NO: C, 72.08; H, 3.91; N, 4.94.
Found: C, 71.78; H, 3.92; N, 4.91.
(OAH), 1591 (C@O) cmꢂ1 1H NMR (CDCl3, DMSO-d6) d 3.79 (s,
;
3H, CH3), 6.71–6.77 (m, 1H, pyrrolyl H-4), 6.91–7.11 (m, 5H, phenyl
H-2, H-5, H-6, benzoyl H-4, and pyrrolyl H-5), 7.27–7.39 (m, 3H,
benzoyl H-2, H-5, H-6), 7.41–7.46 (m, 1H, pyrrolyl H-2), 9.25 (br
s, 1H, OH); Anal. Calcd for C18H14FNO3ꢀ0.06CH2Cl2: C, 68.56; H,
4.50; N, 4.43. Found: C, 68.22; H, 4.49; N, 4.31.
4.1.2.2.
none (7).
[1-(2-Hydroxyphenyl)-1H-pyrrol-3-yl]phenylmetha-
Prepared from 236 and benzoyl chloride according
4.1.2.8. [1-(3-Fluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl](4-(tri-
to general procedure l with a reaction time of 48 h (120 mg,
fluoromethoxy)phenyl)methanone (13).
Prepared from 536
80%). Mp 137–138 °C; IR (KBr) 3120 (OAH), 1592 (C@O) cmꢂ1
;
and 4-trifluoromethoxybenzoyl chloride according to general pro-
cedure l with a reaction time of 48 h (144 mg, 70%). Mp 138.5–
1H NMR (CDCl3) d 6.80–6.85 (m, 1H, pyrrolyl H-4), 6.95–7.02 (m,
2H, phenyl H-3, H-6), 7.18–7.33 (m, 3H, pyrrolyl H-5, and phenyl
H-4, H-5), 7.37 (br s, 1H, OH, exchangeable with D2O), 7.44–7.61
(m, 3H, benzoyl H-3, H-4, H-5), 7.76–7.82 (m, 1H, pyrrolyl H-2),
7.91 (d, J = 8.1 Hz, 2H, benzoyl H-2, H-6); Anal. Calcd for
139.5 °C; IR (KBr) 3167 (OAH), 1610 (C@O) cmꢂ1 1H NMR (CDCl3,
:
DMSO-d6) d 6.70–6.77 (m, 1H, pyrrolyl H-4), 6.91–7.12 (m, 4H,
phenyl H-2, H-5, H-6, and pyrrolyl H-5), 7.26 (d, J = 8.6 Hz, 2H, ben-
zoyl H-3, H-5), 7.40–7.47 (m, 1H, pyrrolyl H-2), 7.86 (d, J = 8.6 Hz,
2H, benzoyl H-2, H-6); Anal. Calcd for C18H11F4NO3ꢀ0.03CH2Cl2: C,
58.87; H, 3.03; N, 3.81. Found: C, 58.52; H, 3.00; N, 3.64.
C17H13NO2: C, 77.55; H, 4.98; N, 5.32. Found: C, 77.58; H, 4.98;
N, 4.95.
4.1.2.3.
none (8).
[1-(3-Hydroxyphenyl)-1H-pyrrol-3-yl]phenylmetha-
Prepared from 336 and benzoyl chloride according
4.1.2.9.
[1-(3-Fluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]
(4-bromo-2-fluorophenyl)methanone (14).
Prepared from
to general procedure l with a reaction time of 48 h (110 mg,
536 and 4-bromo-2-fluorobenzoyl chloride34 according to general
73%). Mp 148–149.5 °C; IR (KBr) 3182 (OAH), 1606 (C@O) cmꢂ1
;
procedure l with a reaction time of 72 h (135 mg, 71%). Mp 181–
1H NMR (CDCl3) d 6.80–6.90 (m, 2H, pyrrolyl H-4 and phenyl H-
4), 6.92–7.03 (m, 2H, phenyl H-2, H-6), 7.03–7.11 (m, 1H, pyrrolyl
H-5), 7.24–7.35 (m, 1H, phenyl H-5), 7.42–7.66 (m, 4H, benzoyl H-
3, H-4, H-5, and pyrrolyl H-2), 7.90 (d, J = 6.9 Hz, 2H, benzoyl H-2,
H-6); Anal. Calcd for C17H13NO2: C, 77.55; H, 4.98; N, 5.32. Found:
C, 77.54; H, 4.96; N, 4.98.
183 °C; IR (KBr) 3178 (OAH), 1610 (C@O) cmꢂ1 1H NMR (CDCl3,
;
DMSO-d6) d 6.62–6.71 (m, 1H, pyrrolyl H-4), 6.87–7.08 (m, 4H,
pyrrolyl H-5, and phenyl H-2, H-5, H-6), 7.30–7.43 (m, 4H, benzoyl
H-3, H-5, H-6, and pyrrolyl H-2), 9.15 (br s, 1H, OH); Anal. Calcd for
C17H10BrF2NO2: C, 53.99; H, 2.67; N, 3.70. Found: C, 53.69; H, 2.68;
N, 3.58.
4.1.2.4.
none (9).
[1-(4-Hydroxyphenyl)-1H-pyrrol-3-yl]phenylmetha-
Prepared from 436 and benzoyl chloride according
4.1.2.10. [1,10-Biphenyl]-4-yl(1-(3-fluoro-4-hydroxyphenyl)-1H-
pyrrol-3-yl)methanone (15).
Prepared from 536 and [1,10-
to general procedure l with a reaction time of 48 h (114 mg,
biphenyl]-4-carbonyl chloride according to general procedure l
with a reaction time of 24 h (157 mg, 77%). Mp 195–197 °C; IR
76%). Mp 190–191 °C; IR (KBr) 3185 (OAH), 1611 (C@O) cmꢂ1
;

M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
2203
ꢂ1
1H NMR (CDCl3, DMSO-d6) d
saturated NaCl solution, dried over MgSO4 and the solvents were
removed under reduced pressure. The residue was flashed chro-
matographed with petroleum ether/EtOAc (6:1–5:1). Analytical
samples were obtained by recrystallization from CH2Cl2/petroleum
ether.
@
(KBr) 3245 (O H), 1602 (C@O) cm
:
@
6.77–6.82 (m, 1H, pyrrolyl H-4), 6.94–7.03 (m, 3H, pyrrolyl H-5,
and phenyl H-2, H-5), 7.10 (d, J = 11.2 Hz, 1H, phenyl H-6), 7.31–
7.38 (m, 1H, biphenyl H0-4), 7.39–7.46 (m, 2H, biphenyl H0-3, H0-
5), 7.47–7.51 (m, 1H, pyrrolyl H-2), 7.54–7.69 (m, 4H, biphenyl
H0-2, H0-6, H-2, H-6), 7.90 (d, J = 8.3 Hz, 2H, biphenyl H-3, H-5);
Anal. Calcd for C23H16FNO2ꢀ0.22CH2Cl2: C, 74.16; H, 4.41; N, 3.72.
Found: C, 73.87; H, 4.39; N, 3.63.
4.1.4.1. 3-(3-Benzoyl-1H-pyrrol-1-yl)-2-hydroxybenzaldehyde
(18).
(92 mg, 63%). Mp 71–71.5 °C; IR (KBr) 2850 (CAHald), 1636 (C@O)
cmꢂ1 1H NMR (CDCl3) d 6.88–6.93 (m, 1H, pyrrolyl H-4), 7.08–
Prepared from 7 according to general procedure 3
4.1.3. General procedure 2. Friedel–Crafts aroylation reaction.
;
Formation of both
a
- and b-pyrrolyl derivatives
7.12 (m, 1H, pyrrolyl H-5), 7.15 (t, J = 7.8 Hz, 1H, phenyl H-5),
7.44–7.57 (m, 3H, benzoyl H-3, H-4, H-5), 7.63 (d, J = 7.8 Hz, 3H,
phenyl H-4, H-6, pyrrolyl H-2), 7.91 (d, J = 6.8 Hz, 2H, benzoyl
H-2, H-6), 10.00 (s, 1H, OH), 11.63 (s, 1H, CHO); Anal. Calcd for
In a suspension of anhydrous AlCl3 (82 mg, 0.615 mmol) in 1,2-
dichloroethane (5 mL) the appropriate aroyl chloride (1.149 mmol)
was added slowly and stirred at room temperature and under a
nitrogen atmosphere. After 15 min, a solution of 536 (0.513 mmol)
in 1,2-dichloroethane (2.5 mL) was added and the reaction was
stirred for 4.5 (11/17) or 24 h (10/16). The reaction was then
quenched with ice and water and the product was extracted with
CHCl3 (2 ꢃ 20 mL). The combined organic extracts were washed
with saturated NaCl solution, dried over anhydrous Na2SO4 and
the solvents were removed under reduced pressure. Hydrolysis
proceeded as described above in general procedure 1. The residue
was flash chromatographed with petroleum ether/EtOAc (7:1–3:1)
so that both regio-isomers could be isolated. Analytical samples
were obtained by recrystallization from CH2Cl2/petroleum ether.
C
18H13NO3ꢀ0.1CH2Cl2: C, 72.51; H, 4.44; N, 4.67. Found: C, 72.22;
H, 4.42; N, 4.62.
4.1.4.2. 4-(3-Benzoyl-1H-pyrrol-1-yl)-2-hydroxybenzaldehyde
(19).
(75 mg, 51%). Mp 138–140 °C; IR (KBr) 3445 (OAH), 2923 (CAHald),
1636 (C@O) cmꢂ1 1H NMR (CDCl3) d 6.90–6.95 (m, 1H, pyrrolyl
Prepared from 8 according to general procedure 3
;
H-4), 7.04–7.07 (m, 1H, phenyl H-3), 7.11 (dd, J = 8.4, 2.1 Hz, 1H,
phenyl H-5), 7.19–7.24 (m, 1H, pyrrolyl H-5), 7.47–7.72 (m, 5H,
phenyl H-6, benzoyl H-3, H-4, H-5, and pyrrolyl H-2), 7.89 (d,
J = 6.9 Hz, 2H, benzoyl H-2, H-6), 9.91 (s, 1H, OH), 11.32 (s, 1H,
CHO); Anal. Calcd for C18H13NO3ꢀ0.01CH2Cl2: C, 74.04; H, 4.49; N,
4.79. Found: C, 73.77; H, 4.65; N, 4.64.
4.1.3.1. [1-(3-Fluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]phenyl-
methanone (10).
Prepared from 536 and benzoyl chloride
according to general procedure 2 (59 mg, 41%). Analytical data
were similar to the sample obtained by general procedure 1.
4.1.4.3. 5-(3-Benzoyl-1H-pyrrol-1-yl)-2-hydroxybenzaldehyde
(20).
(60 mg, 41%). Mp 107–107.5 °C; IR (KBr): 3447 (OAH), 2850
(CAHald), 1666, 1652, 1631 (C@O) cmꢂ1 1H NMR (CDCl3) d 6.87–
Prepared from 9 according to general procedure 3
4.1.3.2. [1-(3-Fluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl]phenyl-
;
methanone (16).
according to general procedure 2 (52 mg, 36%). Mp 129–130 °C;
IR (KBr) 3199 (OAH), 1609 (C@O) cmꢂ1 1H NMR (CDCl3)
Prepared from 536 and benzoyl chloride
6.92 (m, 1H, pyrrolyl H-4), 7.02–7.08 (m, 1H, pyrrolyl H-5), 7.14
(d, J = 9.7 Hz, 1H, phenyl H-3), 7.46–7.65 (m, 6H, phenyl H-4, H-
6, benzoyl H-3, H-4, H-5, and pyrrolyl H-2), 7.90 (d, J = 8.0 Hz,
2H, benzoyl H-2, H-6), 9.97 (s, 1H, OH), 11.01 (s, 1H, CHO); Anal.
Calcd for C18H13NO3ꢀ0.03CH2Cl2: C, 73.70; H, 4.48; N, 4.77. Found:
C, 73.74; H, 4.44; N, 4.58.
;
d 6.26–6.43 (m, 2H, pyrrolyl H-4, OH, exchangeable with D2O),
6.83–7.13 (m, 5H, phenyl H-2, H-5, H-6, and pyrrolyl H-3, H-5),
7.40–7.64 (m, 3H, benzoyl H-3, H-4, H-5), 7.91 (d, J = 7.0 Hz, 2H,
benzoyl H-2, H-6); Anal. Calcd for C17H12FNO2: C, 72.59; H, 4.30;
N, 4.98. Found: C, 72.41; H, 4.30; N, 4.64.
4.1.5. General procedure 4. Formation of salicylaldoximes
To a suspension of 18–20 (70 mg, 0.24 mmol) in EtOH (4.6 mL) a
solution of NH2OHꢀHCl (33 mg, 0.48 mmol) in water (1.1 mL) was
added and the resulting mixture was heated to 50 °C and under a
nitrogen atmosphere for 1 h. The solvents were then removed
under reduced pressure and the residue was diluted with water
and filtered. After washing the filtrate with water repeatedly it
was dried under reduced pressure and an analytical sample was
obtained by recrystallization from iPrOH (21) or iPrOH/H2O (22
and 23).
4.1.3.3.
[1-(3-Fluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]
Prepared from 536 and
(4-methoxyphenyl)methanone (11).
4-methoxybenzoyl chloride according to general procedure 2
(65 mg, 41%). Analytical data were similar to the sample obtained
by general procedure 1.
4.1.3.4.
[1-(3-Fluoro-4-hydroxyphenyl)-1H-pyrrol-2-yl]
Prepared from 536 and
(4-methoxyphenyl)methanone (17).
4-methoxybenzoyl chloride according to general procedure 2
(48 mg, 30%). Mp 182–184 °C; IR (KBr) 3134 (OAH), 1605 (C@O)
4.1.5.1.
ybenzaldoxime (21).
(E)-3-(3-Benzoyl-1H-pyrrol-1-yl)-2-hydrox-
Prepared from 18 according to general
cmꢂ1
;
1H NMR (CDCl3, DMSO-d6) d 3.82 (s, 3H, CH3), 6.19–6.26
(m, 1H, pyrrolyl H-4), 6.72–6.77 (m, 1H, phenyl H-5), 6.82–6.99
(m, 6H, phenyl H-2, H-6, benzoyl H-3, H-5, and pyrrolyl H-3, H-
5), 7.81 (d, J = 8.9 Hz, 2H, benzoyl H-2, H-6), 8.81 (br s, 1H, OH).
Anal. Calcd for C18H14FNO3ꢀ0.04CH2Cl2: C, 68.85; H, 4.51; N, 4.45.
Found: C, 68.74; H, 4.56; N, 4.35.
procedure 4 (37 mg, 50%). Mp 159–160 °C; IR (KBr) 3225 (OAH),
1617 (C@O) cmꢂ1 1H NMR (acetone-d6) d 6.74–6.83 (m, 1H, pyrr-
;
olyl H-4), 7.09 (t, J = 7.8 Hz, 1H, phenyl H-5), 7.18–7.24 (m, 1H,
pyrrolyl H-5), 7.42–7.62 (m, 5H, phenyl H-4, H-6, and benzoyl
H-3, H-4, H-5), 7.68–7.73 (m, 1H, pyrrolyl H-2), 7.90 (d,
J = 6.7 Hz, 2H, benzoyl H-2, H-6), 8.49 (s, 1H, CHN), 10.90 (br s,
2H, OH, NOH); Anal. Calcd for C18H14N2O3ꢀ0.2 C3H8O: C, 70.18; H,
4.94; N, 8.80. Found: C, 69.89; H, 4.58; N, 8.58.
4.1.4. General procedure 3. Formylation reaction
A mixture of the appropriate [1-(hydroxyphenyl)-1H-pyrrol-3-
yl]phenylmethanone 7–9 (132 mg, 0.5 mmol), anhydrous MgCl2
(71 mg, 0.75 mmol), dry (Na) Et3N (190 mg, 1.875 mmol) and dry
(P2O5) paraformaldehyde (149 mg, 3.375 mmol) in dry (CaH2, MS
3 Å) CH3CN (2.5 mL) was refluxed under a nitrogen atmosphere
for 72 h. Then 10% HCl (0.5 mL) was added followed by extraction
with AcOEt (3 ꢃ 10 mL). The organic extracts were washed with
4.1.5.2.
ybenzaldoxime (22).
(E)-4-(3-Benzoyl-1H-pyrrol-1-yl)-2-hydrox-
Prepared from 19 according to general
procedure 4 (37 mg, 50%). Mp 178–179 °C; IR (KBr) 3334 (OAH),
1625 (C@O) cmꢂ1 1H NMR (acetone-d6) d 6.79–6.85 (m, 1H, pyrr-
olyl H-4), 7.17–7.22 (m, 1H, phenyl H-3), 7.26 (dd, J = 8.3, 2.3 Hz,
;

2204
M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
1H, phenyl H-5), 7.41–7.65 (m, 5H, phenyl H-6, benzoyl H-3, H-4,
H-5 and pyrrolyl H-5), 7.82–7.95 (m, 3H, pyrrolyl H-2 and benzoyl
H-2, H-6), 8.42 (s, 1H, CHN), 10.41 (br s, 1H, OH), 10.66 (br s, 1H,
NOH); Anal. Calcd for C18H14N2O3ꢀ0.2H2O: C, 69.76; H, 4.68; N,
9.04. Found: C, 69.55; H, 4.68; N, 8.86.
cmꢂ1 1H NMR (acetone-d6) d 6.85–6.92 (m, 1H, pyrrolyl H-4),
;
7.48–7.66 (m, 4H, benzoyl H-3, H-4, H-5, pyrrolyl H-5), 7.93 (d,
J = 6.9 Hz, 2H, benzoyl H-2, H-6), 8.03–8.17 (m, 3H, phenyl H-4,
H-5, and pyrrolyl H-2), 8.32–8.39 (m, 1H, phenyl H-3), 8.50 (s,
1H, CHN), 11.37 (s, 1H, OH); Anal. Calcd for
C18H13N3O4
0.07C3H8O: C, 64.42; H, 4.03; N, 12.38. Found: C, 64.54; H, 3.99;
N, 11.99.
4.1.5.3.
ybenzaldoxime (23).
(E)-5-(3-Benzoyl-1H-pyrrol-1-yl)-2-hydrox-
Prepared from 20 according to general
procedure 4 (55 mg, 75%). Mp 148–149 °C; IR (KBr) 3316 (OAH),
4.1.7.2. (E)-5-(3-Benzoyl-1H-pyrrol-1-yl)-2-nitrobenzaldoxime
1613 (C@O) cmꢂ1 1H NMR (acetone-d6) d 6.73–6.83 (m, 1H, pyrr-
;
(29).
Prepared from 27 according to general procedure 6
olyl H-4), 6.99–7.09 (m, 1H, phenyl H-3), 7.21–7.33 (m, 1H, pyrrol-
yl H-5), 7.39–7.75 (m, 6H, phenyl H-4, H-6, benzoyl H-3, H-4, H-5,
and pyrrolyl H-2), 7.78–7.92 (m, 2H, benzoyl H-2, H-6), 8.46 (s, 1H,
CHN), 10.17 (br s, 1H, OH), 10.81 (br s, 1H, NOH); Anal. Calcd for
(63 mg, 60%). Mp 183–185 °C; IR (KBr) 3224 (OAH), 1612 (C@O)
cmꢂ1; 1H NMR (acetone-d6/MeOH-d4) d 6.88–6.93 (m, 1H, pyrrolyl
H-4), 7.50–7.68 (m, 4H, benzoyl H-3, H-4, H-5, and pyrrolyl H-5),
7.85–7.94 (m, 3H, phenyl H-4, benzoyl H-2, H-6), 7.96–7.99 (m,
1H, pyrrolyl H-2), 8.14 (d, J = 2.6 Hz, 1H, phenyl H-6), 8.23 (d,
J = 8.9 Hz, 1H, phenyl H-3), 8.60 (s, 1H, CHN). Anal. Calcd for
C
18H14N2O3ꢀ0.25C3H8O: C, 70.08; H, 5.02; N, 8.72. Found: C,
69.75; H, 4.82; N, 8.31.
C
18H13N3O4ꢀ0.02C3H8O: C, 64.46; H, 3.94; N, 12.49. Found: C,
4.1.6. General procedure 5. CAN bond formation with Pd(OAc)2
64.24; H, 4.10; N, 12.10.
and xanthphos
A mixture of the appropriate 2-nitrobenzaldehyde (24,41 2542
)
4.1.8. General procedure 7. Microwave-assisted pyrrole
acylation
(378 mg, 1.65 mmol), K2CO3 (622 mg, 4.5 mmol), phenyl(1H-pyr-
rol-3-yl)methanone62 (257 mg, 1.5 mmol), Pd(OAc)2 (3.37 mg,
0.015 mmol) and xanthphos (26 mg, 0.045 mmol) in p-xylene
(4 mL) was degased and heated to 120 °C under a nitrogen atmo-
sphere for 24 h. Then it was cooled, water (10 mL) was added,
followed by extraction with EtOAc (2 ꢃ 25 mL). The combined
organic extracts were washed with brine, dried over MgSO4 and
the solvents were removed under reduced pressure. The residue
was flashed chromatographed with petroleum ether/EtOAc [2:1
(26), 4:1 (27)]. Analytical samples were obtained by recrystalliza-
tion from EtOAc/petroleum ether (26) or EtOAc (27).
A
solution of [1-(3-fluoro-4-hydroxyphenyl)-1H-pyrrol-3-
yl]phenylmethanone (10) (100 mg, 0.36 mmol) and the appropri-
ate fluoroacetic anhydride (3.88 mmol) in 1,2-dichloroethane
(1 mL) were added to a 10-mL vial, sealed with a crimp cap and
placed in a CEM Discover Lab Mate reactor microwave cavity. After
irradiation at 180 °C for 10 ꢃ 30 min (Power: 300 Watt), with cool-
ing in the intermediate stages between two cycles, the reaction
mixture was quenched with ice/water, followed by extraction with
CH2Cl2 (2 ꢃ 5 mL). The organic extracts were washed with satu-
rated NaCl solution, dried over Na2SO4. The solvents were evapo-
rated under reduced pressure and the residue was flash
chromatographed with petroleum ether/EtOAc [7:1 (30), 7:2
(31)]. Analytical samples were obtained by recrystallization from
CH2Cl2/petroleum ether.
4.1.6.1.
(26).
4-(3-Benzoyl-1H-pyrrol-1-yl)-2-nitrobenzaldehyde
Prepared from 2441 according to general procedure 5
(265 mg, 55%). Mp 134–135 °C; IR (KBr) 2876 (CAHald), 1696
(C@Oald), 1636 (C@Oket) cmꢂ1 1H NMR (CDCl3) d 6.96–7.01 (m,
;
1H, pyrrolyl H-4), 7.24–7.30 (m, 1H, pyrrolyl H-5), 7.49–7.65 (m,
3H, benzoyl H-3, H-4, H-5), 7.73–7.77 (m, 1H, pyrrolyl H-2), 7.85
(dd, J = 8.4, 2.2 Hz, 1H, phenyl H-5), 7.91 (d, J = 6.9 Hz, 2H, benzoyl
H-2, H-6), 8.12–8.18 (m, 2H, phenyl H-3, H-5), 10.45 (s, 1H, CHO).
Anal. Calcd for C18H12N2O4ꢀ0.05EtOAc: C, 67.32; H, 3.85; N, 8.63.
Found: C, 66.92; H, 3.79; N, 8.37.
4.1.8.1. 1-[4-Benzoyl-1-(3-fluoro-4-hydroxyphenyl)-1H-pyrroly-
2-yl]-2,2,2-trifluoroethanone (30).
Prepared from trifluoro-
acetic anhydride according to general procedure 7 (98 mg, 73%).
Mp 142.5–143.5 °C; IR (KBr): 3220 (OAH), 1702 (C@OCF3CO), 1630
(C@Obenzoyl) cmꢂ1 1H NMR (CDCl3) d 5.51 (br s, 1H, OH), 6.99–
;
7.06 (m, 1H, pyrrolyl H-3), 7.08–7.17 (m, 2H, phenyl H-5, H-6),
7.43–7.64 (m, 3H, benzoyl H-3, H-4, H-5), 7.65–7.70 (m, 1H, phenyl
H-2), 7.76–7.79 (m, 1H, pyrrolyl H-5), 7.89 (d, J = 6.9 Hz, 2H, ben-
zoyl H-2, H-6); 13C NMR (CDCl3) d 114.2 (d, J = 21.0 Hz), 117.5 (d,
J = 2.7 Hz), 118.8 (dd, J = 350.9, 235.6 Hz, CF3), 122.7 (d,
J = 3.6 Hz), 124.8 (dd, J = 7.7, 3.8 Hz), 125.5, 125.8, 128.7, 128.9,
131.2 (d, J = 8.6 Hz), 132.6, 137.7, 138.2, 144.8 (d, J = 14.0 Hz),
150.3 (d, J = 241.3 Hz, phenyl C-F), 170.3 (dd, J = 35.3, 3.5 Hz,
CF3CO), 189.1; Anal. Calcd for C19H11F4NO3ꢀ0.03CH2Cl2: C, 60.17;
H, 2.93; N, 3.69. Found: C, 59.81; H, 2.92; N, 3.61.
4.1.6.2.
(27).
5-(3-Benzoyl-1H-pyrrol-1-yl)-2-nitrobenzaldehyde
Prepared from 2542 according to general procedure 5
(326 mg, 68%). Mp 178–181 °C; IR (KBr) 2890 (CAHald), 1696
(C@Oald), 1631 (C@Oket
)
cmꢂ1 1H NMR (CDCl3-DMSO-d6)
;
d 6.81–6.88 (m, 1H, pyrrolyl H-4), 7.26–7.33 (m, 1H, pyrrolyl
H-5), 7.40–7.54 (m, 3H, benzoyl H-3, H-4, H-5), 7.74–7.93 (m,
5H, phenyl H-4, H-6, benzoyl H-2, H-6, and pyrrolyl H-2), 8.24 (d,
J = 8.8 Hz, 1H, phenyl H-3), 10.40 (s, 1H, CHO); Anal. Calcd for
C18H12N2O4: C, 67.50; H, 3.78; N, 8.75. Found: C, 67.33; H, 3.86;
N, 8.40.
4.1.8.2. 1-(4-Benzoyl-1-(3-fluoro-4-hydroxyphenyl)-1H-pyrrol-
4.1.7. General procedure 6. Formation of nitroaldoximes
To a solution of the appropriate (3-benzoyl-1H-pyrrol-1-yl)-2-
nitrobenzaldehyde 26 or 27 (100 mg, 0.31 mmol) in dry (MS 4 Å)
EtOH (4 mL), NH2OHꢀHCl (67 mg, 0.96 mmol) and CH3COONa
(104 mg, 1.26 mmol) were added and the mixture was stirred un-
der a nitrogen atmosphere for 4 h. The solvent was then removed
under reduced pressure, water was added and the product was iso-
lated by filtration after washing repeatedly with water. Analytical
samples were obtained by recrystallization from iPrOH.
2-yl)-2,2-difluoroethanone (31).
Prepared from difluoroace-
tic anhydride according to general procedure 7 (80 mg, 63%). Mp
153–155 °C; IR (KBr): 3154 (OAH), 1695 (C@OCHF2CO), 1617
(C@Obenzoyl
)
cmꢂ1
;
1H NMR (CDCl3-DMSO-d6)
d
6.11 (t,
J = 53.9 Hz, 1H, CHF2), 6.92–6.98 (m, 1H, pyrrolyl H-3), 7.00–7.11
(m, 2H, phenyl H-5, H-6), 7.47–7.65 (m, 4H, benzoyl H-3, H-4,
H-5 and phenyl H-2), 7.56–7.82 (m, 1H, pyrrolyl H-5), 7.87 (d,
J = 7.0 Hz, 2H, benzoyl H-2, H-6); 13C NMR (CDCl3-DMSO-d6) d
110.4 (t, J = 253.7 Hz, CHF2–), 114.4 (d, J = 21.3 Hz), 117.8 (d,
J = 3.5 Hz), 122.1 (d, J = 3.4 Hz), 123.4 (t, J = 4.6 Hz), 125.0, 127.2,
128.5, 128.8, 130.3 (d, J = 8.5 Hz), 132.3, 137.0, 138.5, 146.0 (d,
J = 12.7 Hz), 150.8 (d, J = 244.0 Hz, phenyl C-F), 177.2 (t,
4.1.7.1. (E)-4-(3-Benzoyl-1H-pyrrol-1-yl)-2-nitrobenzaldoxime
(28).
Prepared from 26 according to general procedure 6
(54 mg, 47%). Mp 225–227 °C; IR (KBr) 3282 (OAH), 1617 (C@O)

M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
2205
J = 25.3 Hz, CHF2CO), 189.2; Anal. Calcd for C19H12F3NO3ꢀ0.08
short term cultivations were preferred to avoid substantial perme-
ability changes of the eye lenses. The washed lenses were kept
deep-frozen for sorbitol determination which was performed as
described before.31 Details on the procedure can be found in Sd.
CH2Cl2: C, 62.60; H, 3.35; N, 3.83. Found: C, 62.54; H, 3.43; N, 3.99.
4.1.9. General procedure 8. Pyrrole acylation at the a-position
To a solution of 536 (91 mg, 0.513 mmol) in 1,2-dichloroethane
(1.5 mL), the appropriate fluoroacetic anhydride (5.61 mmol) was
added and the mixture was refluxed for 4 h under a nitrogen atmo-
sphere. The reaction mixture was then quenched with ice/water,
followed by extraction with CH2Cl2 (3 ꢃ 10 mL). The organic ex-
tracts were washed with saturated NaCl solution, dried over Na2
SO4. The solvents were evaporated under reduced pressure and
the residue was flash chromatographed with petroleum ether/
EtOAc [6:1 (32), 16:3 (33)]. Analytical samples were obtained by
recrystallization from CH2Cl2/petroleum ether (32) or CH2Cl2 (33).
4.3. Computational methods, docking simulations
MarvinSketch [MarvinSketch 5.11 2012, ChemAxon (http://
www.chemaxon.com)] was used for drawing the chemical struc-
tures, which were then converted in 3D-Structures with OpenBabel
2.3.2,64 employing MMFF94 force field.65
The structure of human ALR2, complexed with several inhibi-
tors, was used in the docking calculations. Although the in vitro
inhibition assays of our compounds were conducted on rat ALR2,
the use of the human ALR2 crystal structure for docking is justified
by the facts, that the crystal structure of rat ALR2 co-crystalized
with inhibitors/ligands is not yet available and human and rat
ALR2 sequences share 85% identity.44
Although a great number of ALR2 co-crystallized structures are
available from PDB, many of them reproduce the same complex
with the same ligand compound and many others show several
unresolved residues. So to speed-up the assessment procedure
and avoiding redundancy a set of 14 structures of ALR2 (1PWL,
1PWM, 1US0, 1Z3N, 1Z89, 2FZ8, 2FZD, 2PZN, 3RX2, 3RX3, 3RX4,
3S3G, 3T42, and 3VQ9) were chosen, among the highest resolution
and with the less unresolved residues, to be retrieved from PDB
(Table 4, Sd).
Protein setup was performed using the UCSF Chimera soft-
ware.66 Energy minimizations were carried out with the molecular
simulation toolkit GROMACS 4.567 using the AMBER99SB-ILDN
force field.68 ACPYPE,69 a tool based on ANTECHAMBER70 for gener-
ating topologies and parameters for chemical compounds, was em-
ployed for the parametrization of the ligands.
4.1.9.1.
pyrrol-2-yl)ethanone (32).
anhydride according to general procedure 8 (110 mg, 78%). Mp
95–97 °C; IR (KBr): 3356 (OAH), 1659 (C@OCF3CO) cmꢂ1 1H NMR
2,2,2-Trifluoro-1-(1-(3-fluoro-4-hydroxyphenyl)-1H-
Prepared from trifluoroacetic
;
(CDCl3) d 5.59 (br s, 1H, OH), 6.46 (dd, J = 4.2, 2.5 Hz, 1H, pyrrolyl
H-4), 6.95–7.12 (m, 3H, phenyl H-5, H-6, H-2), 7.14–7.20 (m, 1H,
pyrrolyl H-3), 7.35–7.43 (m, 1H, pyrrolyl H-5); Anal. Calcd for
C12H7F4NO2: C, 52.76; H, 2.58; N, 5.13. Found: C, 52.39; H, 2.59;
N, 5.04.
4.1.9.2. 2,2-Difluoro-1-(1-(3-fluoro-4-hydroxyphenyl)-1H-pyr-
rol-2-yl)ethanone (33).
dride according to general procedure 8 (95 mg, 72%). Mp 146–
147.5 °C; IR (KBr): 3305 (OAH), 1650 (C@OCHF2CO) cmꢂ1 1H NMR
Prepared from difluoroacetic anhy-
;
(CDCl3-DMSO-d6) d 6.07 (t, J = 54.1 Hz, 1H, CHF2), 6.28–6.38 (m,
1H, pyrrolyl H-4), 6.87–6.98 (m, 2H, phenyl H-5, H-6), 6.77–6.86
(m, 1H, phenyl H-2), 6.99–7.06 (m, 1H, pyrrolyl H-3), 7.22–7.28
(m, 1H, pyrrolyl H-5), 9.02 (br s, 1H, OH); Anal. Calcd for C12H8F3
NO2ꢀ0.04CH2Cl2: C, 55.92; H, 3.15; N, 5.42. Found: C, 55.86; H,
3.21; N, 5.51.
Molecular Docking simulations were performed with Autodock
Vina 1.1.2.71 The MGLTools72 python scripts were used to generate
the docking input files and to analyze the docking results. The de-
fault Vina settings were used, except for the following: exhaustive-
ness was set to 32, the box was set as a cube with edge of 29.15 Å
and center of mass corresponding to that of a single pdb file com-
posed from all the experimental co-crystallized ligands. The side
chain flexibility feature of Autodock Vina was not used. To assess
docking application, a cross-docking procedure54 was set-up in or-
der to take into consideration some discreet protein flexibility.
4.2. Biological evaluation
4.2.1. ALR2/ALR1 in vitro assays
The authors have previously reported the ALR2 and ALR1
in vitro assays and as such they are synoptically described in Sd.
4.2.2. Inhibition of sorbitol formation in rat lenses
4.2.2.1. Eye lenses sorbitol assay.
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 Pharma-
cology and Toxicology, Dobra Voda (Slovak Republic). The study
was approved by the Ethics Committee of the Institute and per-
formed in accordance with the Principles of Laboratory Animal
Care (NIH publication 83–25, revised 1985) and the Slovak law reg-
ulating animal experiments (Decree 289, Part 139, July 9th 2003).
The animals in light ether anesthesia were killed by exsanguin-
ations of the carotid artery and the eye globes were excised. The
lenses were quickly dissected and rinsed with saline.
The compounds studied (30 and 31) dissolved in DMSO were
added into the tubes containing freshly dissected eye lenses (1 lens
per tube) in M-199 medium (Sigma M 3769) at pH 7.4, bubbled at
37 °C with pneumoxid (5% CO2, 95% O2), to the final concentrations
as reported, 30 min before adding glucose. The final concentration
of DMSO in all incubations was 1%. 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 pneum-
oxid. The incubations were terminated after a 3-h period by cool-
ing the mixtures in an ice bath, followed by washing the lenses
three times with ice-cold phosphate buffered saline (1 mL). The
4.4. Physicochemical properties
4.4.1. Solutes and reagents for the logP, pKa, and logKw
experimental calculations
n-Octanol was extra pure and was purchased from Panreac Qui-
mica, Spain. Acetonitrile was HPLC grade and was purchased from
Lab-Scan Science Ltd, Ireland. Sodium hydrogen phosphate, potas-
sium dihydrogen phosphate, potassium chloride and sodium chlo-
ride were purchased from Merck, Darmstadt, Germany. Water was
deionised and further purified by means of a Milli-Q Plus Water
purification system, Millipore Ltd.
4.4.2. Octanol/water partitioning experiments
Octanol/water distribution coefficient values were measured by
the shake-flask method, using a standard procedure as described in
Vrakas et al.73 Briefly, the protocol is as follows: the compound was
first dissolved in DMSO at a concentration of 5 mg/mL (stock solu-
tion). Aqueous solutions were then prepared by the addition of
appropriate volume of stock solutions in order for a concentration
approximately 10ꢂ5 M to be obtained. The final concentration of
DMSO in the aqueous solution did not exceed 0.1%. The pH of the

2206
M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
aqueous solutions was controlled by phosphate buffer at a range
from 7.0 to 12.0.
of rat jejunum were stretched on the inserts for diffusion chambers
with apertures of 1 cm.2 The experiment conforms to the Law for
the protection of animals (Republic of Slovenia) and the use of
the tissue samples from previously sacrificed animals for experi-
mental purposes is registered at the Veterinary Administration of
the Republic of Slovenia.
Both n-octanol and aqueous phases were mutually saturated
before use. The volume ratio of the two phases was chosen, so that
adequate amount of the solute remained in the aqueous phase
after equilibration. In fact, due to the high lipophilicity of the com-
pounds, as computationally estimated, the volume ratios Vaq/Voct
was in the range 20:1–200:1, depending on the pH. An equilibra-
tion time of 1 h was chosen, since further shaking until 5 h did
not affect the partitioning. Centrifugation followed for 15 min at
2500 rpm and 25 °C.
The aqueous phase, before and after equilibration, was analyzed
by UV–Vis spectroscopy at 240 nm and by HPLC using the chro-
matographic system described below. The distribution coefficient
was calculated according to Eq. 3.
2.5 mL of Ringer buffer with 10 mM D-glucose or 10 mM manni-
tol on serosal and mucosal side of the tissue, respectively, was used
as an incubation saline. The tissue was kept at 37 °C during the
experiment. Incubation salines were oxygenated and circulated
by bubbling with carbogen gas mixture containing 95% O2 and
5% CO2. The experiment started by replacing the mucosal incuba-
tion medium with a donor solution, which also included 100
of compound 31 as the tested compound and 10 M fluorescein
as an internal standard. Five samples of 250 L were withdrawn
lM
l
l
from the acceptor (serosal) compartment in 20 min intervals. This
volume was replaced by the appropriate fresh incubation saline.
The concentrations of tested drugs in the samples were determined
by HPLC immediately after the experiment. Degasser, binary pump,
well plate sampler, column thermostat and a diode-array detector,
D ¼ ½ðA0 ꢂ A1Þ=A1ꢁðVaq=Voct
A0 and A1 being the absorption or peak area before and after equil-
ibration and Vaq, Voct the volumes of aqueous and n-octanol phase,
respectively.
Þ
ð3Þ
Each determination was performed at least in triplicate and
mean logD values obtained using both UV/Vis spectroscopy and
HPLC measurements are presented in Table 3 of Sd.
all Agilent 1100 series using a Phenomenex, Kinetex 2.6
l XB-C18
50 ꢃ 3.0 mm column thermostated at 55 °C and operating at a flow
rate of 2.5 mL/min of mobile phase (55% 50 mM phosphate buffer
pH=3.5 and 45% acetonitrile), 100
280 nm detection wavelength.
lL injection volumes and
4.5. Permeability indices
The rat jejunal tissue viability and integrity were controlled
throughout the experiment by monitoring the trans-tissue poten-
tial difference and the short circuit current with a multi-channel
voltage-current clamp (model VCC MC8, Physiologic Instru-
ments).74 The tissue segments in the diffusion chambers were con-
sidered viable if the negativity of the trans-tissue electrical
4.5.1. IAM chromatography
The HPLC isocratic pumping system consisted of a GBC Model
1120 pump and a Rheodyne Model 7725i GBC injector with a
20 lL loop, which were coupled to a GBC Model LC1210 UV–Vis
detector (GBC Scientific Equipment Pty Ltd, Dandenong, Victoria,
Australia). The detector operated at 254 nm (30) or 240 nm (31).
Data acquisition was performed using Empower Built 1154, Waters
Corporation 2002 software package, implemented in the chro-
matographic system.
The stationary phase consisted of an IAMPC DD2 column
(3 cm ꢃ 4.6 mm), Regis Technology, Morton Grove, IL, USA. Mix-
tures of acetonitrile (HPLC grade) and Phosphate Buffer Saline
(PBS) were used as the mobile phase. Acetonitrile fractions varied
from 20% to 30% using a 5% step. The eluent flow rate was 1 mL/
min.
Injections were performed using the samples obtained from
octanol–water partitioning experiments before and after equilibra-
tion, as well as samples obtained after dilution of the compound in
the mobile phase.
Retention times (tr) were measured at least from three separate
injections and converted to the logarithm of isocratic retention fac-
tors via the Eq. 4:
potential measured at the mucosal side after the addition of D-glu-
cose to the mucosal compartment (final concentration was 25 mM)
at the end of experiment was at least ꢂ1.1 mV. The tissue integrity
was additionally evaluated by the trans-tissue electrical resistance,
which must be greater than 18
experiment.
X
cm2 during the entire
The Papp (apparent permeability coefficients) were calculated by
the following equation:
Papp ¼ ½dQ=dtꢁ½1=AC0ꢁ½cm=sꢁ
ð5Þ
where dQ/dt is the steady state appearance rate of the investigated
substance on the acceptor side of the tissue, A is the exposed tissue
area and C0 is the donor concentration of the dissolved investigated
substance. dQ/dt is determined as the slope of the linear part (min-
imal accepted Pearson coefficient values were 0.98) in the ‘amount
permeated versus time of the experiment’ plot, which was drawn
for each tissue segment.
logk ¼ log½ðtr ꢂ t0Þ=t0ꢁ
t0 being the retention time of sodium dichromate. All measure-
ments were performed at room temperature.
Retention time was not affected by the use of octanol saturated
buffer or mobile phase as the solvent medium. For samples ob-
tained from octanol to water partitioning experiments before and
after equilibration, peak areas were calculated and used in Eq. 3.
ð4Þ
4.5.3. Statistical analysis
SPPSS v. 17.0 was used for linear regression and non-linear
fitting.
Acknowledgments
M.C. would like to thank the Greek State Scholarship Founda-
tion (IKY) and Aristotle University’s Research Committee for fund-
ing. This work was partially supported by VEGA Grant No. 2/0067/
11 (M.S.).
4.5.2. ‘In vitro’ permeability evaluation
Rat jejunum from a male Sprague–Dawley rat (200–300 g) was
obtained, prepared and mounted in side by side diffusion chambers
(Easy Mount; Physiologic Instruments, San Diego, CA, USA) as also
described previously.74 Briefly, the animal was denied access to
food and had water ad libitum 18 h before it was sacrificed by
decapitation. The intestine was quickly excised and placed in the
ice-cold oxygenated Ringer buffer. Two to three cm long segments
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.016.

M. Chatzopoulou et al. / Bioorg. Med. Chem. 22 (2014) 2194–2207
2207
37. Adogla, E. A.; Janser, R. F. J.; Fairbanks, S. S.; Vortolomei, C. M.; Meka, R. K.;
Janser, I. Tetrahedron Lett. 2012, 53, 11.
References and notes
38. Hofslokken, N. U.; Skattebol, L. Acta Chem. Scand. 1999, 53, 258.
39. Minutolo, F.; Bertini, S.; Granchi, C.; Marchitiello, T.; Prota, G.; Rapposelli, S.;
Tuccinardi, T.; Martinelli, A.; Gunther, J. R.; Carlson, K. E.; Katzenellenbogen, J.
A.; Macchia, M. J. Med. Chem. 2009, 52, 858.
40. Wu, Y.; Lawson, P. V.; Henary, M. M.; Schmidt, K.; Bredas, J. L.; Fahrni, C. J. J.
Phys. Chem. A 2007, 111, 4584.
41. Imming, P.; Imhof, I.; Zentgraf, M. Synth. Commun. 2001, 31, 3721.
42. Hu, Y. Z.; Zhang, G.; Thummel, R. P. Org. Lett. 2003, 5, 2251.
43. Rakowitz, D.; Muigg, P.; Schroder, N.; Matuszczak, B. Arch. Pharm. (Weinheim)
2005, 338, 419.
44. Chung, S.; LaMendola, J. J. Biol. Chem. 1989, 264, 14775.
45. Gui, T.; Tanimoto, T.; Kokai, Y.; Nishimura, C. Eur. J. Biochem. 1995, 227, 448.
46. Böhm, H.-J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Müller, K.; Obst-
Sander, U.; Stahl, M. ChemBioChem 2004, 5, 637.
47. Vulpetti, A.; Dalvit, C. Drug Discovery Today 2012, 17, 890.
48. Dalvit, C.; Vulpetti, A. ChemMedChem 2011, 6, 104.
49. DiMagno, S. G.; Sun, H. R. Curr. Top. Med. Chem. 2006, 6, 1473.
50. Rees-Milton, K. J.; Jia, Z.; Green, N. C.; Bhatia, M.; El-Kabbani, O.; Flynn, T. G.
Arch. Biochem. Biophys. 1998, 355, 137.
51. Barski, O. A.; Tipparaju, S. M.; Bhatnagar, A. Drug Metab. Rev. 2008, 40, 553.
52. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
53. Keseru, G. M.; Makara, G. M. Nat. Rev. Drug Disc. 2009, 8, 203.
54. Musmuca, I.; Caroli, A.; Mai, A.; Kaushik-Basu, N.; Arora, P.; Ragno, R. J. Chem.
Inf. Model. 2010, 50, 662.
55. Ragno, R.; Frasca, S.; Manetti, F.; Brizzi, A.; Massa, S. J. Med. Chem. 2004, 48, 200.
56. Skagerberg, B.; Bonelli, D.; Clementi, S.; Cruciani, G.; Ebert, C. Quant. Struct.-Act.
Relat. 1989, 8, 32.
1. Hers, H. G. Biochim. Biophys. Acta 1956, 22, 202.
2. Obrosova, I. G.; Kador, P. F. Curr. Pharm. Biotechnol. 2011, 12, 373.
3. Oates, P. J. Curr. Drug Targets 2008, 9, 14.
4. van Dijk, C.; Berl, T. Rev. Endocr. Metab. Dis. 2004, 5, 237.
5. Chatzopoulou, M.; Alexiou, P.; Kotsampasakou, E.; Demopoulos, V. J. Expert
Opin. Ther. Pat. 2012, 22, 1303.
6. Alexiou, P.; Pegklidou, K.; Chatzopoulou, M.; Nicolaou, I.; Demopoulos, V. J.
Curr. Med. Chem. 2009, 16, 734.
7. Obrosova, I. G.; Minchenko, A. G.; Vasupuram, R.; White, L.; Abatan, O. I.;
Kumagai, A. K.; Frank, R. N.; Stevens, M. J. Diabetes 2003, 52, 864.
8. Chatzopoulou, M.; Pegklidou, K.; Papastavrou, N.; Demopoulos, V. J. Expert Opin.
Drug Dis. 2013, 8, 1365.
9. Ramana, K. V.; Srivastava, S. K. Int. J. Biochem. Cell Biol. 2010, 42, 17.
10. Pandey, S.; Srivastava, S. K.; Ramana, K. V. Exp. Opin. Invest. Drug 2012, 21, 329.
11. Ramana, K. V. Biomol. Concepts 2011, 2, 103.
12. Tammali, R.; Srivastava, S. K.; Ramana, K. V. Curr. Cancer Drug Targets 2011, 11,
560.
13. Del Corso, A.; Cappiello, M.; Mura, U. Curr. Med. Chem. 2008, 15, 1452.
14. Gabbay, K. Curr. Diab. Rep. 2004, 4, 405.
15. Hotta, N.; Akanuma, Y.; Kawamori, R.; Matsuoka, K.; Oka, Y.; Shichiri, M.;
Toyota, T.; Nakashima, M.; Yoshimura, I.; Sakamoto, N.; Shigeta, Y. Diabetes
Care 2006, 29, 1538.
16. Bocker, A.; Bonneau, P. R.; Hucke, O.; Jakalian, A.; Edwards, P. J. ChemMedChem
2010, 5, 2102.
17. Richon, A. B.; Maragoudakis, M. E.; Wasvary, J. S. J. Med. Chem. 1982, 25, 745.
18. Sarges, R.; Schnur, R. C.; Belletire, J. L.; Peterson, M. J. J. Med. Chem. 1988, 31,
230.
19. Steuber, H.; Heine, A.; Podjarny, A.; Klebe, G. J. Mol. Biol. 2008, 379, 991.
20. Bril, V.; Hirose, T.; Tomioka, S.; Buchanan, R. Diabetes Care 2009, 32, 1256.
21. Kurono, M.; Itogawa, A.; Noguchi, H.; Sanjoba, M. J. Pharm. Sci. 2008, 97, 1468.
22. Ko, K.; Won, H.; Won, Y. Bioorg. Med. Chem. 2006, 14, 3090.
23. Mylari, B. L.; Armento, S. J.; Beebe, D. A.; Conn, E. L.; Coutcher, J. B.; Dina, M. S.;
O’Gorman, M. T.; Linhares, M. C.; Martin, W. H.; Oates, P. J.; Tess, D. A.;
Withbroe, G. J.; Zembrowski, W. J. J. Med. Chem. 2005, 48, 6326.
24. Rao, V. R.; Muthenna, P.; Shankaraiah, G.; Akileshwari, C.; Babu, K. H.; Suresh,
G.; Babu, K. S.; Chandra Kumar, R. S.; Prasad, K. R.; Yadav, P. A.; Petrash, J. M.;
Reddy, J. B.; Rao, J. M. Eur. J. Med. Chem. 2012, 57, 344.
25. Maccari, R.; Ciurleo, R.; Giglio, M.; Cappiello, M.; Moschini, R.; Corso, A. D.;
Mura, U.; Ottanà, R. Bioorg. Med. Chem. 2010, 18, 4049.
26. Maccari, R.; Ottanà, R.; Ciurleo, R.; Rakowitz, D.; Matuszczak, B.; Laggner, C.;
Langer, T. Bioorg. Med. Chem. 2008, 16, 5840.
27. Rakowitz, D.; Maccari, R.; Ottanà, R.; Vigorita, M. G. Bioorg. Med. Chem. 2006,
14, 567.
28. La Motta, C.; Sartini, S.; Mugnaini, L.; Simorini, F.; Taliani, S.; Salerno, S.; Marini,
A. M.; Da Settimo, F.; Lavecchia, A.; Novellino, E.; Cantore, M.; Failli, P.; Ciuffi,
M. J. Med. Chem. 2007, 50, 4917.
29. Nicolaou, I.; Demopoulos, V. J. J. Med. Chem. 2003, 46, 417.
30. Pegklidou, K.; Koukoulitsa, C.; Nicolaou, I.; Demopoulos, V. J. Bioorg. Med. Chem.
2010, 18, 2107.
31. Chatzopoulou, M.; Mamadou, E.; Juskova, M.; Koukoulitsa, C.; Nicolaou, I.;
Stefek, M.; Demopoulos, V. J. Bioorg. Med. Chem. 2011, 19, 1426.
32. Nicolaou, I.; Zika, C.; Demopoulos, V. J. J. Med. Chem. 2004, 47, 2706.
33. Shen, C.; Sigman, D. S. Arch. Biochem. Biophys. 1991, 286, 596.
34. Kotsampasakou, E.; Demopoulos, V. J. Bioorg. Med. Chem. 2013, 21, 869.
35. Cardinaud, I.; Gueiffier, A.; Debouzy, J. C.; Milhavet, J. C.; Chapat, J. P.
Heterocycles 1993, 36, 2513.
57. Kotecha, J.; Shah, S.; Rathod, I.; Subbaiah, G. Int. J. Pharm. 2007, 333, 127.
58. Taillardat-Bertschinger, A.; Martinet, C. A.; Carrupt, P. A.; Reist, M.; Caron, G.;
Fruttero, R.; Testa, B. Pharm. Res. 2002, 19, 729.
59. Zakelj, S.; Berginc, K.; Roskar, R.; Kraljic, B.; Kristl, A. Curr. Drug Metab. 2013, 14,
21.
60. Terashima, H.; Hama, K.; Yamamoto, R.; Tsuboshima, M.; Kikkawa, R.;
Hatanaka, I.; Shigeta, Y. J. Pharm. Exp. Ther. 1984, 229, 226.
61. Sawicki, E. J. Org. Chem. 1956, 21, 376.
62. Anderson, H. J.; Loader, C. E.; Xu, R. X.; Lê, N.; Gogan, N. J.; McDonald, R.;
Edwards, L. G. Can. J. Chem. 1985, 63, 896.
63. Ma, F.-P.; Li, P.-H.; Li, B.-L.; Mo, L.-P.; Liu, N.; Kang, H.-J.; Liu, Y.-N.; Zhang, Z.-H.
Appl. Catal., A 2013, 457, 34.
64. O’Boyle, N. M.; Banck, M.; James, C. A.; Morley, C.; Vandermeersch, T.;
Hutchison, G. R. J. Cheminform. 2011, 3, 33.
65. Halgren, T. A. J. Comput. Chem. 1999, 20, 730.
66. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.;
Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605.
67. Pronk, S.; Pall, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M.
R.; Smith, J. C.; Kasson, P. M.; van der Spoel, D.; Hess, B.; Lindahl, E.
Bioinformatics 2013, 29, 845.
68. Lindorff-Larsen, K.; Piana, S.; Palmo, K.; Maragakis, P.; Klepeis, J. L.; Dror, R. O.;
Shaw, D. E. Proteins 2010, 78, 1950.
69. Da Sillva, A. W. S.; Vranken, W. F. BMC Res. Notes 2012, 5, 367.
70. Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A. J. Mol. Graph. Model. 2006, 25,
247.
71. Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455.
72. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D.
S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785.
73. Vrakas, D.; Tsantili-Kakoulidou, A.; Hadjipavlou-Litina, D. QSAR Comb. Sci. 2003,
22, 622.
36. Chatzopoulou, M.; Kotsampasakou, E.; Demopoulos, V. Synth. Commun. 2013,
43, 2949.
74. Zakelj, S.; Legen, I.; Veber, M.; Kristl, A. Int. J. Pharm. 2004, 272, 173.