Chalcones and their pyrazine analogs: synthesis, inhibition of aldose reductase, antioxidant activity, and molecular docking study

Article abstract of DOI:10.1007/s00706-018-2146-6

Abstract: Chalcones and their pyrazine analogs synthesized by Claisen–Schmidt condensation were tested for inhibition of aldose reductase, which is the key enzyme in the development of secondary diabetic complications. The most active compounds exerted IC₅₀ values within the micromolar scale, and their interactions with the enzyme were described in a molecular docking study. Antioxidant activity of several representative compounds was explored in DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, revealing significant scavenging for 4-hydroxy-substituted derivatives endowed with electron-donating methoxy substituent in position 3 of the ring B. To conclude, the novel chalcones hydroxylated and methoxylated in the B-ring and their pyrazine analogs exhibited significant aldose reductase inhibition activity, albeit lower in comparison with the reference epalrestat. Medium antioxidant activity (not exceeding the antioxidant efficacy of the standard Trolox) was shown by the representative compounds tested.

Full text of DOI:10.1007/s00706-018-2146-6

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ORIGINAL PAPER
Chalcones and their pyrazine analogs: synthesis, inhibition of aldose
reductase, antioxidant activity, and molecular docking study
Marta Kucerova-Chlupacova1
Martin Dosedel2
Jiri Kunes3
Marta Soltesova-Prnova4
•
•
•
•
Magdalena Majekova4
Milan Stefek4
•
Received: 1 November 2017 / Accepted: 8 January 2018
Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
Chalcones and their pyrazine analogs synthesized by Claisen–Schmidt condensation were tested for inhibition of aldose
reductase, which is the key enzyme in the development of secondary diabetic complications. The most active compounds
exerted IC50 values within the micromolar scale, and their interactions with the enzyme were described in a molecular
docking study. Antioxidant activity of several representative compounds was explored in DPPH (2,2-diphenyl-1-picryl-
hydrazyl) assay, revealing significant scavenging for 4-hydroxy-substituted derivatives endowed with electron-donating
methoxy substituent in position 3 of the ring B. To conclude, the novel chalcones hydroxylated and methoxylated in the
B-ring and their pyrazine analogs exhibited significant aldose reductase inhibition activity, albeit lower in comparison with
the reference epalrestat. Medium antioxidant activity (not exceeding the antioxidant efficacy of the standard Trolox) was
shown by the representative compounds tested.
Graphical abstract
Keywords Aldol reactions Á Enones Á Molecular modeling Á Structure–activity relationships
Introduction
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00706-018-2146-6) contains supplementary
material, which is available to authorized users.
Chalcones (Fig. 1), chemical derivatives of 1,3-diphenyl-
prop-2-en-1-ones, can be obtained by isolation from plants
& Marta Kucerova-Chlupacova
kucerom@faf.cuni.cz
through isolation or by means of laboratory synthesis
[1, 2]. The natural chalcones belong to the group of fla-
1
Department of Pharmaceutical Chemistry and Pharmaceutical
Analysis, Faculty of Pharmacy, Charles University,
Hradec Kralove, Czech Republic
vonoids and they are biosynthetically related to them.
Therefore, a range of bio-activities is associated with var-
ious chalcone derivatives and their analogs [3, 4]. Despite
2
Department of Social and Clinical Pharmacy, Faculty of
Pharmacy, Charles University, Hradec Kralove, Czech
Republic
being natural products, chalcones are easily synthesized,
thus enabling a multiplicity of substitutions [5].
Aldose reductase (EC 1.1.1.21 [6], AKR1B1 [7], ALR2)
3
Department of Organic and Bioorganic Chemistry, Faculty of
Pharmacy, Charles University, Hradec Kralove, Czech
Republic
acts as the first enzyme of the polyol metabolic pathway
catalyzing the reduction of glucose to sorbitol with
NADPH as a coenzyme [8]. Osmolyte sorbitol accumulates
intracellularly, which may result in disruption of the
4
Institute of Experimental Pharmacology and Toxicology,
Slovak Academy of Sciences, Bratislava, Slovak Republic
123

M. Kucerova-Chlupacova et al.
activity. Inhibitory activity on ALR2 was expected, espe-
cially with chalcones containing an acidic functional group
in the ring (phenolic hydroxyl). Additionally, one repre-
sentative with an electron-donating group (methoxyl) and
one derivative with an electron-withdrawing group (nitro
group) were included in the study. A pyrazine ring in place
of a benzene ring A of chalcones represents an interesting
alternative in the search for new aldose reductase inhibi-
tors. Pyrazine is a stable and sufficiently aromatic analog
ring to benzene in comparison with other diazines
(pyrimidine and pyridazine) [16]. It is very slightly basic
(pKa 0.65, - 6.6) [17] and might help to improve the
water-solubility of chalcones. A comparison of two series
with identical substitution could also provide information
about the importance of the ring analogy. The antioxidant
effect of the synthesized compounds was also explored as a
beneficial side effect of potential ALR2 inhibitors [13].
E/Z-configuration
O
2´
5´
2
1
1´
3´
4´
3
4
6
6´
5
ring A
ring B
s-cis/s-trans-conformation
Fig. 1 General pattern of chalcone derivatives with rings and their
numbering indicated; the depicted isomer represents (E)-(s-cis)-
chalcone
osmotic homeostasis in tissues exposed to hyperglycemia.
The polyol pathway thus represents one of the key
molecular mechanisms contributing to the glucose toxicity
responsible for the development of chronic diabetic com-
plications such as diabetic neuropathy, nephropathy,
retinopathy and cataracts. Carboxylic acid derivatives (e.g.,
drug epalrestat used for the prophylaxis of diabetic
nephropathy and retinopathy, Fig. 2), spirohydantoins and
related cyclic amides and phenolic derivatives (related to
benzopyran-4-one and chalcones) belong to the chemical
pattern exhibiting the inhibition of ALR2 [9]. Synthetic
chalcones inhibiting ALR2 were the subject of a patent
application [10]; several previous studies have been
reviewed in Refs. [11, 12].
Results and discussion
Chemistry
Chalcones 1–14 were synthesized by Claisen–Schmidt
condensation combining aryl/pyrazine-2-yl methyl ketone
and variously substituted aldehyde under different condi-
tions (Scheme 1, for substitution see Table 1). Method A—
methanol with a sodium hydroxide pellet [18–20] was used
for synthesis of 1,3-diphenylprop-2-en-1-ones 2, 3, 5, and
6. The procedure differed from the published ones in the
working-up (acidification necessary) and purifying process
(column chromatography). Method B—pyridine with
diethylamine was applied for synthesis of substituted
3-phenyl-1-pyrazin-2-ylprop-2-en-1-ones 7–14, as was
previously shown [21–24], and for the synthesis of (E)-3-
(2-hydroxyphenyl)-1-phenylprop-2-en-1-one (1) as well.
We faced some difficulties throughout the synthesis of
3-(4-hydroxy-3-methoxyphenyl)-1-phenylprop-2-en-1-one
(4). Neither method A nor method B resulted in the desired
product, despite method A being modified in the amount of
solvent or sodium hydroxide and working-up (column
chromatography). Using potassium hydroxide as a basic
catalyst [25] helped obtain the compound 4. All the
methods used a basic catalysis. The best yields were
obtained with method A (31–67%) in comparison with
methods C (40%) and B (10–44%). Method B is more
suitable for synthesis of 3-phenyl-1-pyrazinylprop-2-en-1-
ones, with the lower yields being caused by the chro-
matographic cleaning process. In all cases, E-isomers were
obtained. This was confirmed by the value of interaction
An antioxidant effect is advantageous for ALR2 inhi-
bitors, as increased activity of ALR2 in the polyol pathway
leads to oxidative stress [13]. Some natural chalcones
(Fig. 3) perform this type of activity, e.g., isoliquritigenin,
licochalcone B and D (all from Glycyrrhiza spp., Faba-
ceae), broussochalcone A (Broussonetia papyrifera L.,
Moraceae), xanthohumol (Humulus lupulus L., Cannaba-
ceae) [8] and other natural prenylated chalcones [14]. At
least one hydroxy substitution and a,b-unsaturated bond
seem to be essential for phenoxy radical formation and its
stabilization [8]. Concurrently, isoliquiritigenin is an ALR2
inhibitor [15].
The goal of this work was the synthesis and study of a
group of chalcone derivatives and their pyrazine analogs
(Scheme 1) as potential inhibitors of aldose reductase.
These compounds have never been tested on this type of
1
constants J in H NMR spectra.
Fig. 2 Structure of epalrestat, drug used for the prophylaxis of
diabetic nephropathy and retinopathy
123

Chalcones and their pyrazine analogs: synthesis, inhibition of aldose reductase, antioxidant…
Fig. 3 Natural chalcones with
antioxidant activity
Scheme 1
M
Table 1 Inhibition of ALR2
Compound
X
Ring A substitution R1
Ring B substitution R2
Inhibition of ALR2 IC50/lM
isolated from rat eye lenses by
the synthesized compounds in
comparison with the reference
epalrestat
1
CH
CH
CH
CH
CH
CH
N
–
2-OH
[ 100
2
–
3-OH
[ 100
3
–
4-OH
25 \ IC50 \ 50
[ 100
4
–
4-OH-3-OCH3
4-OCH3
4-NO2
2-OH
5
–
[ 50
6
–
[ 50
7
–
[ 100
8
N
–
3-OH
25 \ IC50 \ 50
25 \ IC50 \ 50
25 \ IC50 \ 50
18.59 1.81
[ 100
9
N
–
4-OH
10
N
CH2CH2CH3
4-OH
11
N
CH(CH3)2
4-OH
12
N
–
–
–
–
4-OH-3-OCH3
4-OCH3
4-NO2
–
13
N
[ 100
14
N
[ 50
0.23 0.02
Epalrestat
–
123

M. Kucerova-Chlupacova et al.
Table 2 Antiradical activity
measured by the initial velocity
of DPPH absorbance decrease at
518 nm
Enzyme inhibition
Compound
(- DA/s)
3
\ 0.001
All synthesized compounds were tested for inhibition of
aldose reductase isolated from rat eye lenses. The inhibi-
tory concentrations corresponding to 50% inhibition (IC50)
in comparison with the reference epalrestat are provided in
Table 1. From the results, it is apparent that in the series of
chalcone derivatives 1–6 a significant inhibition was
recorded only for derivative 3, with 4-hydroxy substituent
at the B-ring. In the group of the pyrazine analogs, sig-
nificant inhibition activity was recorded for derivatives
hydroxylated in the 3- or 4-position of the B-ring; for
4-hydroxy-substituted derivatives, the highest inhibition
was recorded for compound 11 with an iso-propyl sub-
stituent in position 5 of the A-ring. Based on IC50 values, a
one to two orders lower inhibition efficacy was recorded
for the tested compounds in comparison to previously
published chalcones hydroxylated in the 4- and 2-positions
in ring A [26–28].
4
0.0032 0.0002
\ 0.001
9
12
0.0023 0.0004
0.0157 0.0006
Trolox
absorbance decrease, corresponding to the transfer of the
most labile H atoms, was followed by a much slower
absorbance decline. This represented the residual antirad-
ical activity of the antioxidant degradation products. The
initial velocity of DPPH decolorization was used as a
marker of the antiradical activity and is shown in Table 2.
As shown in Fig. 4 and Table 2, significant antioxidant
activity was recorded for 4-hydroxy-substituted derivatives
4 and 12 endowed with electron-donating methoxy sub-
stituent in position 3. Nonetheless, the efficacy was lower
than that of standard Trolox.
The ethanolic solution of the DPPH radical (50 lM) was
incubated in the presence of the 100 lM compound tested.
Absorbance decrease at 518 nm during the first 70-s
interval (3, 4 and 9, 12) or 30-s interval (Trolox) was
determined. Results are mean values SD of the three
measurements.
Antioxidant activity
Four representative compounds of the tested group were
inspected for antioxidant action in the DPPH (2,2-diphe-
nyl-1-picrylhydrazyl) test. As a weak hydrogen atom
abstractor, DPPH is considered a good kinetic model for
peroxyl ROOÁ radicals [29]. DPPH assay is routinely used
as a primary screening test for antiradical efficacy. The
time-dependent decrease of the characteristic absorbance
of the ethanolic solution of DPPH at 518 nm in the pres-
ence of the compounds tested and in comparison with the
reference Trolox is shown in Fig. 4. The initial fast
Molecular modeling
To explain the experimental ALR2 inhibition data obtained,
molecular modeling in silico was performed. Conformation
analysis revealed that, owing to stronger conjugation in
pyrazine-based chalcones, their molecules are strictly pla-
nar. In the chalcone group, rotation of the benzene ring is
looser, resulting in slight distortion of the ring. This con-
clusion is in accordance with our previous results concerning
conformation analysis of 3-(2-hydroxyphenyl)-1-pyrazin-2-
ylprop-2-en-1-ones [30]. Other authors concerned with con-
formational analysis calculations [31–33] or crystallographic
studies [34–40] on substituted 1,3-diphenylprop-2-en-1-ones
or their analogs [41–43] revealed that the dihedral angle
between the aromatic rings amounts to 0°–20°. Another
stereochemical characteristic of chalcones represents the
conformation of a single bond connecting carbonyl func-
tional group and double bond in the linkage chain [31]
(indicated in Fig. 1). In solid phase, the chalcones usually
exert in (E)-s-cis form, whereas in solution, a minor part of
the compound can occur as (E)-s-trans-conformer [33].
Based on the IR spectra, we have concluded that the
structures characterized here were in s-cis conformation
due to the missing band around 1636 cm-1. Compounds 7,
9, and 12 described earlier have shown the presence of both
conformers [30].
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0
500
1000
1500
t / s
Fig. 4 Continual absorbance decrease of ethanolic solution of DPPH
radical (50 lM) in the presence of 100 lM concentration of the
compounds tested (filled square) 3; (filled circle) 4; (open square) 9;
(open circle) 12; (filled triangle) Trolox
123

Chalcones and their pyrazine analogs: synthesis, inhibition of aldose reductase, antioxidant…
(A)
(B)
Fig. 5 a Key interactions of compound 3 with the active binding site
of aldose reductase: O interacts with N of LEU300 as H-acceptor at a
- 2.9 kJ/mol, and ring A interacts with 6-ring of TRP111 via p–p
˚
interaction at a distance of 3.49 A. b Key interactions of compound 4
˚
distance of 2.75 A and energy - 11.7 kJ/mol, ring B interacts with
with the active binding site of aldose reductase: ring A interacts with
˚
˚
PHE122 via p–H interaction at a distance of 4.02 A and energy
6-ring of TRP111 via p–p interaction at a distance of 3.45 A
(A)
(B)
˚
interaction at a distance of 3.35 A. b Key interactions of compound
Fig. 6 a Key interactions of compound 9 with the active binding site
of aldose reductase: O interacts with N of LEU300 as an H-acceptor
˚
at a distance of 3.04 A and energy - 3.8 kJ/mol, ring B interacts with
12 with the active binding site of aldose reductase: ring B interacts
˚
with PHE122 via p–H interaction at a distance of 4.08 A, and ring A
˚
PHE122 via p–H interaction at a distance of 3.85 A and energy
interacts with ring A of TRP111 via p–p interaction at a distance of
˚
3.41 A
- 2.9 kJ/mol, and ring A interacts with 6-ring of TRP111 via p–p
Molecular docking of the compounds tested into a
human aldose reductase (pdb code: 1z3n) binding site
revealed a common feature in the binding of the com-
pounds. All derivatives were found in the cavity of the
active site anchored by the p–p interaction of a pyrazine or
benzene ring with TRP111. Lacking negatively charged
group, the compounds were not visibly attracted to the
positively charged nicotinamide. Derivatives 3 and 9 with a
benzene ring substituted only by a hydroxyl group were
able to approach LEU300 by carbonyl oxygen to create a
hydrogen bond (Figs. 5, 6).
bond. However, 12 was bound via p–H interaction with
PHE122, as was 9 (Fig. 6).
Considering the binding mode of our compounds, we
detected an interaction with TRP111 which is able to
interact with the hydrophilic (pyrazine in 9 and 12) as well
with the hydrophobic part (benzene in 3 and 4) of chalcone
derivatives. TRP111 and LEU300 are shared by the cat-
alytic anionic pocket and by the hydrophobic pocket
[44, 45]. Although some authors claim that LEU300 tends
to bind to lipophilic scaffold of inhibitors [46], in the case
of our compounds it interacted slightly with carbonyl
oxygen via hydrogen bonding. Generally, amino acids
TYR48, HIS110 [9, 46, 47] and TRP111 [46] are
Methoxy-substituted derivatives 4 and 12 were not able
to adopt a proper position for the formation of the hydrogen
123

M. Kucerova-Chlupacova et al.
Fig. 7 Positions of 3 (cyan), 4
(dark green), 9 (light pink), and
12 (dark pink) anchored by the
p–p interaction with TRP111
highlighted for interaction with inhibitory ligands in the
active binding site. However, PHE122 was included in the
amino acids of the specific ALR2 pocket [48]. PHE122 was
reported to interact with the more lipophilic ring of chal-
cone derivatives [49]; in the case of chalcones synthesized
in frame of this work, PHE122 always interacted with the
ring B. Superposition of all four compounds in the binding
site of the enzyme is shown in Fig. 7.
(Prague, Czech Republic). 1-(Pyrazin-2-yl)ethan-1-one was
available in our laboratory prepared previously according to
Refs. [50, 51]. Silica gel 0.040–0.063 nm (Merck, Prague,
Czech Republic) was used for column chromatography. The
purity of the products was checked by TLC on aluminium
sheets, silica gel 60 F254 (Merck, Prague, Czech Republic).
Analytical samples were dried over anhydrous phosphorus
pentoxide under reduced pressure at room temperature.
¨
Melting points were determined either on a Boetius appa-
ratus or Stuart SMP20 and were corrected. Elemental anal-
yses (C, H, N, S) were conducted using an EA 1110 CHNS
instrument (CE Instruments, Milano, Italy) or Vario Micro
Cube Elemental Analyzer (Elementar Analysensysteme
GmbH, Hanau, Germany). Their results were found to be in
good agreement ( 0.3%) with the calculated values.
Infrared spectra were recorded either in KBr pellets with a
Nicolet Impact 400 IR spectrophotometer (Thermo Scien-
tific, Waltham, MA, USA) or on germanium crystal using
ATR method with a Nicolet Impact 6700 IR spectropho-
tometer (Thermo Scientific). 1H and 13C NMR spectra were
corroborated at ambient temperature with a Varian Mercury-
Vx BB 300 spectrometer operating at 300 MHz for 1H and
75 MHz for 13C or with a VNMR S500 spectrometer oper-
ating at 500 MHz for 1H NMR and 125 MHz for 13C NMR
(both from Varian Corp., Palo Alto, CA, USA). Chemical
shifts are indirectly referenced to tetramethylsilane (TMS)
via the solvent signal (2.49 ppm for 1H, 39.7 ppm for 13C in
DMSO-d6 and 7.26 ppm for 1H, 77.0 ppm for 13C in CDCl3).
Conclusion
To conclude, the synthesized chalcones hydroxylated and
methoxylated in ring B and their pyrazine (ring A) analogs
exhibited significant aldose reductase inhibition activity,
albeit lower in comparison with the reference drug epalre-
stat. Generally, the pyrazine analogs of chalcone showed
higher inhibition of aldose reductase than chalcones
(diphenylpropenones) and presence of 4-hydroxy group in
the ring B seems to be essential for the inhibitory activity.
The molecular modeling study confirmed interaction with
the same amino acid residues involved in the binding mode
of other inhibiting compounds described in literature. Med-
ium antioxidant activity was demonstrated for both deriva-
tives with 4-hydroxy-3-methoxy substitution, but it did not
exceed the antioxidant efficacy of the standard Trolox.
Experimental
Synthetic procedures
Acetophenone, vanillin, 2-, 3-, and 4-hydroxybenzaldehyde,
4-methoxybenzaldehyde, and 4-nitrobenzaldehyde were
used as starting materials and were purchased from Merck
Syntheses and characteristics of pyrazine analogs of chal-
cone have already been described in our papers and
123

Chalcones and their pyrazine analogs: synthesis, inhibition of aldose reductase, antioxidant…
corresponded to the previously published data: (E)-3-(2-
hydroxyphenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (7), (E)-
3-(4-hydroxyphenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (9),
(E)-3-(4-hydroxyphenyl)-1-(5-propylpyrazin-2-yl)prop-2-
en-1-one (10), and (E)-3-(4-hydroxy-3-methoxyphenyl)-1-
(pyrazin-2-yl)prop-2-en-1-one (12) in Ref. [21], (E)-3-(3-
hydroxyphenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (8) in
Ref. [22], (E)-3-(4-hydroxyphenyl)-1-(5-isopropylpyrazin-
2-yl)-2-en-1-one (11) in Ref. [24], and (E)-3-(4-nitro-
phenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (14) in Ref. [23].
the desired compounds were combined, evaporated, and
recrystallized (repeatedly if needed) from absolute ethanol
to obtain the following compounds as analytically pure
crystals:
(E)-3-(2-Hydroxyphenyl)-1-phenylprop-2-en-1-one (1) Yield:
0.76 g (41%); Rf = 0.45 (hexane–ethyl acetate 7:3); m.p.:
152.0–155.0 °C (Ref. [56] 154–156 °C decomp.).
(E)-3-(4-Methoxyphenyl)-1-(pyrazin-2-yl)prop-2-en-1-one
(13, C14H12N2O2) Yield: 0.24 g (10%); Rf = 0.45 (hex-
ane–ethyl acetate 6:4); m.p.: 91.0–93.0 °C; 1H NMR
(300 MHz, CDCl3): d = 9.36 (d, 1H, J = 1.7 Hz, H30),
8.75 (d, 1H, J = 2.5 Hz, H60), 8.68 (dd, 1H, J = 2.5 Hz,
1.7 Hz, H50), 8.06 (d, 1H, J = 15.9 Hz, CH), 7.95 (d, 1H,
J = 15.9 Hz, CH), 7.72–7.64 (m, AA0, BB0, 2H, H2, H6),
6.98–6.90 (m, AA0, BB0, 2H, H3, H5), 3.85 (s, 3H, OCH3)
ppm; 13C NMR (75 MHz, CDCl3): d = 188.4, 162.0,
148.7, 147.2, 145.7, 144.8, 143.3, 130.8, 127.5, 117.6,
Method A
Acetophenone (0.01 mol) and appropriate substituted
benzaldehyde (0.01 mol) were dissolved in 30 cm3
methanol. Five NaOH pellets were added to this solution.
The reaction mixture was stirred at r.t. for 24 h [18–20].
The reaction mixture was then diluted with 100 cm3 of
H2O, acidified by 98% acetic acid to pH 6–7 and cooled in
a refrigerator. The precipitated solid was filtered and
recrystallized from anhydrous ethanol, repeatedly if nee-
ded. The following compounds of analytical purity were
obtained (spectroscopic data for known compounds can be
found in the Supplementary Material):
ꢀ
114.4, 55.4 ppm; IR (ATR-Ge): m = 3045 (CH arom,
alkene), 2958, 2921 (CH aliph), 1661 (C=O), 1591
(CH=CH), 1260 (C–O ether arom), 1167 (C=N pyrazine),
1057 (C=C pyrazine), 991 (C–H trans-alkene) cm-1
.
Method C
(E)-3-(3-Hydroxyphenyl)-1-phenylprop-2-en-1-one (2) Yield:
0.69 g (31%); Rf = 0.56 (hexane–ethyl acetate 6:4); m.p.:
162.0–166.0 °C (Ref. [52] 156–158 °C from ethanol).
Solid KOH (16.8 g. 0.3 mol) was added to a mixture of
1.01 g acetophenone (8.4 mmol) and 1.28 g vanillin
(8.4 mmol) in 16.8 cm3 MeOH and 8.4 cm3 H2O. The
resulting solution was refluxed for 1.5 h, cooled in an ice-
water bath, then acidified with 33.6 cm3 conc. HCl. The
solution was diluted with 80 cm3 water and stored in a
refrigerator overnight. The reaction mixture was extracted
into ethyl acetate and prepared for column chromatogra-
phy. Recrystallization from anhydrous ethanol resulted in
the following product:
(E)-3-(4-Hydroxyphenyl)-1-phenylprop-2-en-1-one (3) Yield:
1.00 g (45%); Rf = 0.50 (hexane–ethyl acetate 6:4); m.p.:
185.0–188.0 °C (Ref. [53] 187–188 °C).
(E)-3-(4-Methoxyphenyl)-1-phenylprop-2-en-1-one (5) Yield:
1.23 g (52%); Rf = 0.45 (hexane–ethyl acetate 8:2); m.p.:
72.0–75.0 °C (Ref. [54] 76 °C).
(E)-3-(4-Nitrophenyl)-1-phenylprop-2-en-1-one (6) Yield:
1.69 g (67%); Rf = 0.48 (hexane–ethyl acetate 8:2); m.p.:
166.0–168.0 °C (Ref. [55] 164.0–164.5 °C).
(E)-3-(4-Hydroxy-3-methoxyphenyl)-1-phenylprop-2-en-1-one
(4) Yield: 0.86 g (40%); Rf = 0.13 (hexane–ethyl acetate
8:2); m.p.: 92.6–94.6 °C (Ref. [57] 86–87 °C).
Method B
Biology
Acetophenone or 1-(pyrazin-2-yl)ethan-1-one (0.01 mol)
and the corresponding substituted benzaldehyde (0.01 mol)
were dissolved in 4.4 cm3 pyridine. Diethylamine
(0.01 mol) was added and the reaction mixture was stirred
at 80–120 °C for 1 h. After cooling, the mixture was
poured into 300 cm3 ice water, acidified to pH 3 with
acetic acid, and then refrigerated for 24 h. The mixture was
extracted with diethyl ether and subjected to column
chromatography. Hexane–ethyl acetate (ratio indicated at
Rf values) was used as the eluent. The fractions containing
Male Wistar rats 8–9 weeks old, weighing 200–230 g,
were used as organ donors. The animals came from the
Breeding Facility of the Institute of Experimental Phar-
macology Dobra Voda (Slovak Republic). The study was
approved by the Ethics Committee of the Institute and
performed in accordance with the Principles of Laboratory
Animal Care (NIH publication 83-25, revised 1985) and
the Slovak law regulating animal experiments (Decree 289,
Part 139, July 9, 2003).
123

M. Kucerova-Chlupacova et al.
enzyme-cofactor complex by the program YASARA [61] by
using YAMBER3 force field and utilizing the flexible
ligands option. The first five clusters (if there) were then
optimized in water. The simulation cell (overlapping the
Inhibition of aldose reductase
Aldose reductase preparation from rat lenses has been
reported previously [58]. Aldose reductase activity was
assayed spectrophotometrically by determining NADPH
consumption at 340 nm and was expressed as a decrease in
optical density (OD)/s/mg protein. The effect of the com-
pounds on enzyme activities was determined by including
the inhibitor in the reaction mixture at required concen-
trations dissolved in DMSO at 1% final concentration. At
the same concentration, the inhibitor was included in the
reference blank. The reference blank contained all the
above reagents except the substrate, in order to correct for
oxidation of NADPH not associated with reduction of the
substrate. The enzyme reaction was initiated by addition of
substrate and monitored for up to 4 min after an initial
period of 1 min. Enzyme activities were adjusted by
diluting the enzyme preparations with distilled water. Thus
0.05 cm3 of the preparation gave an average reaction rate
for the control sample of 0.020 0.005 absorbance units/
min. IC50 values (the concentration of the inhibitor
required to produce 50% inhibition of the enzyme reaction)
were determined both from the least-square analysis of the
linear portion of the semi-logarithmic inhibition curves and
non-linear regression analysis. Each curve was generated
using at least four concentrations of inhibitor, resulting in
an inhibition in the range of 25–75%.
˚
complex by 8 A) was filled by water molecules (final density
0.997 g/cm3) and ions Na? and Cl- in the amount of 0.9% of
the overall water environment. The ratio of ions was chosen
to eliminate the total charge of the complex. Re-calculation
of pKa values of amino acids was performed and a pH of 6.2
was maintained. A standard optimization protocol was used,
consisting of the steepest gradient optimization, molecular
dynamics and simulated annealing. The final geometries
were then analyzed according to the binding place of the
inhibitor and the value of the binding energy.
Acknowledgements This study was funded by the research program
Development and Study of Drugs Progres Q42 (Charles University,
Czech Republic), and the VEGA 2/0033/14 and VEGA 2/0041/15
Grants of the Ministry of Education, Science, Research and Sport of
the Slovak Republic.
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