Design and synthesis of novel series of pyrrole based chemotypes and their evaluation as selective aldose reductase inhibitors. A case of bioisosterism between a carboxylic acid moiety and that of a tetrazole

Article abstract of DOI:10.1016/j.bmc.2010.02.010

Pyrrolyl-propionic and butyric-acid derivatives 1 and 2 were synthesized in order to study the effect of the variation of the methylene chain in comparison to the previously reported pyrrolyl-acetic acid compound I, which was found as potent aldose reductase inhibitor, while the pyrrolyl-tetrazole derivatives 3-5 were prepared as a non-classical bioisosteres of a carboxylic acid moiety. Also, pyrrolyl-tetrazole isomers 6 and 7 without an alkyl chain between the two aromatic rings were synthesized. The in vitro aldose reductase inhibitory activity of the prepared 1-7 compounds were estimated and compared with that of the initial compound (I). Overall, the data indicate that the presented chemotypes 6 and 7 are a promising lead compounds for the development of selective aldose reductase inhibitors, aiming to the long-term complications of diabetes mellitus.

Full text of DOI:10.1016/j.bmc.2010.02.010

Bioorganic & Medicinal Chemistry 18 (2010) 2107–2114
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel series of pyrrole based chemotypes and
their evaluation as selective aldose reductase inhibitors. A case of
bioisosterism between a carboxylic acid moiety and that of a tetrazole
a
Kyriaki Pegklidou a, Catherine Koukoulitsa b, Ioannis Nicolaou a, , Vassilis J. Demopoulos
*
a Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
b Department of Chemistry, University of Athens, Zographou 15784, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrrolyl-propionic and butyric-acid derivatives 1 and 2 were synthesized in order to study the effect of
the variation of the methylene chain in comparison to the previously reported pyrrolyl-acetic acid com-
pound I, which was found as potent aldose reductase inhibitor, while the pyrrolyl-tetrazole derivatives
3–5 were prepared as a non-classical bioisosteres of a carboxylic acid moiety. Also, pyrrolyl-tetrazole iso-
mers 6 and 7 without an alkyl chain between the two aromatic rings were synthesized. The in vitro aldose
reductase inhibitory activity of the prepared 1–7 compounds were estimated and compared with that of
the initial compound (I). Overall, the data indicate that the presented chemotypes 6 and 7 are a promising
lead compounds for the development of selective aldose reductase inhibitors, aiming to the long-term
complications of diabetes mellitus.
Received 15 October 2009
Revised 2 February 2010
Accepted 5 February 2010
Available online 11 February 2010
Keywords:
Antidiabetic aldose reductase inhibitors
Antioxidant activity
Nonclassical bioisosterism
Selectivity
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
converts glucose to sorbitol, was first found to be implicated in
the etiolology of the long-term diabetic complications. Its inhibi-
Diabetes mellitus is a metabolic disease in which the body’s
ability to regulate blood glucose levels goes awry, either from de-
fects in the secretion or in the activity of the hormone insulin. To-
day, the worldwide prevalence of diabetes is taking pandemic
dimensions. In 2003, 194 million people globally, ranging in age
from 20 to 79 years, were suffering from diabetes. By 2025, this
number is projected to increase to 333 million.1
People with diabetes are vulnerable to late onset complications
such as retinopathy, nephropathy and neuropathy that are largely
responsible for the morbidity and mortality observed in patients.2,3
The direct economic cost of diabetes is about 10 percent of the to-
tal health care budget, and approximately 90 percent of the total
direct cost is needed for the treatment of the devastating diabetic
complications.4,5 The chronic complications of the disease are asso-
ciated with the duration and the degree of hyperglycemia. It has
been demonstrated that the more severe and sustained the degree
of hyperglycemia, the more likely it is that the chronic complica-
tions of diabetes will develop.6
tion by aldose reductase inhibitors (ARIs) has been gaining atten-
tion over the last decades from the pharmaceutical community
as a promising pharmacotherapeutic target.8
Recently, in our laboratory, effective ARIs have been synthe-
sized, which are derivatives of the pyrrolyl-acetic acid moiety
(I).9 In the present work, the elongation of the methylene chain
as well as the replacement of the carboxylic acid moiety with that
of the non-classical bioisosteric heterocyclic ring of tetrazole and
their effect on aldose reductase activity were studied. The main
advantage of the tetrazole derivatives in comparison to those of
the carboxylic acid ones is that tetrazoles escape most of the phase
II biotransformation of carboxylic acids.10 In addition, although tet-
razole derivatives are ionized in blood pH in a similar way to that
of carboxylic acids, they are 10 times more lipophilic.11 This in-
crease in the lipophilicity could be relevant to their increased per-
meability through biomembranes.11 Thus, a series of pyrrolyl-acids
1 and 2 and pyrrolyl-tetrazole 3–5 derivatives as well as pyrrolyl-
tetrazole isomers 6 and 7 without an alkyl chain between the two
heteroaromatic rings (Fig. 1) were designed synthesized and tested
for their potential to inhibit the enzyme ALR2. In addition, the
inhibitory activity of compounds 1–7 towards aldehyde reductase
(ALR1) was studied, in order to define the selectivity between these
two enzymes with similar amino acid sequence (65%).12,13
Under hyperglycemic conditions, there is an increased flux
through the polyol pathway, accounting for greater than 30% of
glucose metabolism.7 Aldose reductase (ALR2; AR; EC 1.1.1.21)
which is the first enzyme of the polyol metabolic pathway that
Furthermore, for selected more active ones the antioxidant
potential was assessed in a homogeneous 2,2-diphenyl-1-pic-
* Corresponding author. Tel.: +30 2310 998670; fax: +30 2310 997852.
E-mail address: inikolao@pharm.auth.gr (I. Nicolaou).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.010

2108
K. Pegklidou et al. / Bioorg. Med. Chem. 18 (2010) 2107–2114
Figure 1. Structures of pyrrolyl-acids and pyrrolyl-tetrazole derivatives.
rylhydrazyl (DPPH) assay system,14 as well as in a heterogeneous
of compounds 3–5 was a two-step procedure (Scheme 1). The first
step included the formation of the intermediates pyrrolyl-nitrile
derivatives via a nucleophilic substitution under a phase transfer
catalysis for the preparation of compounds 3 and 5 and via a Mi-
chael reaction for compound 4.18 The second step included the
conversion of pyrrolyl-nitrile derivatives to the final pyrrolyl-tetra-
zole compounds using trimethylsilyl azide in the presence of a
unilamellar L-a-phosphatidylcholine dioleoyl (DOPC) liposomes’
assay system.14 Oxidative stress is well-established as an impor-
tant biochemical factor implicated in the long-term complications
of diabetes mellitus.15,16
2. Results and discussion
2.1. Chemistry
catalytic amount of dibutyltin oxide in refluxing toluene.19
A
number of different conditions was studied for the preparation of
the intermediates pyrrolyl-nitrile derivatives 3i and 5i, using
benzyltributylammonium chloride (BTAC) or tris-[2-(2-methoxy-
ethoxy)ethyl]amine (TDA-1) as phase transfer catalysts in tetrahy-
drofuran (THF) or toluene, respectively. Best results were obtained
with BTAC in THF as solvent. The synthesis of the isomeric com-
pounds 6 and 7 was based on a Friedel–Crafts aroylation of
1-(1H-pyrrol-1-yl)-1H-tetrazole (6i), which was synthesized from
5-amino-tetrazole by a modified Clauson–Kaas reaction.9 The Fri-
edel–Crafts aroylation was performed according to Nicolaou and
Demopoulos20 using nitromethane as a cosolvent. Both compounds
6 and 7 were isolated from the above reaction.
The preparation of the compounds 1 and 2 was based on the re-
ported methodology17,9 and it is shown in Scheme 1. The synthesis
O
O
O
(b)
(a)
N
N
N
H
(CH2)kCOOEt
(CH2)kCOOH
k= 2 (1i)
k= 3 (2i)
k= 2 (1)
(2)
k= 3
2.2. In vitro and in silico results
2.2.1. Aldose reductase inhibitory activity
The synthesized compounds 1–7 were evaluated in vitro for
their ability to inhibit partially purified rat lens AR. It has been
shown that there is an approximately 85% sequence similarity be-
tween rat lens and human ALR2, while the proposed active sites of
both enzymes are identical.21 The performed assay was based on a
spectrometric measurement, which is proven to be a reliable meth-
od,22 with DL-glyceraldehyde as the substrate and NADPH as the
cofactor. Sorbinil was used as a positive control. Inhibitor IC50 val-
ues were determined for those compounds displaying greater than
50% percent inhibition at the high concentration of 10ꢀ4 M. Results
O
(c)
N
(4i)
O
O
(CH2)2CN
(e )
(d)
N
N
H
(CH2)m
m = 1 (3)
m = 2 (4)
m = 3 (5)
NH
O
N
N
N
n =1 (3i)
(5i)
N
n =3
(CH2)nCN
Table 1
Aldose reductase inhibitory activity data
Compound
% Inhibition ( SDa) 10ꢀ4
M
IC50 ( SDa)
lM
O
1
2
3
4
5
6
7
I
9.7 ( 0.15)
6.04 ( 0.11)
NH2
(g)
(f)
N
N
N
+
40 ( 4.1)
6 ( 0.4)
41( 0.39)
N
N
N
NH
O
N
N
N
N
N
N
N
NH
N
NH
NH
N
(6i)
18.0 ( 1.49)
20.0 ( 0.96)
1.97 ( 0.03)b
(6 )
(7 )
0.25 ( 0.006)c
Scheme 1. Reagents and conditions: (a) ethyl bromopropanate or ethyl bromobu-
tanate, NaH, TDA-1, toluene; (b) 5% aq. NaOH, dioxane; (c) acrylonitrile, Triton B;
(d) 2-bromoacetonitrile or 4-bromobutanenitrile, NaH, BTAC, THF; (e) Me3SiN3,
nBu2Sn=O, toluene; (f) 2,5-bimethoxy-tetrahydrofuran, 4-chloropyridine hydro-
chloride, 1,4-dioxane; (g) AlCl3/CH3NO2, PhCOCl, CH2Cl2.
Sorbinil
a
b
c
n = 3
Reported data by Nicolaou and Demopoulos.9
Reported IC50: 0.07 l
V by Kador and Sharpless.23

K. Pegklidou et al. / Bioorg. Med. Chem. 18 (2010) 2107–2114
2109
are presented in Table 1 and compared with compound I, which
has been previously shown that is an effective ARI.17,9
squared deviation). The crystallographic structure of IDD 594 was
docked into ALR2 with GLUE provided excellent result as shown in
our previous work.26
It was found that pyrrolyl-propionic or butyric-acid derivatives
1 and 2 have IC50 values a little higher than their acetate counter-
part (compound I). This is in accordance to the well-established
pharmacophore role of the acetic acid functionality in the majority
of the carboxylate ARIs.24 It is also, worth noting that the butyrate
derivative 2 is more potent than the propionate derivative 1. In a
similar example Sun et al noticed that a number of indolobutanoic
acid derivatives have been shown to be active in submicromolar
range as ARIs.25 The replacement of the carboxylic acid moiety
with that of the bioisosteric heteroaromatic ring of tetrazole in
cases of compounds 3 and 5, dramatically reduce the inhibitory
activity, while compound 4 was found to be completely inactive
at the same concentration. This is also in agreement with that pre-
viously referred about the propionate counterpart. On the other
hand, the lack of the methylene chain between pyrrole and tetra-
zole aromatic rings in compounds 6 and 7 improves noticeably
the total inhibition, in comparison to tetrazole derivatives which
have an alkyl chain. Inspection of low energy conformations of
all tetrazole derivatives reveals that the two heterocyclic aromatic
rings of compounds 3–5 are not in the same plane as they are, in
compounds 6 and 7 (Fig. 2). This is also in accordance with that
previously reported by Nicolaou and Demopoulos.9
It is observed that the most active compound (6) exhibits the
higher binding energy (ꢀ21.65 Kcal/mol). Compound 7 possess
similar binding energy (ꢀ21.14 Kcal/mol), while the inactive com-
pound 3 exhibits the lower binding energy (ꢀ15.76 Kcal/mol). As
illustrated in Figure 3a–c, the compounds were placed in the same
location as IDD 594 in the crystal structure. In particular, the tetra-
zole moiety of compounds 6 and 7 is anchored in the anionic bind-
ing site forming polar interactions with Tyr48 and electrostatic
interactions with the positively charged nicotinamide ring of
NADP+. Compound 7 forms polar interactions also between the
phenyl oxygen and Trp111. Further stabilization of the molecules
is attributed to the aromatic interactions of the phenyl ring with
Trp79, Trp111, Trp219 and Van der Waals interactions between
phenyl ring and residues Phe122, and Leu300. Compound 3 en-
tered the anionic binding site with the phenyl ring, and can not
establish strong interactions with the key aminoacid residues
and NADP+. The results from the docking calculations are in consis-
tence with the lower estimated binding energy and the observed
inactivity of compound 3 against aldose reductase.
In Figure 3d is presented the superimposition of the docked com-
pounds 6 and 7 and IDD594 in the active site of ALR2. It should be
noticed that the phenyl ring entered in the specificity pocket local-
ized between Phe122, Trp111, Leu300, and Ala299 as IDD594, sug-
gesting a selectivity for aldose versus aldehyde reductase.
In order to shed light on their interactions in the active site of
ALR2, docking simulations were performed. The human aldose
reductase holoenzyme complexed 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 docking simula-
tions in the active site of ALR2 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
2.2.2. Aldehyde reductase inhibitory activity
An important feature of pharmacologically applicable ARIs is
their selectivity of action. The co-inhibition of structurally related
physiological oxidoreductases might have unwanted side effects.
In the present study, the target compounds 1–7 and I were also
tested for their selectivity. In testing for selectivity we used the en-
zyme aldehyde reductase (ALR1, EC 1.1.1.1.2). The assay was based
on a spectrometric measurement, using D-glucuronate as a sub-
strate in the presence of NADPH as a cofactor.27 ALR1 and ALR2
share a high degree of sequence (ꢁ65%) and structural homology.28
ALR1 is one of the significant enzymes for the reduction of many
aldehydes, counteraction and excretion of drugs, reduction of 3-
deoxyglucosone (3-DG), which is an intermediate for advanced
glycation end-products (AGEs), and metabolism of methylgly-
oxal.29 Valproic acid was used as a positive control. Results are
shown in Table 2. As it could be seen, compounds 1, 2, 6, 7 and I
have high selectivity for aldose versus aldehyde reductase since
they possess very low inhibitory activity towards ALR1 at the high
concentration of 10ꢀ4 M.
2.2.3. DPPH scavenging activity
In this part of our work, three of the most active and selective
pyrrolyl ARIs I, 6 and 7 were assessed in vitro for the radical scav-
enging potential of them, by using the model reaction with the sta-
ble free radical of 2,2-diphenyl-1-picrylhydrazyl (DPPH). In this
homogeneous system of the ethanol solution of DPPH, antioxidant
activity stems from an intrinsic chemical reactivity towards radicals.
DPPH, as a weak hydrogen radical atom abstractor, could be consid-
ered asa good kineticmodelfor peroxylROOÅ radicals.30 Resultsindi-
cated a slight antioxidant potential at the equimolar concentrations
(0.2 mM) of the tested compounds and DPPH (ꢁ10% scavenging
activity). Trolox which was used as a reference compound had
shown 98% scavenging activity at the same concentration.
2.2.4. Lipid peroxidation inhibition
In membranes, the relative antioxidant reactivity is probably
different from a homogeneous system since it is determined by
additional factors, such as the location of the antioxidant and rad-
Figure 2. Representative low energy conformations of tetrazole-derivative
(down) versus tetrazole-derivative 6 (up).
3

2110
K. Pegklidou et al. / Bioorg. Med. Chem. 18 (2010) 2107–2114
Figure 3. Docked orientations of (a) compound 6, (b) compound 7, and (c) compound 3, with additional depiction of the key aminoacids of ALR2 active site. Polar interactions
are shown as dotted lines; (d) Superimposition of the docked compounds 6 (red), 7 (green), and IDD594 (orange) in the active site of ALR2.
Table 2
Lipinski’s ‘rule of five’, and moreover the topological polar surface
area (TPSA) as additional molecular descriptor32–34 (Table 3). For
Aldehyde reductase inhibitory activity data
Compound
% Inhibition ( SD)a 10ꢀ4
M
all the drug candidates studied, none of the criteria was violated,
thus predicting their good oral bioavailability.
1
20 ( 6.5)
2
3
4
5
6
7
I
22 ( 1.53)
23 ( 2.52)
41 ( 0.58)
35 ( 2.52)
18 ( 1.75)
18 ( 1.00)
34 ( 3.51)
3. Conclusion
Pyrrolyl-acids 1, 2 and I as well as pyrrolyl-tetrazoles 6 and 7
have been found to be promising selective aldose reductase inhib-
itors. It should be noted at this point that the selective activity for
aldose versus aldehyde reductase is considered as a great advan-
tage for a compound because of reduction of the risk for toxic
Valproic acid
IC50b: 50.1 ( 3.0)
lM
a
n = 3.
b
Reported IC50: 56.1 ( 2.7) by Stefek and co-workers.14
Table 3
icals and is ruled predominantly by the partition ratios between
water and lipophilic compartments. As an indicative heteroge-
neous assay, the antioxidant inhibitory efficiency of compounds I,
6 and 7 was evaluated in the system of unilamellar DOPC lipo-
somes oxidatively stressed by peroxyl radical generated in the
aqueous phase by thermal decomposition of the hydrophilic azo
initiator 2,2-azobis(2-amidinopropane) hydrochloride (AAPH). Re-
Drug-likeness of the synthesized compounds
Compound MW
(<500)
NH/OH
(<5)
N/O
(<10)
Clog P
(<5)
TPSA
(Å2)
1
2
3
4
5
6
7
243.28
257.31
253.29
267.32
281.35
239.26
239.26
1
1
1
1
1
1
1
4
4
6
6
6
6
6
2.04
2.40
0.96
1.06
1.44
1.07
1.07
59.30
59.30
76.46
76.46
76.46
76.46
76.46
sults indicated
a weak antioxidant potential of compounds
(IC50 >300 M). Trolox in an identical assay was used as the posi-
l
tive control (IC50 = 93.5 l
V).31
MW, molecular weight; OH/NH, number of H-bond donors; O/N, number of H-bond
acceptors; Clog P v. 4.0 (Biobyte), based on Leo-Hansch fragmental system (Clog P);
TPSA, topological polar surface area calculated by Pallas v.3.3.2.4 (CompuDrug
Chemistry Ltd.
2.2.5. ‘Drug-likeness’
The ‘drug-likeness’ of the novel aldose reductase inhibitors was
assessed on the basis of their structural properties by applying the

K. Pegklidou et al. / Bioorg. Med. Chem. 18 (2010) 2107–2114
2111
effects. Furthermore, properties implicit to ‘drug-likeness’, inferred
from simple molecular descriptors, rendered novel aldose reduc-
tase inhibitors potential drugs with expected good bioavailability
and with a prospect of further optimization. Additionally, com-
pounds 6 and 7 as tetrazole-derivatives might have a better phar-
macokinetic profile than pyrrolyl-acids derivatives. Thus, we
propose that compounds 6 and 7 could comprise the basis for
the design of novel chemotypes, as pharmacotherapeutic agents
for the treatment of the long-term diabetic complications.
COOH). Anal. Calcd for C14H13O3N: C, 69.12; H, 5.39; N, 5.76.
Found: C, 69.13; H, 5.39; N, 5.72.
4.2.2.2. 4-(3-Benzoyl-1H-pyrrol-1-yl)butanoic acid (2). Yield
79%, mp 106 °C, IR (Nujol): 1730, 1540 cmꢀ1 1H NMR (CDCl3/
,
DMSO-d6): d 1.92–2.42 (m, 4Y, CH2CH2CO), 4.10 (t, 2H, N–CH2),
6.60–6.82 (m, 2H, pyrrolyl-4,5H), 7.20–7.60 (m, 3H, phenyl-H),
7.70–8.00 (m, 3H, phenyl-H and pyrrolyl-2H), 9.30 (br s, 1H,
COOH). Anal. Calcd for C15H15O3N: C, 70.02; H, 5.88; N, 5.44.
Found: C, 70.07; H, 5.83; N, 5.37.
4. Experimental section
4.1. General notes
4.2.3. General procedure for the preparation of 2-(3-benzoyl-
1H-pyrrol-1-yl)acetonitrile (3i) and 4-(3-benzoyl-1H-pyrrol-1-
yl)butanenitrile (5i)
To a cold (ice bath) stirred under a nitrogen atmosphere mix-
ture of phenyl(1H-pyrrol-3-yl)methanone (2.92 mmol), 2-bromo-
acetonitrile or 4-bromobutanenitrile (2.92 mmol), respectively,
and a small quantity of benzyltributylammonium chloride (BTAC)
(0.292 mmol) in tetrahydrofuran (30 mL), was added NaH (60% dis-
persion in mineral oil) (4.38 mmol). The resulting mixture was stir-
red at room temperature for 24 h. Afterwards a 30% of the total
amount of the above reagents were added and the reaction was
stirred overnight. After this period, the mixture was poured into
a stirred ice cold mixture of H2O (40 mL) and CH2Cl2 (40 mL).
The two phases were separated and the aqueous phase was ex-
tracted with CH2Cl2 (2 ꢂ 50 mL). The combined organic extracts
were washed with saturated brine and dried over anhydrous
sodium sulfate. The solvents were evaporated under reduced pres-
sure and the residue was flashed chromatographed with petroleum
ether/EtOAc 1:4 for compound 3i and 2:3 for compound 5i. Analyt-
ical sample for compound 3i was prepared by recrystallization
from CH2Cl2/petroleum ether.
Unless otherwise stated, all commercial reagents were avail-
able from Aldrich, Sigma or Fluka. Melting points were deter-
mined in open glass capillaries using a Mel-TempII apparatus.
UV spectra were recorded either on a Perkin–Elmer 554 or on a
Hitachi U-2001 spectrophotometer. IR spectra were obtained on
a Shimadzu spectrophotometer, and 1H NMR spectra on a Bruker
AW-80 spectrometer with internal TMS standard. LC–MS/MS
spectra were obtained using an Acquity Waters UPLC–MS/MS
(Triple quandropole) system, while a Shimadgu 2010-EV LC/MS
was used for obtaining LC/MS spectra. Elemental analyses were
performed in School of Chemistry, Department of Organic Chem-
istry of Aristotle University. Statistical analyses were performed
using SPSS v.12.0.
4.2. Chemistry
4.2.1. General procedure for the preparation of the ethyl
propanoic and ethyl butanoic acid esters derivatives (1i and 2i)
To a cold (ice bath) stirred and under a nitrogen atmosphere
mixture of phenyl(1H-pyrrol-3-yl)methanone (12.86 mmol), ethyl
bromopropanate or ethyl bromobutanate (19.29 mmol), respec-
tively, and TDA-1 (2.06 mmol) in toluene (200 mL) was added
NaH (60% dispersion in mineral oil) (19.29 mmol). The resulting
mixture was stirred at room temperature for 24 h. After this peri-
od, it was poured into a stirred, ice cold mixture of Et2O (100 mL)
and 5% HCl (100 mL). The two phases were separated, and the
aqueous phase was extracted with Et2O (2 ꢂ 100 mL). The com-
bined organic extracts were washed with 10% NaHCO3 and satu-
rated brine and dried over anhydrous sodium sulfate. The
solvents were evaporated under reduced pressure and the residue
was flashed chromatographed with petroleum ether/EtOAc 4:1 to
afford 1i and 2i (yellow oils) and used without further purification
for the next step of the reaction.
4.2.3.1. 2-(3-Benzoyl-1H-pyrrol-1-yl)acetonitrile (3i). Yield 68%,
mp 108–109 °C, IR (Nujol): 3410, 1619 cmꢀ1 1H NMR (CDCl3): d
,
5.10 (s, 2H, CH2), 6.95–7.05 (m, 2H, pyrrolyl-4,5H), 7.40–7.80 (m,
4H, phenyl-H and pyrrolyl-2H), 7.80–8.12 (m, 2H, phenyl-H). Anal.
Calcd for C13H10N2O(0.01CH2Cl2): C, 74.27; H, 4.79; N, 13.33. Found:
C, 74.03; H, 4.78; N, 13.27.
4.2.3.2. 4-(3-Benzoyl-1H-pyrrol-1-yl)butanenitrile (5i). Yield
60%, yellow oil, IR (Nujol): 3517, 1637 cmꢀ1 1H NMR (CDCl3): d
,
2.00–2.50 (m, 4H, CH2CH2CN), 4.10 (t, 2H, N–CH2), 6.64–6.89 (m,
2H, pyrrolyl-4,5H), 7.18–7.61 (m, 4H, phenyl-H and pyrrolyl-2H),
7.70–7.90 (m, 2H, phenyl-H). LC–MS calcd for C15H14N2O: m/z
238.11 (100%), found: (M+23): 261.45.
4.2.4. 3-(3-Benzoyl-1H-pyrrol-1-yl)propanenitrile (4i)
4.2.2. General procedure for the preparation of the propanoic
and butanoic acid derivatives (1 and 2)
A
mixture of phenyl(1H-pyrrol-3-yl)methanone (0.290 g,
1.7 mmol) and 0.015 mL of Triton B (40%) was stirred under a
nitrogen atmosphere. Acrylonitrile (0.12 mL, 0.096 g, 1.8 mmol)
was added dropwise over 5 min. The reaction temperature was
maintained below 4 °C with occasional cooling by means of an
ice-water bath. After stirring at room temperature overnight, the
reaction mixture was diluted with dichloromethane (20 mL),
washed with water and saturated brine, and dried over anhydrous
sodium sulfate and evaporated. The residue was flashed chromato-
graphed with petroleum ether/EtOAc 2:3 to provide compound 4i
(0.229 g, yield 60%). An analytical sample was prepared by recrys-
tallization from CH2Cl2/petroleum ether.Mp 69–70 °C, IR (Nujol):
A mixture of 1i or 2i (4.61 mmol), dioxane (80 mL), and 5%
NaOH (80 mL) were stirred at room temperature for 1 h. After this
period, it was concentrated to half of its volume, H20 (80 mL) was
added, and the mixture was cooled (ice bath) and acidified with
concentrated HCl. The formed precipitate was collected and the fil-
trate was extracted with CH2Cl2 (2 ꢂ 50 mL). The organic phase
was washed with saturated brine and evaporated. The residue
was combined with the precipitate and recrystallized from tolu-
ene/petroleum ether.
4.2.2.1. 3-(3-Benzoyl-1H-pyrrol-1-yl)propanoic acid (1). Yield
3517, 1632 cmꢀ1 1H NMR (CDCl3): d 2.82 (t, 2H, CH2CN), 4.20 (t,
,
85%, mp 143–144 °C, IR (Nujol): 1731, 1540 cmꢀ1
,
1H NMR
2H, N-CH2), 6.64 -6.87 (m, 2H, pyrrolyl-4,5H), 7.20–7.95 (m, 4H,
phenyl-H and pyrrolyl-2H), 7.72–7.92 (m, 2H, phenyl-H). Anal.
Calcd for C14H12N2O(0.001EtOAc): C, 74.98; H, 5.39; N, 12.49.
Found: C, 74.97; H, 5.39; N, 12.49.
(CDCl3/DMSO-d6): d 2.80 (t, 2Y, CH2CO), 4.20 (t, 2H, N–CH2),
6.60–6.82 (m, 2H, pyrrolyl-4,5H), 7.25–7.60 (m, 3H, phenyl-H),
7.70–8.00 (m, 3H, phenyl-H and pyrrolyl-2H), 9.30 (br s, 1H,

2112
K. Pegklidou et al. / Bioorg. Med. Chem. 18 (2010) 2107–2114
4.2.5. General procedure for the preparation of methyl-, ethyl-
and propyl-tetrazole derivatives (3–5)
the stirred mixture for 15 min at room temperature under a nitro-
gen atmosphere. After this period, benzoyl chloride (0.588 g,
4.2 mmol) was added and the reaction was stirred for 1.5 h under
a nitrogen atmosphere. The reaction was quenched with ice and
water and the product was extracted to EtOAc (70 mL). The com-
bined organic extracts were washed with water and saturated
brine, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure. The residue was flashed chromato-
graphed with ether/acetic acid (0.1–0.5%) to provide compounds
6 and 7.
To a solution of nitriles 3i–5i (3.33 mmol), respectively, and tri-
methylsilyl azide (0.88 mL, 6.66 mmol) in toluene (10 mL) was
added dibutyltin oxide (0.333 mmol), and the mixture was re-
fluxed under a nitrogen atmosphere until the nitriles were con-
sumed (TLC analysis). The reaction mixture was concentrated in
vacuo. The residue was dissolved in methanol (10 mL) and recon-
centrated. The residue was treated with 10% NaHCO3 (50 mL)
and extracted with EtOAc (2 ꢂ 50 mL). The aqueous phase was
cooled (ice bath) and acidified with 10% HCl, and extracted with
EtOAc (2 ꢂ 50 mL). The combined organic extracts were washed
with brine, dried over magnesium sulfate and concentrated under
reduced pressure to give compounds 3–5. An analytical sample of
compounds 3 and 4 were prepared by recrystallization from EtOAc
/petroleum ether.
4.2.7.1. 1-(2H-Tetrazole-5-yl)-1H-pyrrol-3-yl)(phenyl)metha-
none (6). Yield 10%, mp 189–191 °C, IR (Nujol): 3182, 2773,
1622 cmꢀ1, 1H NMR (CDCl3/DMSO-d6): d 6.84–7.04 (m, 1H, pyrrol-
yl-4H), 7.34–7.71 (m, 4H, phenyl-H and pyrrolyl-5H), 7.79–7.95 (m,
2H, phenyl-H), 7.98–8.09 (m, 1H, pyrrolyl-2H), 8.36 (br s, 1H). Anal.
Calcd for C12H9N5O(0.001CH3COOH): C, 60.25; H, 3.79; N, 29.27.
Found: C, 60.24; H, 3.79; N, 29.27.
4.2.5.1. (1-(2H-Tetrazol-5-yl)methyl)-1H-pyrrol-3-yl)(phenyl)-
methanone (3). Yield 66%, mp 113–115 °C, IR (Nujol): 3453,
1615 cmꢀ1
,
1H NMR (CDCl3/DMSO-d6): d 5.50 (s, 2H, CH2), 6.57–
4.2.7.2. 1-(2H-Tetrazole-5-yl)-1H-pyrrol-2-yl)(phenyl)metha-
none (7). Yield 36%, mp 103–105 °C, IR (Nujol): 3182, 2773,
1622 cmꢀ1, 1H NMR (CDCl3/DMSO-d6): d 6.38–6.58 (m, 1H, pyrrol-
yl-4H), 6.84–7.06 (m, 1H, pyrrolyl-3H), 7.32–7.69 (m, 4H, phenyl-H
and pyrrolyl-5H), 7.78–8.08 (m, 2H, phenyl-H). Anal. Calcd for
C12H9N5O: C, 60.25; H, 3.79; N, 29.27. Found: C, 60.03; H, 3.88;
N, 29.46.
6.99 (m, 2H, pyrrolyl-4,5H), 7.35–7.65 (m, 4H, phenyl-H and pyrr-
olyl-2H), 7.72–7.91 (m, 2H, phenyl-H). Anal. Calcd for C13H11
N5O(0.001EtOAc): C, 61.65; H, 4.38; N, 27.65. Found: C, 61.65; H,
4.38; N, 27.64.
4.2.5.2. (1-(2-(2H-Tetrazol-5-yl)ethyl)-1H-pyrrol-3-yl)(phenyl)-
methanone (4). Yield 63%, mp 143–145 °C, IR (Nujol): 3432,
1605 cmꢀ1, 1H NMR (CDCl3/DMSO-d6): d 3.39 (t, 2H, CH2-tetrazole),
4.41 (t, 2H, N–CH2), 6.55–6.82 (m, 2H, pyrolyl-4,5H), 7.11–7.27
(m, 1H, pyrrolyl-2H), 7.30–7.63 (m, 3H, phenyl-H), 7.65–7.92
(m, 2H, phenyl-H). Anal. Calcd for C14H13N5O: C, 62.91; H, 4.90;
N, 26.20. Found: C, 62.86; H, 4.78; N, 26.28.
4.3. In vitro assays
4.3.1. In vitro aldose reductase enzyme assay
The target compounds 1–7 as well as the reference compound
sorbinil (C11H9FN2O3, Phizer, Inc., Central Research Division, Gro-
ton, CT, USA) were dissolved in aqueous solution of NaHCO3.
Lenses were quickly removed from Fischer-344 rats of both sexes
following euthanasia and homogenized. The experiments conform
to the law for the protection of experimental animals (Republic of
Greece) and are registered at the Veterinary Administration of the
Republic of Greece. The enzyme preparation and assay were per-
formed as previously described.35,9 All compounds except 3–5
were tested at five concentrations, the log (dose)–response curves
were then constructed from the inhibitory data, and the IC50 values
were calculated by least-square analysis of the linear portion of the
log (dose) versus response curves (0.952 < r2 < 0.994). The experi-
ments were performed in triplicate.
4.2.5.3. (1-(3-(2H-Tetrazol-5-yl)propyl)-1H-pyrrol-3-yl)(phenyl)-
methanone (5). Yellow oil, yield 78%, IR (Nujol): 3069, 1612 cm
ꢀ1, 1H NMR (CDCl3): d 2.31 (m, 2H, CH2), 2.93 (t, 2H, CH2-tetrazole),
4.03 (t, 2H, N–CH2), 6.51–6.75 (m, 2H, pyrrolyl-4,5H), 7.20–7.75(m,
4H, phenyl-H and pyrrolyl-2H), 7.75–7.95 (m, 2H, phenyl-H), 9.95
(br s, 1H). LC–MS–MS calcd for C15H15N5O: m/z 281.127660
(100%), found: 281.123463.
4.2.6. 5-(1Y-Pyrrol-1-yl)-1Y-tetrazole (6i)
2,5-Bimethoxy-tetrahydrofuran (5.36 g, 40.6 mmol) and 4-chlo-
ropyridine hydrochloride (6.79 g, 45.3 mmol) were added in a
solution of 5-aminotetrazole (2.5 g, 29.4 mmol) in 1,4-dioxane
(210 mL), and the mixture was refluxed under a nitrogen atmo-
sphere for 3 h. After removal of the solvent under reduced pres-
sure, the mixture was treated with 50 mL EtOAc and filtered.
EtOAc was removed under reduced pressure from the filtrate and
the residue was treated with 10% NaHCO3 (20 mL) and extracted
with EtOAc (2 ꢂ 50 mL). The aqueous phase was cooled (ice bath)
and acidified with 10% HCl, and extracted with EtOAc (2 ꢂ 50
mL). The combined organic extracts were washed with brine, dried
over anhydrous sodium sulfate, and concentrated under reduced
pressure. Recrystallization from EtOAc/petroleum ether provided
6i as a white solid. yield 63%, mp 214 °C, IR (Nujol) 3154, 2748–
4.3.2. In vitro aldehyde reductase enzyme assay
ALR1 from rat kidney was partially purified according to the re-
ported procedure of Constantino et al.16 as follows: kidneys were
quickly removed from rats following euthanasia and homogenized
in a knife homogenizer followed by processing in a glass homoge-
nizer with a teflon pestle in 3 vol of 10 mM sodium phosphate buf-
fer, pH 7.2, containing 0.25 M sucrose, 2.0 mM EDTA dipotassium
salt, and 2.5 mM b-mercaptoethanol. The homogenate was centri-
fuged at 10,000 rpm at 0–4 °C for 20 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 concentration
of approx. 20 mg/mL. DEAE DE 52 resin was added to the solution
(33 mg/mL) and after gentle mixing for 15 min removed by centri-
fugation. 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.
2347, 1627 cmꢀ1 1H NMR (CDCl3/DMSO-d6) d 6.36 (s, 2H, pyrrol-
;
yl-3,4H), 7.47 (s, 2H, pyrrolyl-2,5H); Anal. Calcd for C5H5N5: C,
44.44; H, 3.73; N, 51.83. Found: C, 44.80; H, 3.42; N, 51.65.
4.2.7. 1-(2H-Tetrazole-5-yl)-1H-pyrrol-3-yl)(phenyl)methanone
(6) and 1-(2H-tetrazole-5-yl)-1H-pyrrol-2-
yl)(phenyl)methanone (7)
Aluminium chloride (0.6 g, 4.5 mmol) was dissolved in the
minimum amount of nitromethane (1 mL) and a suspension of
6i (0.5 g, 3.7 mmol) in dichloromethane (25 mL) was added to

K. Pegklidou et al. / Bioorg. Med. Chem. 18 (2010) 2107–2114
2113
4.3.3. DPPH assay
group. Conversely, steric hindrance occurs whenever part of the li-
gand clashes into the binding site: in this case, the clashing part is
accommodated along the site if possible; otherwise, the orienta-
tion is excluded. Indeed, this refinement allows only reliable orien-
tations to be processed in the next step: each orientation is
optimized within the cavity by means of successive torsions and
translations. These are driven by the ligand-target interaction en-
ergy computed by the GRID force field: each little movement is fol-
lowed by an energy reassessment according to the GRID standard
equation applied over the whole ligand and the active site:
To investigate the antiradical activity of the title compounds 6,
7 and I in a homogeneous system, a method based on the scaveng-
ing of the stable free radical DPPH was used.14 Compounds 6, 7 and
I and the reference compound 6-hydroxy-2,5,7,8-chroman-2-car-
boxylic acid (trolox) were dissolved in 1 mL of absolute ethanol
(0.4–0.025 mM) and added to 1 mL solution of DPPH in absolute
ethanol (0.4 mM) to give final concentrations of 0.2–0.0125 mM
and 0.2 mM for the tested compounds and DPPH, respectively.
The continual absorbance decrease of the ethanol solution of the
stable free radical at 517 nm, in the presence of the tested com-
pounds, was measured. The experiments were performed in
triplicate.
EGRID ¼ ELJ þ EEL þ EHB þ EENTROPY
The optimized orientations represent possible binding modes of
the ligand within the site. The interaction energy between the en-
tire ligand and the protein binding site is calculated by using the
GLUE equation, which provides an energy scoring function (EGLUE)
composed by four contributions: ESR = steric repulsion energy,
EES = electrostatic energy, ERHB = hydrogen bonding charge rein-
forcement, and EDRY = hydrophobic energy.
4.3.4. Liposome preparation, incubation and LOOH determi-
nation
The experimental protocol is based on a previously described
methodology.14 A suspension of unilamellar
L-a-phosphatidylcho-
line dioleoyl (C18:1,[cis]-9; DOPC; 99% grade) liposomes (1 mM) in
phosphate buffer (20 mL, 20 mM, pH 7.4) was prepared. The lipo-
somes (final concentration 0.8 mM DOPC) were incubated in the
EGLUE ¼ ESR þ EES þ ERHB þ EDRY
The final output of the docking procedure is a set of solutions
ranked according to the corresponding scoring function values,
each defined by the 3D coordinates of its atoms and expressed as
a PDB file. PyMol molecular graphics system was used in order to
visualize the results of the docking.43
presence of different concentrations of compound 4 (10–300 lM)
with the water-soluble initiator AAPH (final concentration
10 mM) at 50 °C for 80 min. Aliquots (1 mL) of the incubation mix-
tures 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 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).36 The thiocyanate reagent was prepared by mixing equivalent
volumes of a methanolic solution of KSCN (3%) and a ferrous
ammonium sulfate solution (45 mM in 0.2 mM HCl). After the mix-
ture 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 experiment was performed in
triplicate.
Acknowledgements
Many thanks to Professor J. Stephanidou-Stephanatou, Depart-
ment of Organic Chemistry, School of Chemistry, Aristotle Univer-
sity of Thessaloniki, Professor K. Fytianos, Enviromental Pollution
Control Laboratory, School of Chemistry, Aristotle University of
Thessaloniki for their important contribution on the reception of
LC/MS and LC–MS/MS spectra, respectively and Professor Anna
Tsantili-Kakoulidou, School of Pharmacy, University of Athens for
the permission to use the Pallas program. The authors are also
grateful to Professor Gabriele Cruciani (Laboratory for Chemomet-
rics, School of Chemistry, University of Perugia, Italy) for kindly
providing us the GRID package, to Assoc. Professor Thomas Mavro-
moustakos (Department of Chemistry, University of Athens)
for kindly allowing us access to the SYBYL molecular modelling
package.
4.4. Computational methods
The molecules 3, 6, and 7 were constructed using SYBYL molecu-
lar modeling package37, and their energies were minimized using
the Powell method with a convergent criterion provided by the Tri-
pos force field.38 The X-ray structure of human aldose reductase
holoenzyme (PDB 1US0) was used in our docking calculations after
deletion of the inhibitor IDD 594 from the PDB file, obtained from
the Brookhaven Protein Data Bank.39
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