1-Hydroxypyrazole as a bioisostere of the acetic acid moiety in a series of aldose reductase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.06.062

Therapeutic intervention with aldose reductase inhibitors appears to be promising for major pathological conditions (i.e., long-term diabetic complications and inflammatory pathologies). So far, however, clinical candidates have failed due to adverse side-effects (spiroimides) or poor bioavailability (carboxylic acids). In this work, we succeeded in the bioisosteric replacement of an acetic acid moiety with that of 1-hydroxypyrazole. This new scaffold appears to have a superior physicochemical profile, while attaining inhibitory activity in the submicromolar range.

Full text of DOI:10.1016/j.bmc.2013.06.062

Bioorganic & Medicinal Chemistry 21 (2013) 4951–4957
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1-Hydroxypyrazole as a bioisostere of the acetic acid moiety
in a series of aldose reductase inhibitors
⇑
⇑
Nikolaos Papastavrou, Maria Chatzopoulou , Kyriaki Pegklidou, Ioannis Nicolaou
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Therapeutic intervention with aldose reductase inhibitors appears to be promising for major pathological
conditions (i.e., long-term diabetic complications and inflammatory pathologies). So far, however, clinical
candidates have failed due to adverse side-effects (spiroimides) or poor bioavailability (carboxylic acids).
In this work, we succeeded in the bioisosteric replacement of an acetic acid moiety with that of 1-hydrox-
ypyrazole. This new scaffold appears to have a superior physicochemical profile, while attaining inhibi-
tory activity in the submicromolar range.
Received 17 May 2013
Revised 22 June 2013
Accepted 26 June 2013
Available online 9 July 2013
Keywords:
Aldose reductase inhibitors
Bioisosterism
Ó 2013 Elsevier Ltd. All rights reserved.
Inflammatory pathologies
Long-term diabetic complications
Molecular obesity
1. Introduction
Over the years, continuous efforts on the development of aldose
reductase inhibitors (ARIs) afforded mainly two chemical classes,
Aldose reductase (ALR2) is a cytosolic enzyme, which plays a
pivotal role in a variety of pathologies.1 It first gained attention
for its implication in the onset and progression of diabetes melli-
tus’ long-term complications2 through the ‘polyol pathway’, where
ALR2 acts as an alternative route to glucose metabolism. ALR2 cat-
alyzes the first and rate-limiting reaction of this pathway, the
NADPH dependent reduction of glucose to sorbitol, which is subse-
quently oxidized by sorbitol dehydronase (SDH) in the presence of
NAD+. Excessive flow through this pathway triggers a chain of reac-
tions leading to high levels of osmotic and especially oxidative
stress that damage the cells.3,4 More recently, though, ALR2 has
been identified as a key mediator of various inflammatory diseases
and cancer.5–7 Mechanistic studies show that ALR2 reduces gluta-
thione conjugates with lipid-derived aldehydes, which in turn
through a cascade of events induce several inflammatory signals.
Therefore, ALR2 remains a compelling target for therapeutic
intervention.
spiroimids (e.g., sorbinil, fidarestat) and carboxylic acid derivatives
(e.g., epalrestat, tolrestat). However, adverse side-effects and poor
bioavailability, mainly due to low pKa values, respectively, led few
of their representatives to advanced clinical trials and just one,
epalrestat, is currently commercially available in Japan, whereas
its efficiency is considered marginal.8 Thus, the need for potent
and selective ARIs remains a problem to be solved.
In our previous work, effective ARIs have been synthesized,
which are derivatives of the pyrrolyl acetic acid moiety.9,10 In order
to overcome the disadvantages of carboxylic acid derivatives men-
tioned above, we have successfully introduced the 2,6-difluorophe-
nol and tetrazole rings as bioisosteres of the acetic acid moiety.11,12
On this basis, in this study we introduce the 1-hydroxypyrazole
ring as a new acetic acid bioisostere (Fig. 1). The 1-hydroxypyraz-
ole moiety has been used as a carboxylic acid bioisostere of glu-
tamic acid and aspartic acid resulting in analogues of the
glutamate ligands.13 The advantage of the 1-hydroxypyrazole
derivatives in comparison to the carboxylic acid and tetrazole is
the higher pKa values, which can lead to more efficient tissue per-
meation. Thus, a series of pyrrol-1-yl-1-hydroxypyrazole deriva-
tives (compounds 4a–c and 8a–c, Fig. 2) were designed,
synthesized, and tested for their inhibitory potency towards the
ALR2 enzyme. Additionally, carboxylic acids IIb–c were synthe-
sized and evaluated on ALR2 in the scope of a more wholesome
comparison. The selectivity index of all the compounds towards
the closely related aldehyde reductase (ALR1) enzyme was then
determined. Structure–activity relationships and comparison with
Abbreviations: AGEs, advanced glycation end-products; ALR1, aldehyde reduc-
tase; ALR2, aldose reductase; ARI, aldose reductase inhibitor; BEI, binding efficiency
index; DHN, 1,4-dihydroxynonene; GSH, glutathione; HNE, 4-hydroxynonenal; LE,
ligand efficiency; LELP, ligand efficiency-dependent lipophilicity; LLE, lipophilic
ligand efficiency; ROS, reactive oxygen species; SDH, sorbitol dehydrogenase.
⇑
Corresponding authors. Tel.: +30 2310997629; fax: +30 2310997852 (M.C.);
tel.: +30 2310998670; fax: +30 2310997852 (I.N.).
E-mail addresses: chatzom@pharm.auth.gr (M. Chatzopoulou), inikolao@pharm.
auth.gr (I. Nicolaou).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.062

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N. Papastavrou et al. / Bioorg. Med. Chem. 21 (2013) 4951–4957
reaction between 1 and the sterically accessible pyrrole, which
O
O
gave compound 5. A regiospecific Friedel–Crafts reaction, de-
scribed in a relative work,15 was, then, employed for the prepara-
tion of compounds 7a–c. Treatment of 5 with the appropriate
Ar
Ar
N
N
aroyl chlorides (6a–c) afforded both the
(3a–c) in various ratios, all however favoring the
ucts. In the final stage, the desired compounds 8a–c were obtained
after deprotection of compounds 7a–c by hydrogenolysis.
a
-(7a–c) and b-isomers
-isomeric prod-
OH
a
N
N
O
OH
The
a-substituted pyrrole 10c synthesis was based on the al-
Figure 1. Bioisosteric replacement of the acetic acid moiety with 1-
hydroxypyrazole.
ready published method used for compound 10b.9,16 Both com-
pounds reacted further to give the corresponding acetic acid
derivatives IIb–c, according to our previously reported work9
(Scheme 3).
the corresponding acetic acid derivatives (Fig. 2) are discussed,
along with a computer-based physicochemical profiling of the
compounds, and calculations of molecular obesity indices.
2.2. ALR2/ALR1 inhibitory activity
2. Results and discussion
2.1. Chemistry
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 ALR2,17 while the catalytic active sites of
both enzymes are considered identical.18 ALR2 inhibitory activity
is expressed as IC50 values and the results are shown in Table 1.
Listed in Table 1 are also the IC50 values of the respective acetic
acid analogues, for comparison’s sake, along with sorbinil that
was used as the reference compound.
Two different synthetic routes were followed for the synthesis
of compounds 4a–c and 8a–c, as depicted in Schemes 1 and 2,
respectively. The starting material in both routes was 1-(benzyl-
oxy)-4-iodo-1H-pyrazole (1), which was prepared via a three-step
procedure from commercially available pyrazole, according to a
known method.13
In the first synthetic pathway (Scheme 1) compounds 1 and 2a–
c reacted in a modified Ullman type coupling reaction, as used in
our previous work,14 giving the respective benzyl ethers, 3a–c.
The benzyl group was, then, reductively removed by hydrogenoly-
sis under mild conditions to selectively afford target compounds
4a–c.
The same pathway was also used in an unsuccessful effort to
synthesize compounds 8a–c. Coupling reactions between 1 and
the respective a-substituted pyrroles gave no product after 24 h,
possibly due to steric hindrance. Thus, an alternative synthetic
route (Scheme 2) was followed. The first step involved a coupling
From the obtained results it becomes apparent that all the com-
pounds are potent ARIs in the micro- or submicromolar range. Sub-
stitution on the b-position of the pyrrole ring appears favored both
in the 1-hydroxypyrazole (4a–c) and in the acetic acid series (Ia–c).
However, this pattern was not observed in our previous works on
bioisosteric replacement of the acetic acid moiety with a tetrazole
and a 2,6-difluorophenol ring.11,14 In the tetrazole series the same
inhibitory activity was exhibited for both a
- and b-regioisomers,11
though the limited number of compounds (2) disallows a definitive
conclusion. On the other hand, in a series of substituted 1-(3,5-di-
fluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]phenylmethanones the
most active derivative bore a 4-methoxyphenylacetyl substituent
on the
a
-position of the pyrrole ring.14 Supposedly, in the latter
R
O
O
R
R
R
N
N
O
N
N
O
OH
HO
a:
R=H
b: R=OCH3
c:
N
N
N
N
OH
O
O
OH
R= C6H5
4a-c
8a-c
Ia-c
IIa-c
Figure 2. Chemical structures of the studied 1-hydrozypyrazole derivatives and the respective acetic acid derivatives.
O
O
O
R
R
I
R
i
ii
N
N
+
a:
R=H
N
H
N
N
b: R=OCH3
c:
OBn
N
N
N N
R= C6H5
OH
4a-c
OBn
3a-c
1
2a-c
Scheme 1. Synthesis of the b-substituted 4-(1H-pyrrol-1-yl)-1H-pyrazol-1-ol derivatives 4a–c.

N. Papastavrou et al. / Bioorg. Med. Chem. 21 (2013) 4951–4957
4953
O
R
R
I
N
N
i
ii
N
+
O
+
N
H
N
N
O
Cl
R
OBn
R
N
N
N
N
iii
N
N
N
OBn
OBn
OBn
3a-c
6a-c
1
5
7a-c
O
a:
R=H
b: R=OCH3
c:
8a-c
N
N
R= C6H5
OH
Scheme 2. Synthesis of the
a
-substituted 4-(1H-pyrrol-1-yl)-1H-pyrazol-1-ol derivatives 8a–c.
R
R
R
N
H
O
N
iii
ii
R
N
N
O
O
i
N
H
CH2COOH
CH2COOEt
O
b
: R=-OCH3
c: R= -C6H5
9b-c
10b-c
11b-c
IIb-c
Scheme 3. Synthesis of acetic acid derivatives IIb–c.
Table 1
Inhibitory activity of compounds against rat lens ALR2, rat kidney ALR1 and the
calculated selectivity index
each series is the b-biphenyl derivative (namely 4c and Ic),
although in the 1-hydroxypyrazole series the respective methoxy
derivative (4b) exhibited a similar effect.
Compd
IC50 SEMa
(lM)
Selectivity indexb
ALR2
ALR1
The synthesized compounds were also assayed for their selec-
tivity on ALR1. ALR2 belongs to the aldoketoreductase (AKR) super-
family 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.19 Since
the closely related AKRs are related with the detoxification of toxic
aldehydes,20 parallel inhibition could cause unwanted effects. ALR1
inhibitory activity was estimated on purified rat kidney ALR1 and
is expressed as IC50 values in Table 1. Also presented in Table 1
is the calculated selectivity index, along with the reference com-
pound for ALR1, valproic acid (VA). From the obtained results it be-
comes clear that all compounds tested are less active on ALR1, and
some of the compounds to a great extent. This observation was
also reproduced in our previous studies on bioisosteric replace-
ment of the acetic acid moiety.11,14 In the present study, the biphe-
nyl moiety in the b-position of the pyrrole ring appears to increase
selectivity for ALR2 as the most selective compounds are deriva-
tives 4c and Ic.
4a
4b
4c
8a
8b
8c
Ia
Ib
1.136 0.020
0.708 0.003
0.698 0.031
13.489 0.013
15.126 0.141
9.808 0.127
1.97c
1.602 0.023
0.344 0.008
22d
10.788 0.125
10.782 0.644
62.063 2.466
>100
>100
>100
>100
>100
52.516 0.233
>100
>100
9.46
11.23
88.92
>7.41
>6.67
>10.20
>50.76
>62.42
152.66
>4.54
>21.87
>14.28
Ic
IIa
IIb
IIc
4.573 0.411
7.002 0.460
0.25e
>100
Sorbinil
VAe
56.100f
a
b
c
n = 3.
Defined as IC50[ALR1]/IC50
IC50 = 1.97 0.03 reported in Ref. 10, IC50 is expressed in
IC50 = 22 (8.5–49) reported in Ref. 10, IC50 is expressed in
.
[ALR2]
lM
SEM.
d
lM and in paren-
theses are the 95% confidence limits.
e
VA, valproic acid.
Reported in Refs. 11,14.
f
2.3. Physicochemical properties and molecular obesity indices
Focusing on potency, the resulting molecules from drug optimi-
zation programs frequently come out as too large and too lipo-
philic. This process has been described as property inflation21
and has been the main reason that drives the ‘syndrome’ called
molecular obesity,22 which represents one of the reasons for drug
candidates’ failures. In order for healthier candidates to be discov-
ered, there have been introduced certain metrics that incorporate
potency and size or lipophilicity in the same index. We proceeded
series, the bulkier six-membered ring of 2,6-difluorophenol signif-
icantly increased the intramolecular distance between the acidic
part of the molecule and its aromatic counterpart, rendering the
less extended a-derivative the most active.
Further substitution on the benzoyl moiety led to an increase in
potency in all but one 1-hydroxypyrazole derivative (8b) and in all
acetic acid derivatives as compared to the unsubstituted deriva-
tives 4a, 8a and Ia, IIa respectively. The most active compound in

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N. Papastavrou et al. / Bioorg. Med. Chem. 21 (2013) 4951–4957
Table 2
Molecular obesity indices calculated for the synthesized compounds
a
a
Compd
pIC50
logD7.4
logPa
logD7.4/logPa
pKa
MW (kDa)
LEb
BEIc
LLEd
LELPe
4a
4b
4c
8a
8b
8c
Ia
Ib
Ic
IIa
IIb
IIc
5.94
6.15
6.16
4.87
4.82
5.01
5.71
5.80
6.46
4.66
5.34
5.15
1.55
0.34
3.53
0.75
0.48
2.11
2.53
3.67
2.02
2.26
3.80
2.16
2.45
3.85
2.16
2.42
3.78
0.73
0.13
0.96
0.37
0.21
6.98
5.21
7.82
6.15
5.63
5.64
4.37
3.97
3.98
4.33
3.96
4.00
0.253
0.283
0.329
0.253
0.283
0.329
0.229
0.259
0.305
0.229
0.259
0.305
0.44
0.41
0.34
0.36
0.32
0.28
0.47
0.43
0.39
0.38
0.39
0.31
23.47
21.71
18.69
19.23
17.02
15.21
24.89
22.36
21.17
20.32
20.60
16.87
3.83
3.62
2.49
2.85
2.56
1.21
3.55
3.35
2.62
2.50
2.92
1.37
4.82
6.16
10.64
5.63
7.03
13.56
4.59
5.73
9.77
5.63
6.15
12.06
2.03
0.53
ꢀ0.87
ꢀ0.98
0.43
ꢀ0.91
ꢀ1.02
0.38
ꢀ0.40
ꢀ0.40
0.11
ꢀ0.42
ꢀ0.42
0.10
a
LogD and logP values were computationally calculated with MedChem Designer™ Version 1.0.1.15, Copyright Ó 2011 Simulations Plus, Inc. [www.simulations-plus.com]
and correspond to the S + logD and S + logP values respectively, logD represents the logarithm of the octanol-buffer[pH 7.4] distribution coefficient, pKa values were
extrapolated from the respective logD and logP values at pH = 7.4.
b
Calculated as LE = ꢀ1.4log(IC50)/N, N: number of heavy atoms.
c
Calculated as BEI = pIC50/MW.
d
Calculated as LLE = pIC50–logP.
Calculated as LELP = logP/LE.
e
to perform such calculations for the synthesized compounds and
the results are shown in Table 2.
An easily calculated index that can be used in order to depict
membrane permeation is the ratio of logD7.4/logP. As can be seen
in Table 2 the 1-hydroxypyrazole derivatives are evidently supe-
rior from the respective acetic acid derivatives. Compounds 4a
and 4c have the optimal values for membrane permeation.
Healthy leads should attain ligand efficiency (LE) values higher
than 0.3 and these values should be maintained in the optimization
process.23 As shown in Table 2 all of the synthesized compounds
fall into these boundaries with the exception of compound 8c,
which is though in close proximity. Moreover, all the compounds
fulfill the requirement of lead compounds for a binding efficiency
index (BEI) over 12.4, and have values above 14.7, the range in
which 90% of drugs are observed.24
Another metric that has recently emerged is lipophilic ligand
efficiency (LLE). LLE facilitates the discrimination between specific
and non-specific interactions during binding and as such enables
the discovery of lead compounds with less non-specific/hydropho-
bic binding. According to Perola,24 the mean LLE value for success-
ful lead compounds is 3.8. Specific binding is favored with higher
LLE values. From the synthesized compounds 4b and Ia are close
to the mean value of 3.8, and only 4a shows a slightly higher value.
From the results can also be deduced that the significantly low IC50
value of 4c, that could have rendered it as a potential lead com-
pound, is mainly attributed to non-specific interactions, stemming
from the highly lipophilic biphenyl ring system, as denoted by the
low LLE value of 2.49. As a result, in order to discover inhibitors
with highly specific binding, such moieties should be avoided dur-
ing the early stages of drug discovery in order to avoid mislead-
ingly high IC50 values.
Finally, the use of an index that incorporates both ligand effi-
ciency and lipophilicity was introduced.25 Ligand efficiency-depen-
dent lipophilicity depicts the price of LE paid in logP and is
calculated as logP/LE. Higher values of this metric correspond to
less drug-like compounds, while successful leads appear in the
range of 0 < LELP < 7.5.25 Apart from the two biphenyl derivatives
(4c and 8c), all the 1-hydroxypyrazole derivatives are in this range.
From another perspective, ALR2 is a cytosolic enzyme and the
target tissues for long-term diabetic complications require the po-
tent ARI to be highly permeable through biological barriers.26,27 As
a result, the search for efficient ARIs requires the incorporation of
parameters such as membrane permeability apart from potency.8
In order to overcome the known problems of carboxylic acid deriv-
atives in terms of membrane permeation, in the present work this
moiety was replaced by 1-hydroxypyrazole. Being less acidic (Ta-
ble 2), 1-hydroxypyrazole derivatives have a greater potential to
permeate more freely through biological barriers, while, at the same
time, retaining a minimal acidic profile, required for ALR2 inhibition.
3. Conclusions
In this work 1-hydroxypyrazole was identified as a bioisostere
of the acetic acid moiety that also bears the potential to induce
physicochemical improvements. Its derivatives exhibit improved
membrane permeation, while retaining significant inhibitory activ-
ity. Moreover, we found out that molecular obesity indices are fa-
vored in this series of compounds and compound 4a emerged as a
particularly ‘healthy’ lead compound. Additionally, 4a has
a
logD7.4/logP ratio close to 1 and as such is considered to have opti-
mal membrane permeation. Concluding we propose that com-
pound 4a could serve as
a potent lead compound in a
physicochemically superior new class of ARIs.
4. Experimental
4.1. Chemistry
General: All reagents were purchased from Sigma–Aldrich Co.
and used without further purification, except for the solvents used
for flash chromatography and recrystallization. IR spectra were ta-
ken with a Perkin–Elmer FT-IR System Spectrum BX. 1H NMR spec-
tra were recorded on a Bruker AM 300 at 300 MHz and chemical
shifts are given in d referenced to the residual solvent peak. UV–
vis measurements were taken with a UV/vis spectrometer Lambda
20. Melting points are uncorrected and were determined in open
glass capillaries using a Mel-Temp II apparatus. A Shimadzu
2010-EV LC/MS was used for obtaining LC/MS spectra. Elemental
analyses were performed by a Perkin–Elmer 2400 CHN analyzer
(in the Department of Organic Chemistry, School of Chemistry,
Aristotle University of Thessaloniki). Flash column chromatogra-
phy 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 and/or recrystallization were
routinely distilled prior to use. Petroleum ether refers to the
fraction with bp 40–60 °C. The known compounds Ia–IIa and Ib–
Ic were synthesized as previously described9,10 for the purpose of
their evaluation on the ALR2 and/or ALR1 assays.

N. Papastavrou et al. / Bioorg. Med. Chem. 21 (2013) 4951–4957
4955
4.1.1. General procedure 1: modified Ullman type coupling
reaction
ring under a nitrogen atmosphere overnight (11 h). The reaction
mixture was, then, quenched with ice and water and extracted
with CH2Cl2 (3 ꢂ 50 mL). The combined organic phases were
washed with a mildly alkaline buffer, pH = 8, (3 ꢂ 25 mL) to re-
move any traces of arylcarboxylic acid, washed with saturated
NaCl solution, dried over Na2SO4 and concentrated under reduced
pressure. The resulting residue was purified by silica gel flash col-
umn chromatography, using an appropriate mixture of petroleum
ether/ethyl acetate, (ratios varied from 10:1.5 to 10:2), as eluent,
to afford both isomers.
To a stirred solution of 1 (500 mg, 1.66 mmol) in dry toluene
(2 mL), the appropriate 3-aroyl-(1H)-pyrrole (1.39 mmol), trans-
tetramethylcyclohexane-1,2-diamine (40 mg, 0.28 mmol) and
K3PO4 (620 mg, 2.92 mmol) were added. A catalytic amount of
CuI (13 mg, 0.07 mmol) was, then, suspended and the mixture
was refluxed for 24 h under a nitrogen atmosphere. The reaction
mixture was cooled at room temperature, diluted with 4 mL of
CH2Cl2 and filtered through a plug of silica. Additional CH2Cl2
(70–90 mL) were used to wash the silica and the solvents were re-
moved under reduced pressure. Further purification of the result-
ing residue was achieved by silica gel flash column
chromatography, using an appropriate mixture of petroleum
ether/ethyl acetate (ratios varied from 25:1 to 10:3) as eluent.
4.1.2.1. (1-(1-(Benzyloxy)-1H-pyrazol-4-yl)-1H-pyrrol-2-yl)(phe-
nyl) methanone (7a).
Prepared from 6a according to general
procedure 2: Flash column chromatography yielded 47 mg (33%, col-
orless oil) of the title compound and 32 mg (22%) of 3a. IR (neat):
1636 cmꢀ1 (C@O). 1H NMR (CDCl3) d (ppm): 5.36 (s, 2H, benzyloxy
CH2), 6.26–6.32 (m, 1H, pyrrolyl H-4), 6.77–6.84 (m, 1H, pyrrolyl
H-3), 6.92–6.99 (m, 1H, pyrrolyl H-5), 7.30–7.61 (m, 10H, benzyl
H, pyrazolyl H, benzoyl H-3, H-4, H-5), 7.84 (d, J = 8.4 Hz, 2H, benzoyl
H-2, H-6). LC–MS m/z: 344.3 (M+H)+.
4.1.1.1. (1-(1-(Benzyloxy)-1H-pyrazol-4-yl)-1H-pyrrol-3-yl)(phe-
nyl) methanone (3a).
Prepared from 2a according to general pro-
cedure 1: Flash column chromatography yielded 334 mg (70%,
colorless viscous oil) of the title compound. IR (neat): 1631 cmꢀ1
(C@O). 1H NMR (CDCl3) d (ppm): 5.35 (s, 2H, benzyloxy CH2), 6.72–
6.81 (m, 2H, pyrrolyl H-4, H-5), 7.12–7.20 (m, 1H, pyrrolyl H-2),
7.26–7.61 (m, 10H, benzyl H, benzoyl H-3, H-4, H-5, pyrazolyl H),
7.86 (d, J = 7.9 Hz, 2H, benzoyl H-2, H-6). LC–MS m/z: 344.3 (M+H)+,
366.3 (M+Na)+.
4.1.2.2. (1-(1-(Benzyloxy)-1H-pyrazol-4-yl)-1H-pyrrol-2-yl)(4-
methoxyphenyl)methanone (7b).
Prepared from 6b accord-
ing to general procedure 2: Flash column chromatography yielded
98 mg (63%, yellow oil) of the title compound and 31 mg (20%) of
3b. IR (neat): 1633 cmꢀ1 (C@O). 1H NMR (CDCl3) d (ppm): 3.90
(s, 3H, OCH3), 5.35 (s, 2H, benzyloxy CH2), 6.25–6.33 (m, 1H, pyrr-
olyl H-4), 6.75–6.81 (m, 1H, pyrrolyl H-3), 6.91–7.01 (m, 3H, ben-
zoyl H-3, H-5, pyrrolyl H-5) 7.33–7.48 (m, 7H, benzyl H,
pyrazolyl H), 7.86 (d, J = 8.9 Hz, 2H, benzoyl H-2, H-6). LC–MS m/
4.1.1.2.
(1-(1-(Benzyloxy)-1H-pyrazol-4-yl)-1H-pyrrol-3-yl)(4-
Prepared from 2b according
methoxyphenyl)methanone (3b).
to general procedure 1: Flash column chromatography yielded
353 mg (68%, orange solid) of the title compound. Mp 104–107 °C.
IR (KBr): 1620 cmꢀ1 (C@O). 1H NMR (CDCl3) d (ppm): 3.91 (s, 3H,
OCH3), 5.36 (s, 2H, benzyloxy CH2), 6.73–6.79 (m, 2H, pyrrolyl H-4,
H-5), 6.99 (d, J = 8.6 Hz, 2H, benzoyl H-3, H-5), 7.14–7.20 (m, 1H,
pyrrolyl H-2), 7.26–7.46 (m, 7H, benzyl H, pyrazolyl H), 7.90 (d,
J = 8.6 Hz, 2H, benzoyl H-2, H-6). Anal. Calcd. for C22H19N3O3-
ꢁ0.05CH2Cl2: C, 70.13; H, 5.10; N, 11.13. Found: C, 70.14; H, 5.03;
N, 11.08.
z: 374.4 (M+H)+, 396.4 (M+Na)+
.
4.1.2.3. [1,10-Biphenyl]-4-yl(1-(1-(benzyloxy)-1H-pyrazol-4-yl)-
1H-pyrrol-2-yl)methanone (7c). Prepared from 6c according
to general procedure 2: Flash column chromatography yielded
66 mg (38%, yellow oil) of the title compound and 29 mg (17%) of
3c. IR (neat): 1631 cmꢀ1 (C@O). 1H NMR (CDCl3) d (ppm): 5.36 (s,
2H, benzyloxy CH2), 6.28–6.36 (m, 1H, pyrrolyl H-4), 6.84–6.90
(m, 1H, pyrrolyl H-3), 6.94–7.00 (m, 1H, pyrrolyl H-5), 7.34–7.54
(m, 10H, benzyl H, pyrazolyl H, biphenyl H-30, H-40, H-50), 7.62–
7.74 (m, 4H, biphenyl H-10, H-20, H-2, H-6), 7.93 (d, J = 8.3 Hz, 2H,
biphenyl H-3, H-5). LC–MS m/z: 420.4 (M+H)+, 442.4 (M+Na)+.
4.1.1.3. [1,10-Biphenyl]-4-yl(1-(1-(benzyloxy)-1H-pyrazol-4-yl)-
1H-pyrrol-3-yl)methanone (3c).
Prepared from 2c according
to general procedure 1: Flash column chromatography yielded
323 mg (56%, white solid) of the title compound. Mp 126–129 °C.
IR (KBr): 1617 cmꢀ1 (C@O). 1H NMR (CDCl3) d (ppm): 5.37 (s, 2H,
benzyloxy CH2), 6.75–6.87 (m, 2H, pyrrolyl H-4, H-5), 7.14–7.23
(m, 1H, pyrrolyl H-2), 7.32–7.57 (m, 10H, benzyl H, pyrazolyl H,
biphenyl H-30, H-40, H-50), 7.63–7.77 (m, 4H, biphenyl H-20, H-60,
H-2, H-6), 7.94 (d, J = 8.1 Hz, 2H, biphenyl H-3, H-5). Anal. Calcd.
for C27H21N3O2: C, 77.31; H, 5.05; N, 10.02. Found: C, 77.01; H,
5.09; N, 9.92.
4.1.3. General procedure 3: hydrogenolysis reaction
To
a stirred solution of the appropriate benzyl ether
(0.77 mmol) in 14 mL of dry MeOH/THF mixture (1:1), ammonium
formate (271 mg, 4.3 mmol) was added and the reaction mixture
was cooled to 0 °C under a nitrogen atmosphere. Palladium on car-
bon 10% (271 mg, 2.5 mmol) was, then, suspended to the cold mix-
ture, which was stirred further for 1 h. Afterwards, the reaction
mixture was diluted with 10 mL of MeOH and filtered through a
Whatman filter paper, which retained the Pd/C. Additional MeOH
(ca. 50 mL) was used to wash the Pd/C and the combined solvents
were removed under reduced pressure. The residue was dissolved
in equal parts of H2O/ethyl acetate (150 mL), the organic layer was
collected, dried over Na2SO4 and concentrated in vacuo. The result-
ing residue was recrystallized with CH2Cl2/petroleum ether.
4.1.1.4. 1-(Benzyloxy)-4-(1H-pyrrol-1-yl)-1H-pyrazole (5).
Pre-
pared from pyrrole according to general procedure 1: Flash column
chromatography yielded 256 mg (77%, colorless crystals) of the title
compound. Mp 80–82 °C. 1H NMR (CDCl3) d (ppm): 5.34 (s, 2H, ben-
zyloxy CH2), 6.22–6.30 (m, 2H, pyrrolyl H-3, H-4), 6.73–6.82 (m, 2H,
pyrrolyl H-2, H-5), 7.06–7.14 (m, 1H, pyrazolyl H-3), 7.32–7.50 (m,
6H, benzyl H, pyrazolyl H-5). Anal. Calcd. for C14H13N3O: C, 70.28;
H, 5.48; N, 17.56. Found: C, 69.91; H, 5.38; N, 17.36.
4.1.3.1. (1-(1-Hydroxy-1H-pyrazol-4-yl)-1H-pyrrol-3-yl)(phenyl)
4.1.2. General procedure 2: Friedel–Crafts aroylation reaction
To a stirred suspension of AlCl3 (67 mg, 0.50 mmol) in 5 mL of
1,2-dichloroethane at room temperature and under a nitrogen
atmosphere, the corresponding chloride (132 mg, 0.94 mmol)
was added and the mixture was left to stir for 15 min. Afterwards,
5 (100 mg, 0.42 mmol) was dissolved in 1.1 mL of 1,2-dichloroeth-
ane and added to the reaction mixture. The reaction was left stir-
methanone (4a).
Prepared from 3a according to general proce-
dure 3: Recrystallization afforded 113 mg (58%, pink solid) of the
title compound. Mp 122 °C. IR (nujol): 1625 cmꢀ1 (C@O). 1H NMR
(CDCl3–DMSO-d6)
d (ppm): 6.75–6.81 (m, 1H, pyrrolyl H-4),
6.82–6.88 (m, 1H, pyrrolyl H-5), 7.29–7.34 (m, 1H, pyrazolyl H-
3), 7.35–7.40 (m, 1H, pyrrolyl H-2), 7.42–7.60 (m, 4H, benzoyl H-
3, H-4, H-5, pyrazolyl H-5), 7.64 (d, J = 7.1 Hz, 2H, benzoyl H-2,

4956
N. Papastavrou et al. / Bioorg. Med. Chem. 21 (2013) 4951–4957
H-6). Anal. Calcd. for C14H11N3O2: C, 66.40; H, 4.38; N, 16.59.
Found: C, 66.57; H, 4.58; N, 16.31.
4.1.4. [1,10-Biphenyl]-4-yl(1H-pyrrol-2-yl)methanone (10c)
mixture of [1,10-biphenyl]-4-yl(morpholino)methanone
A
(529 mg, 2 mmol) and phosphoryl chloride (0.4 mL, 4.3 mmol)
was stirred at room temperature for 6 h. A solution of pyrrole
(0.14 mL, 4.3 mmol) in 1,2-dichloroethane (10 mL) was added
and the reaction mixture was stirred for a further 14 h at room
temperature. After hydrolysis with 10% aqueous sodium carbonate
solution (10 mL), the organic layer was separated and the aqueous
layer was extracted with CH2Cl2 (2 ꢂ 20 mL). The combined organ-
ic layers were dried (Na2SO4) and the solvents were removed un-
der reduced pressure. Further purification of the resulting residue
was achieved by silica gel flash column chromatography, using a
mixture of petroleum ether/ethyl acetate 7:1 as eluent. Flash col-
umn chromatography and consequent recrystallization of the res-
idue from CH2Cl2/petroleum ether yielded 247 mg (50% yellow
solid) of the title compound. Mp 184–187 °C. IR (KBr): 1606 cmꢀ1
(C@O). 1H NMR (CDCl3) d (ppm): 6.34–6.43 (m, 1H, pyrrolyl H-4),
6.93–7.03 (m, 1H, pyrrolyl, H-3), 7.20–7.24 (m, 1H, pyrrolyl H-5),
7.37–7.57 (m, 3H, biphenyl H-30, H-40, H-50), 7.63–7.80 (m, 4H,
biphenyl H-20, H-60, H-2, H-6), 8.03 (d, J = 8.5 Hz, 2H, biphenyl H-
3, H-5), 10.00 (br, 1H, N-H). Anal. Calcd. for C17H13NOꢁ0.05CH2Cl2:
C, 81.41; H, 5.25; N, 5.57. Found: C, 81.55; H, 5.35; N, 5.55.
4.1.3.2.
(1-(1-Hydroxy-1H-pyrazol-4-yl)-1H-pyrrol-3-yl)(4-
Prepared from 3b accord-
methoxyphenyl)methanone (4b).
ing to general procedure 3: Recrystallization afforded 110 mg
(51%, red solid) of the title compound. Mp 165–166 °C. IR (KBr):
1606 cmꢀ1 (C@O), 3451 cmꢀ1 (O–H). 1H NMR (CDCl3–DMSO-d6) d
(ppm): 3.82 (s, 3H, OCH3), 6.66–6.73 (m, 1H, pyrrolyl H-4), 6.78–
6.85 (m, 1H, pyrrolyl H-5), 6.91(d, J = 8.8 Hz, 2H, benzoyl H-3, H-
5), 7.20–7.26 (m, 1H, pyrazolyl H-3), 7.30–7.36 (m, 1H, pyrrolyl
H-2), 7.43–7.49 (m, 1H, pyrazolyl H-5), 7.82 (d, J = 8.8 Hz, 2H, ben-
zoyl H-2, H-6). Anal. Calcd. for C15H13N3O3ꢁ0.05CH2Cl2: C, 62.87; H,
4.59; N, 14.61. Found: C, 62.71; H, 4.63; N, 14.49.
4.1.3.3. [1,10-Biphenyl]-4-yl(1-(1-hydroxy-1H-pyrazol-4-yl)-1H-
pyrrol-3-yl)methanone (4c).
Prepared from 3c according to
general procedure 3: Recrystallization afforded 142 mg (56%, white
solid) of the title compound. Mp 166–168 °C. IR (KBr): 1622 cmꢀ1
(C@O), 3412 cmꢀ1 (O–H). 1H NMR (CDCl3–DMSO-d6) d (ppm):
6.67–6.76 (m, 1H, pyrrolyl H-4), 6.78–6.87 (m, 1H, pyrrolyl H-5),
7.18–7.23 (m, 1H, pyrazolyl H-5), 7.26–7.29 (m, 1H, pyrazolyl H-
3), 7.32–7.42 (m, 3H, biphenyl H-30, H-40, H-50), 7.43–7.49 (m, 1H,
pyrrolyl H-2), 7.51–7.67 (m, 4H, biphenyl H-20, H-60, H-2, H-6),
7.83 (d, J = 7.6, 2H, biphenyl H-3, H-5). Anal. Calcd. for C20H15N3-
4.1.5. General procedure 4: preparation of the ethyl acetic acid
ester derivatives
.
O2 0.05CH2Cl2: C, 72.19; H, 4.56; N, 12.60. Found: C, 72.46; H,
To a cold (ice bath), stirred, and under a nitrogen atmosphere
mixture of the corresponding (aroyl-1H-pyrrol-3-yl)methanones
(10b–c) (15 mmol), ethyl bromoacetate (4.05 g, 24 mmol), and
[tris[2-(methoxyethoxy)ethyl]amine [TDA-1] (0.75 mL, 2.4 mmol)
in toluene (225 mL) was added NaH (50% dispersion in mineral
oil) (1.05 g, 22.5 mmol). The resulting mixture was stirred at room
temperature for 24–48 h. After this period, it was poured into a stir-
red, ice cold, mixture of Et2O (150 mL) and 5% HCl (150 mL). The
two phases were separated, and the aqueous phase was extracted
with Et2O (2 ꢂ 100 mL). The combined organic extracts were
washed with 10% NaHCO3 (2 ꢂ 100 mL) and saturated NaCl solu-
tion and dried (Na2SO4). The solvents were evaporated under re-
duced pressure, and the residue was either flash
chromatographed with a suitable mixture of EtOAc/petroleum
ether (11c) followed by recrystallization from Et2O/petroleum
ether (11b).
4.72; N, 12.35.
4.1.3.4. (1-(1-Hydroxy-1H-pyrazol-4-yl)-1H-pyrrol-2-yl)(phenyl)
methanone (8a).
Prepared from 7a according to general proce-
dure 3: Recrystallization afforded 48 mg (66%, white solid) of the
title compound. Mp 160–162 °C. IR (KBr): 1634 cmꢀ1 (C@O),
3451 cmꢀ1 (O–H). 1H NMR (CDCl3) d (ppm): 6.32 (dd, J = 4.0,
1.6 Hz, 1H, pyrrolyl H-4), 6.85 (dd, J = 4.0, 2.6 Hz, 1H, pyrrolyl, H-
3), 7.03–7.12 (m, 1H, pyrrolyl H-5), 7.31–7.39 (m, 1H, pyrazolyl
H-3), 7.41–7.52 (m, 2H, benzoyl H-3, H-4), 7.53–7.62 (m, 1H, ben-
zoyl H-4), 7.63–7.70 (m, 1H, pyrazolyl H-5), 7.82 (d, J = 7.0 Hz, 2H,
benzoyl H-2, H-6). Anal. Calcd. for C14H11N3O2ꢁ0.05CH2Cl2: C,
65,53; H, 4.34; N, 16.32. Found: C, 65.70; H, 4.50; N, 16.35.
4.1.3.5.
(1-(1-Hydroxy-1H-pyrazol-4-yl)-1H-pyrrol-2-yl)(4-
Prepared from 7b accord-
methoxyphenyl)methanone (8b).
ing to general procedure 3: Recrystallization afforded 47 mg (57%,
white solid) of the title compound. Mp 179–180 °C. IR (KBr):
1628 cmꢀ1 (C@O), 3451 cmꢀ1 (O–H). 1H NMR (CDCl3) d (ppm):
3.87 (s, 3H, OCH3), 6.27 (dd, J = 3.9, 2.7 Hz, 1H, pyrrolyl H-4), 6.76
(dd, J = 3.9, 2.7 Hz, 1H, pyrrolyl, H-3), 6.93 (d, J = 8.8 Hz, 2H, ben-
zoyl H-3, H-5), 6.98–7.02 (m, 1H, pyrrolyl H-5), 7.23–7.26 (m, 1H,
pyrazolyl H-3), 7.53–7.61 (m, 1H, pyrazolyl H-5), 7.83 (d,
J = 8.8 Hz, 2H, benzoyl H-2, H-6). Anal. Calcd. for C15H13N3O3-
ꢁ0.05CH2Cl2: C, 62.87; H, 4.59; N, 14.61. Found: C, 62.71; H, 4.50;
N, 14.48.
4.1.5.1. Ethyl 2-(2-(4-methoxybenzoyl)-1H-pyrrol-1-yl)acetate
(11b).
Prepared from 10b according to general procedure 4:
Recrystallization afforded 3707 mg, 86% white solid) of the title
compound. Mp 55 °C. IR (nujol): 1620 cmꢀ1 (C@O), 1756 cmꢀ1
(EtO–C@O). 1H NMR (CDCl3) d (ppm): 1.29 (t, J = 7.1 Hz, 3H, CH3),
3.89 (s, 3H, OCH3), 4.25 (q, J = 7.1 Hz, 2H, OCH2), 5.13 (s, 2H,
COCH2), 6.21–6.28 (m, 1H, pyrrolyl H-4), 6.77–6.84 (m, 1H, pyrrolyl
H-3), 6.90–7.00 (m, 3H, benzoyl H-3, H-5, pyrrolyl H-5), 7.85 (d,
J = 8.5 Hz, 2H, benzoyl H-2, H-6). Anal. Calcd. for C16H17NO4: C,
66.89; H, 5.96; N, 4.88. Found: C, 66.43; H, 6.16; N, 5.05.
4.1.3.6. [1,10-Biphenyl]-4-yl(1-(1-hydroxy-1H-pyrazol-4-yl)-1H-
4.1.5.2. Ethyl 2-(2-([1,10-biphenyl]-4-carbonyl)-1H-pyrrol-1-yl)
pyrrol-2-yl)methanone (8c).
Prepared from 7c according to
acetate (11c).
Prepared from 10c according to general proce-
general procedure 3: Recrystallization afforded 57 mg (60%, white
solid) of the title compound. Mp 153–155 °C. IR (KBr): 1625 cmꢀ1
(C@O), 3406 cmꢀ1 (O–H). 1H NMR (CDCl3) d (ppm): 6.34 (dd,
J = 4.0, 2.6 Hz, 1H, pyrrolyl H-4), 6.92 (dd, J = 4.0, 1.6 Hz, 1H, pyrr-
olyl, H-3), 7.09 (dd, J = 2.6, 1.6 Hz, 1H, pyrrolyl H-5), 7.33–7.39
(m, 1H, pyrazolyl H-5), 7.40–7.54 (m, 3H, biphenyl H-30, H-40, H-
50), 7.59–7.75 (m, 5H, pyrazolyl H-3, biphenyl H-20, H-60, H-2, H-
6), 7.92 (d, J = 8.4 Hz, 2H, biphenyl H-3, H-5). Anal. Calcd. for C20-
H15N3O2ꢁ0.1CH3COOC2H5: C, 72.46; H, 4.71; N, 12.43. Found: C,
72.82; H, 4.72; N, 12.16.
dure 4: Recrystallization afforded 2500 mg, 50% white solid) of
the title compound. Mp 86–88 °C. IR (KBr): 1617 cmꢀ1 (C@O),
1720 cmꢀ1 (EtO–C@O). 1H NMR (CDCl3)
d (ppm): 1.32 (t,
J = 7.1 Hz, 3H, CH3), 4.29 (q, J = 7.1 Hz, 2H, OCH2), 5.15 (s, 2H,
COCH2), 6.30 (dd, J = 4.0, 2.6 Hz, 1H, pyrrolyl H-4), 6.91 (dd,
J = 4.0, 1.6 Hz, pyrrolyl, H-3), 6.97–7.03 (m, 1H, pyrrolyl H-5),
7.36–7.56 (m, 3H, biphenyl H-30, H-40, H-50), 7.62–7.78 (m, 4H,
biphenyl H-20, H-60, H-2, H-6), 7.93 (d, J = 8.4 Hz, 2H, biphenyl H-
3, H-5). Anal. Calcd. for C21H19NO3: C, 75.66; H, 5.74; N, 4.20.
Found: C, 75.41; H, 5.81; N, 4.15.

N. Papastavrou et al. / Bioorg. Med. Chem. 21 (2013) 4951–4957
4957
4.1.6. General procedure 5: preparation of the acetic acid
derivatives
7.2, containing 0.25 M sucrose, 2.0 mM EDTA dipotassium salt, and
2.5 mM b-mercaptoethanol. The homogenate was centrifuged at
10,000g at 0–4 °C for 30 min and the supernatant was subjected
to ammonium sulfate fractional precipitation at 40%, 50%, and
75% salt saturation. The pellet obtained from the last step, possess-
ing ALR1 activity, was redissolved in 10 mM sodium phosphate
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.
A mixture of the previously described ethyl ester derivatives
(6 mmol), dioxane (100 mL), and 5% aqueous NaOH solution
(100 mL) was stirred at room temperature for 1 h. After this period,
it was concentrated to half of its volume, H2O (100 mL) was added,
and the mixture was cooled (ice bath) and acidified with concen-
trated HCl. The formed precipitate was collected by filtration,
and the filtrate was extracted with CHCl3 (2 ꢂ 100 mL). The organic
phase was washed with saturated NaCl solution and evaporated
under reduced pressure. The residue was purified by recrystalliza-
tion from toluene/petroleum ether (IIb) or ethyl acetate/petroleum
ether (IIc).
4.1.6.1. 2-(2-(4-Methoxybenzoyl)-1H-pyrrol-1-yl)acetic acid
(IIb).
Prepared from 11b according to general procedure 5:
Recrystallization afforded 1353 mg, 87% white solid) of the title
compound. Mp 142–143 °C. IR (nujol): 1615 cmꢀ1 (C@O),
1740 cmꢀ1 (HO–C@O), 3300-2580 cmꢀ1 (O–H). 1H NMR (CDCl3) d
(ppm): 3.90 (s, 3H, OCH3), 5.06 (s, 2H, COCH2), 6.28 (dd, J = 3.7,
2.4 Hz, 1H, pyrrolyl H-4), 6.81–6.86 (m, 1H, pyrrolyl H-3), 6.93–
7.04 (m, 3H, benzoyl H-3, H-5, pyrrolyl H-5), 7.87 (d, J = 8.7 Hz,
2H, benzoyl H-2, H-6). Anal. Calcd. for C14H13NO4: C, 64.86; H,
5.05; N, 5.40. Found: C, 64.75; H, 5.26; N, 5.45.
4.2.2.1. Enzyme assays.
solved in 0.2 M NaHCO3. ALR2 and ALR1 activities were assayed
spectrophotometrically by determining NADPH consumption at
340 nm. In order to determine ALR2 inhibitory activity D,L-glyceral-
dehyde was used as a substrate and the measurements took place
The target compounds were dis-
in 30 °C, whereas ALR1 inhibitory activity was determined with
D-glycuronate as a substrate and the measurements took place in
37 °C. All the compounds were tested at five concentrations, the
log(dose)–response curves were then constructed from the inhibi-
tory data, and the IC50 values were calculated by least-square anal-
ysis of the linear portion of the log(dose) versus response curves.
The experiments were performed in triplicate.
4.1.6.2. 2-(2-([1,10-Biphenyl]-4-carbonyl)-1H-pyrrol-1-yl)acetic
acid (IIc).
Prepared from 11c according to general procedure
5: Recrystallization afforded 1686 mg, 92% white solid) of the title
compound. White solid, yield 92%. Mp 207-208 °C. IR (KBr):
1606 cmꢀ1 (C@O), 1673 cmꢀ1 (HO–C@O), 3451 cmꢀ1 (O–H). 1H
NMR (CDCl3–DMSO-d6) d (ppm): 5.07 (s, 2H, COCH2), 6.17–6.23
(m, 1H, pyrrolyl H-4), 6.76–6.82 (m, 1H, pyrrolyl, H-3), 6.90–6.96
(m, 1H, pyrrolyl H-5), 7.30–7.46 (m, 3H, biphenyl H-30, H-40, H-
50), 7.54–7.65 (m, 4H, biphenyl H-20, H-60, H-2, H-6), 7.83 (d,
Acknowledgment
This work was supported by Aristotle University’s Research
Committee under Action Gamma: Support of Basic Research.
.
J = 8.1 Hz, 2H, biphenyl H-3, H-5). Anal. Calcd. for C19H15NO3 0.05-
References and notes
CH2Cl2: C, 73.91; H, 4.92; N, 4.52. Found: C, 74.05; H, 4.98; N, 4.44.
1. Alexiou, P.; Pegklidou, K.; Chatzopoulou, M.; Nicolaou, I.; Demopoulos, V. J.
Curr. Med. Chem. 2009, 16, 734.
2. Ramasamy, R.; Goldberg, I. J. Circ. Res. 2010, 106, 1449.
3. Giacco, F.; Brownlee, M. Circ. Res. 2010, 107, 1058.
4.2. ALR2/ALR1 inhibitory activity
4. Srivastava, S. K.; Ramana, K. V.; Bhatnagar, A. Endocr. Rev. 2005, 26, 380.
5. Yadav, U. C.; Ramana, K. V.; Srivastava, S. K. Chem. Biol. Interact. 2011, 191, 339.
6. Tammali, R.; Srivastava, S. K.; Ramana, K. V. Curr. Cancer Drug Targets 2011, 11,
560.
ALR2 and ALR1 in vitro assays have been previously described
in detail.9,11,12,14
7. Yadav, U. C.; Srivastava, S. K.; Ramana, K. V. Curr. Mol. Med. 2010, 10, 540.
8. Chatzopoulou, M.; Alexiou, P.; Kotsampasakou, E.; Demopoulos, V. J. Expert
Opin. Ther. Pat. 2012, 22, 1303.
9. Nicolaou, I.; Demopoulos, V. J. J. Med. Chem. 2003, 46, 417.
10. Demopoulos, V. J.; Rekka, E. J. Pharm. Sci. 1995, 84, 79.
11. Pegklidou, K.; Koukoulitsa, C.; Nicolaou, I.; Demopoulos, V. J. Bioorg. Med. Chem.
2010, 18, 2107.
12. Nicolaou, I.; Zika, C.; Demopoulos, V. J. J. Med. Chem. 2004, 47, 2706.
13. Petersen, J. G.; Bergmann, R.; Moller, H. A.; Jorgensen, C. G.; Nielsen, B.; Kehler,
J.; Frydenvang, K.; Kristensen, J.; Balle, T.; Jensen, A. A.; Kristiansen, U.; Frolund,
B. J. Med. Chem. 2013, 56, 993.
14. Chatzopoulou, M.; Mamadou, E.; Juskova, M.; Koukoulitsa, C.; Nicolaou, I.;
Stefek, M.; Demopoulos, V. J. Bioorg. Med. Chem. 2011, 19, 1426.
15. Kotsampasakou, E.; Demopoulos, V. J. Bioorg. Med. Chem. 2013, 21, 869.
16. White, J.; McGillivray, G. J. Org. Chem. 1977, 42, 4248.
17. Chung, S. S. M.; Ho, E. C. M.; Lam, K. S. L.; Chung, S. K. J. Am. Soc. Nephrol. 2003,
14, S233.
4.2.1. ALR2 preparation
Lenses were quickly removed from Fischer-344 rats of both
sexes following euthanasia. 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. ALR2 from rat lens was partially purified according to
the reported procedure as follows: lenses were quickly removed
from rats following euthanasia and stored at ꢀ20 °C until used.
The lenses were homogenized in 5 vol of cold distilled water. The
homogenate was centrifuged at 10,000g at 0–4 °C for 15 min. The
supernatant was precipitated with saturated ammonium sulfate
at 40% salt saturation and this solution was centrifuged at
10,000g at 0–4 °C for 15 min. The latter supernatant was either
used directly or stored for maximum 24 h at ꢀ80 °C.
18. Gui, T.; Tanimoto, T.; Kokai, Y.; Nishimura, C. Eur. J. Biochem. 1995, 227, 448.
19. Rees-Milton, K. J.; Jia, Z.; Green, N. C.; Bhatia, M.; El-Kabbani, O.; Flynn, T. G.
Arch. Biochem. Biophys. 1998, 355, 137.
20. Barski, O. A.; Tipparaju, S. M.; Bhatnagar, A. Drug Metab. Rev. 2008, 40, 553.
21. Tarcsay, A.; Keseru, G. M. J. Med. Chem. 2013, 56, 1789.
22. Hann, M. M. MedChemComm 2011, 2, 349.
4.2.2. ALR1 preparation
Kidneys were quickly removed from Fischer-344 rats of both
sexes following euthanasia. 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. ALR1 from rat kidney was partially purified according to
the reported procedure as follows: kidneys were homogenized in a
knife homogenizer followed by processing in a glass homogenizer
with a teflon pestle in 3 vol of 10 mM sodium phosphate buffer, pH
23. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
24. Perola, E. J. Med. Chem. 2010, 53, 2986.
25. Keseru, G. M.; Makara, G. M. Nat. Rev. Drug Disc. 2009, 8, 203.
26. Ko, K.; Won, H.; Won, Y. Bioorg. Med. Chem. 2006, 14, 3090.
27. 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.