Identification of novel pyrazole-rhodanine hybrid scaffolds as potent inhibitors of aldose reductase: Design, synthesis, biological evaluation and molecular docking analysis

Article abstract of DOI:10.1039/c6ra14531k

In an effort to develop a new class of potent aldose reductase inhibitors, a series of 1,3-diarylpyrazole assimilated 3-substituted 4-oxo-2-thioxo-1,3-thiazolidines (9a-n) was designed, and synthesized in good to excellent yields by a pharmacophore integr

Full text of DOI:10.1039/c6ra14531k

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ARTICLE
Identification of novel pyrazole‒rhodanine hybrid scaffolds as
potent inhibitors of aldose reductase: design, synthesis, biological
evaluation and molecular docking analysis
Received 00th January 2016,
Accepted 00th January 20xx
Hina Andleeb,a Yildiz Tehseen,b Syed Jawad Ali Shah,b Imtiaz Khan,a Jamshed Iqbalb* and Shahid
Hameeda*
DOI: 10.1039/x0xx00000x
www.rsc.org/
In an effort to develop a new class of potent aldose reductase inhibitors, a series of 1,3-diarylpyrazole assimilated 3-
substituted 4-oxo-2-thioxo-1,3-thiazolidines (9a‒n) was designed, and synthesized in good to excellent yields by
pharmacophore integration approach. The structures of the newly synthesized pyrazole-rhodanine derivatives were
established by readily available spectroscopic methods (FTIR, 1H and 13C NMR) and mass spectrometry. The hybrid
compounds were evaluated as aldehyde and aldose reductase inhibitors. The biological screening results identified several
compounds as remarkable inhibitors of ALR1 and ALR2. Among them, compounds 9c and 9k showed excellent activity (and
complete selectivity) towards aldose reductase enzyme with IC50 values of 1.22 ± 0.67, and 2.34 ± 0.78 µM, respectively, as
compared to the standard drug (Sorbinil; IC50 = 3.10 ± 0.20 µM). The molecular docking analysis of the most potent
inhibitor 9c was performed in order to identify the putative binding modes inside the active pocket of the enzymes. These
newly discovered aldose reductase inhibitors are believed to represent valuable lead structures to further streamline the
generation of candidate compounds to target a number of pathological conditions, most strikingly long-term diabetic
complications.
effort in this direction has led to the discovery of a large number of
AR inhibitors.11‒14
Introduction
Aldose reductase (AR) has been implicated in the etiology of
diabetic complications that may be attributed to a significant flux of
glucose through the polyol pathway, induced in tissues such as
nerves, retina, lens and kidney at elevated blood glucose levels.
Diabetes, a complex and chronic metabolic disorder, is now
recognized as a public health problem. According to epidemiological
data, 366 million people were diagnosed with diabetes in 2011, and
this number is expected to rise sharply to 552 million people within
the next 20 years.1‒3 The economic cost attributable to the
management of this disease markedly influences countries’ health
budgets, being the highest of any disease category.4,5 Hence, the
inhibition of aldose reductase is emerging as a major therapeutic
strategy for the treatment of hyperglycemia-induced cardiovascular
pathologies.6 Progression of chronic diabetes results in long-term,
debilitating complications such as cataractogenesis and
microangiopathy (including nephropathy, retinopathy and
neuropathy) thought to be linked to excess free glucose in
corresponding tissues.7,8 Numerous observations have provided
evidence of aldose reductase (AR) implication in diabetic
complications.9,10 Inhibition of aldose reductase, the first enzyme of
the polyol pathway, is therefore a useful strategy for prevention
and treatment of complications of chronic diabetes. A considerable
Aldose reductase (ALR2; EC 1.1.1.21) is the first rate-
determining enzyme in the polyol pathway and catalyses the
reduction of glucose to sorbitol with concomitant conversion of
NADPH into NADP+ (Fig. 1). Sorbitol is in turn converted into
fructose with accompanied reduction of NAD+ by sorbitol
dehydrogenase.15 Under normal circumstances, glucose is
predominantly converted to glucose-6-phosphate by hexokinase
and then enters the glycolytic pathway, whereas only a small
amount of glucose is metabolized through the polyol pathway due
to a relatively low affinity of ALR2 for this substrate. During
hyperglycemia, the polyol metabolic pathway is activated and the
increased flux of glucose through the polyol pathway triggers the
accumulation of sorbitol, which mainly happens in tissues
demonstrating insulin-independent uptake of glucose, such as lens,
kidney, retina, and peripheral nerves.16 Because of the excess sugar
alcohol (polyol), the lens imbibes water, causing osmotic imbalance.
Eventually, increased sodium and decreased potassium levels and
decreased glutathione levels lead to cataract formation. Topical
administration of aldose reductase inhibitors has been shown to
prevent the cataract in rats.17
Fig. 1. Polyol pathway for glucose metabolism.
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Fig.2. Representative examples of aldose reductase inhibitor (epalrestat; A) and pyrazole-based antidiabetic agents (B and C).
According to recent reports, AR is up-regulated not only under this speculation, we embarked on the synthesis of target pyrazole-
hyperglycemic conditions but also in other pathological states rhodanine hybrid compounds with structural variations on the
including cardiac disorders (myocardial ischemia and ischemia- aromatic moiety and determined their AR inhibitory potency using
reperfusion injury, congestive heart failure, cardiac hypertrophy an in vitro biological assay. The molecular docking investigations
and cardiomyopathy), inflammation, mood disorders, renal provided the insights on the putative binding mode of the potent
insufficiency, ovarian abnormalities and human cancers such as inhibitor inside the active site of ALR1 and ALR2.
liver, breast, ovarian, cervical and rectal cancers.5,18‒20 These
Results and discussion
pathological processes have become major threats to human health
in the 21st century. Intense efforts have been directed toward the
Chemistry
development of effective aldose reductase inhibitors,21 however,
Synthesis of intermediate pharmacophores 4a‒b and 8a‒g for the
only
a few compounds have reached clinical trials, mostly
formation of target compounds 9a‒n was accomplished according
to the synthetic routes illustrated in Schemes 1 and 2. Synthesis was
performed using modified literature procedure.43a-d,44 3-
Bromoaniline/p-toluidine (1) (1.0 equiv) was reacted with carbon
disulfide (2.0 equiv) in diethyl ether in the presence of triethylamine
thiazolidine-2,4-diones such as glitazones (ciglitazone, troglitazone,
pioglitazone and rosiglitazone).22‒26 So far, a 2-thioxothazolidine-4-
one, epalrestat (Kinedak),27,28 marketed in Japan and China, is the
only commercially available ARI drug. Apart from epalrestat, AR
inhibitors have failed in clinical trials because of poor
pharmacokinetic properties and side effects,29 and even epalrestat
has been withdrawn from the market in some countries because of
its side effects. Thus, it is important to develop novel ARIs with
improved efficacy and less toxic profile.
(2.0 equiv) as
a base to furnish triethylammonium N-aryl
dithiocarbamate (2), which on subsequent reaction with sodium
chloroacetate in ethanol/water (1:1) afforded sodium 2-(N-4-
substituted phenylcarbamothioylthio)acetate (3). The 3-aryl-2-
thioxo-1,3-thiazolidin-4-ones (4a and 4b) were synthesized in up to
85% yield by the cyclization of 3 in boiling hydrochloric acid.
Pyrazoles, key structural fragments in drug discovery, occupy a
significant position in medicinal chemistry due to their diverse
spectrum of biological activities including anti-viral, antitumor,30‒32
anti-inflammatory,33 antihyperglycemic,34,35 and antidiabetic
activities.36‒41 Inspired by these findings, and in a continuation of
our research interests for the development of novel and effective
antidiabetic agents,42a,b we focused our attention on the
heterocyclic core of epalrestat, (thioxothiazolidine) and pyrazole,
exploited previously for numerous pharmacological properties. Idea
was to couple the two bioactive nuclei (thioxothiazolidine and
pyrazole) using a hybrid pharmacophore approach to develop a
novel scaffold with favorable chemical modifications and enhanced
aldose reductase inhibitory activities, which to the best of our
knowledge, has not been reported previously (Fig. 3). Prompted by
One of the most important feature of this synthetic route is the
use
of
3-(4-substituted
phenyl)-1-phenyl-1H-pyrazole-4-
carbaldehyde as a key starting material for further transformation.
The Vilsmeier–Haack cyclization of hydrazones provided an efficient
route for the preparation of 1H-pyrazole-4-carbaldehydes.
Compounds (8a‒g) were prepared in two steps. In the first step,
substituted acetophenones (6) and phenyl hydrazine (5) were
reacted in the presence of glacial acetic acid in ethanol to afford the
hydrazone derivative (7). The treatment of hydrazone (7) under
Vilsmeier–Haack (DMF–POCl3) conditions in the second step
furnished corresponding 4-carboxaldehyde functionalized pyrazole
derivatives (8a‒g).45
Fig. 3. Structural design of pyrazole-thioxothiazolidine hybrid compounds by pharmacophore integration approach.
2 | J. Name., 2012, 00, 1-3
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Scheme 1. Synthetic pathway to 3-aryl-2-thioxo-1,3-thiazolidin-4-ones.
The reaction was carried out at 0–70 °C for 3 h using 3 cm‒1 were attributed to C=N (pyrazole) and C=S functionalities,
equivalents of the Vilsmeier–Haack reagent, followed by treatment respectively. The strong bands for carbonyl stretching frequencies
with base (NaHCO3aq) affording a series of 3-aryl-1H-pyrazole-4- appeared in the region 1710‒1698 cm‒1
carbaldehydes (8a-g) in good yields (Scheme 2).
.
In 1H NMR spectra, the most characteristic =C‒H in the pyrazole
The pyrazole-rhodanine hybrid compounds 3-(4-substituted ring resonated in the range 8.15-8.88 ppm. The olefinic proton (=C‒
phenyl)-5-((1-phenyl-3-aryl-1H-pyrazol-4-yl)methylene)-2-thioxothi- H) appeared in rather deshielded region of 7.66–7.88 ppm
azolidin-4-ones (9a–n) were synthesized by the Knoevenagel confirming the presence of Z-configuration for the exocyclic double
condensation of 3-(4-sustituted phenyl)-2-thioxothiazolidin-4-ones bond as is evident from literature45a-e
.
The reason for this
(4a and 4b) with 1-phenyl-3-(aryl)-1H-pyrazole-4-carbaldehydes deshielding is attributed to the cis position of the carbonyl oxygen
(8a–g).
The feasibility of the condensation reaction was initially
of rhodanine ring to the =CH hydrogen, and hence Z configuration
owing to the high degree of thermodynamic stability credited to
intramolecular hydrogen bond that can be formed between the
hydrogen atom of =CH group and the oxygen atom in rhodanine
carbonyl.45a 13C NMR spectra showed diagnostic chemical shifts
around 193.97–190.54 ppm due to the presence of C=S in the
rhodanine ring, whereas, the C=O (rhodanine ring) and C=N
(pyrazole ring) functional groups resonated in the regions 167.12–
165.63 and 155.63–153.96 ppm, respectively. Finally, the high
resolution mass spectra further confirmed the formation of the
target compounds 9a‒n.
examined using rhodanine (4a) and pyrazole-4-carbaldehyde (8a) as
model substrates (Table 1). The reaction was conducted using acetic
acid as a solvent and NaOAc (1.0 equiv) as a base, leading to the
desired product 9a in 40% yield (entry 1). Other solvents such as
toluene and water in combination with bases (NH4OAc and TBAB)
gave inferior results (entries 2 and 3). The change of solvent to
methanol with the addition of crystalline EDDA as a base produced
compound 9a in 49% yield (entry 4). Notably, EDDA prepared in situ
(from EDA and AcOH) remained a very appropriate base for this
condensation reaction. It was observed that a maximum yield of Enzyme inhibition studies
81% was achieved using chloroform/ethanol as a solvent at room
temperature (entry 5). However, the change of solvent to 1,4-
dioxane gave 9a in 70% yield only (entry 6).
All of the designed compounds (9a‒n) were tested for their
potential inhibition against aldehyde and aldose reductase
enzymes. The isozymes ALR1 and ALR2 belong to the aldo-keto
The scope of the Knoevenagel condensation reaction for the reductases superfamily of enzymes and thus share almost 65%
preparation of a library of pyrazole-rhodanine hybrid compounds similarity on structure as well as sequence homology. As these are
was then explored under the optimized reaction conditions (Table related closely, unwanted effects can be seen with parallel
2). With both the electron-rich and electron-poor substituents on inhibition as ALR1 is normally involved in detoxification of toxic
the aryl rings attached to rhodanine and pyrazole cores, the aldehydes.46 The main objective of the study was to identify most
reaction proceeded smoothly and provided the corresponding potent and selective inhibitors of ALR1 and ALR2 with an aim to
hybrid compounds (9a‒n) in good to excellent yields. Gratifyingly, control long-term diabetic complications using the novel
functional groups such as methoxy, methyl, nitro, fluoro, bromo, synthesized rhodanine derivatives (9a‒n). The inhibitory response
and hydroxyl were very compatible in the present reaction of the synthesized compounds was tested at various concentrations
conditions. Some of the functional groups (halogen & hydroxyl) against aldose reductase and produced significant inhibition. The
offer the opportunity for further functionalization to construct percentage inhibition of each tested compound was calculated and
more complex molecules.
IC50 ± SEM (µM) values of most potent compounds were
determined using graph pad prism. The inhibition data of the tested
series (9a‒n) is presented in Table 3.
All the synthesized compounds were characterized by readily
available analytical methods including FT-IR, 1H and 13C NMR
spectroscopy and mass spectrometry. In the IR spectra, the
stretching vibrations observed around 1600–1560 and 1350–1320
Scheme 2. Synthetic pathway to 1,3-diaryl-1H-pyrazole-4-carbaldehyde derivatives (8a-g).
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Table 1. Optimization of reaction conditions for the synthesis of 9a.[a]
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Entry
Solvent
Base (mmol)
T (°C)
Time (h)
Yield (%)[b]
1
AcOH
NaOAc (1.0 mmol)
120
24
40
2
3
4
5
6
PhMe
NH4OAc (1.0 mmol)
110
100
65
48
36
3
10
13
49
81
70
H2O
TBAB (1.0 mmol)
MeOH
EDDA (1.0 mmol)
CHCl3:MeOH (8:1)
EDA (0.2 mmol) in AcOH (2 mmol)
25
1
1,4-dioxane
EDA (0.2 mmol) in AcOH (2 mmol)
25
3
[a]The reactions were conducted using 4a (1.0 mmol) and 8a (1.2 mmol). [b]Isolated yield. TBAB
= Tetrabutylammonium bromide; EDDA =
Ethylenediammonium diacetate; EDA = Ethylenediamine.
Among the evaluated compounds, the order showing the activity relationship also illustrated that the presence of functional
highest potency of inhibition was 9h > 9m > 9a > 9n > 9e > 9f groups like methoxy, nitro, fluoro (in some cases) at the para-
against ALR1 while the inhibition potency pattern against ALR2 was position of the aryl ring attached to the pyrazole moiety resulted in
9c > 9k > 9i > 9e > 9m > 9h > 9f > 9b. Table 3 clearly demonstrated reduced inhibition of ALR1, whereas when these groups were
that the compounds 9a, 9e, 9h, 9m and 9n possess better activity as replaced with bromo and hydroxyl functionalities, the results
compared to the standard inhibitor (valproic acid) against ALR1 demonstrated higher inhibitory potency towards ALR1.
while compounds 9c and 9k show potent activity as compared to
the standard inhibitor (sorbinil) against ALR2.
In case of ALR2 inhibition, the lead compound 9c features a
combination of electron-withdrawing (bromo) group at the meta-
The structure-activity relationship analysis of the tested 3- position of the aryl ring (attached to rhodanine) and mild electron-
substituted (1,3-diaryl pyrazol-5-yl) methylene rhodanine donating (methyl) group at the para-position of the aryl ring linked
derivatives revealed that the selectivity and inhibitory activity to the pyrazole core. The replacement of bromo group in 9c with a
profile was dependent on the functional groups attached at various methyl substituent and methyl group with a more polarized nitro
positions of the aromatic ring. For example, compound 9h showed substituent furnished compound 9k which is also a potent inhibitor
the highest potency against ALR1 with an IC50 value of 1.72 ± 0.08 with an IC50 value of 2.34 ± 0.78 µM. Furthermore, compounds 9c
µM, thus approximately 30-fold stronger inhibition as compared to and 9k were also identified as selective inhibitors of ALR2. The aryl
the standard inhibitor (valproic acid; IC50 = 57.4 ± 10 µM), while rings substituted only with electron-rich (methyl and methoxy)
compound 9c showed highest potency against ALR2 with an IC50 groups resulted in compound 9i which shows significant inhibitory
value of 1.22 ± 0.67 µM, more than 2-fold stronger inhibition as effect with an IC50 value of 4.24 ± 1.32 µM. In general, the present
compared to the standard inhibitor (sorbinil; IC50 = 3.10 ± 0.20 µM). study explored a new class of aldose reductase inhibitors based on
Compound 9h being the most potent towards ALR1 incorporates pyrazole‒rhodanine compounds.
methyl group at the para-position of the aryl ring attached to the
rhodanine skeleton and an unsubstituted phenyl ring attached to
Molecular Docking
Molecular docking study of compound 9c (z-isomer) was carried out
using FlexX docking utility of LeadIT from BioSolveIT GmbH,
Germany.48 As evident from the in-vitro results, the compound 9c
shows more potent inhibition of aldose reductase (ALR2) as
compared to aldehyde reductase (ALR1). To understand the
molecular basis of such difference in activity, the molecular docking
study of the compound is carried out against both enzyme systems.
the pyrazole core. Compound 9h also demonstrates more
selectivity towards ALR1 as compared to ALR2. Introduction of an
electron-withdrawing halo group on the phenyl ring of pyrazole
produced compound 9m which is approximately 25-fold stronger
inhibitor than valproic acid. The replacement of the methyl group
(rhodanine ring) with an electron-withdrawing bromo group at
meta-position reduces the inhibitory efficacy but, still this affect is
~20-fold higher than the standard inhibitor. Thus, it can be
concluded from the results that both electron-rich and electron-
poor groups at the aryl rings are essential for the recognition of the
enzyme pocket to show the inhibition potential. The structure-
To perform molecular docking study, the parameters were
optimized after successfully reproducing the co-crystallized
reference ligand poses with root mean square deviation of 1.890
and 0.695 Å inside ALR1 and ALR2, respectively.
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Table 2. Scope of Knoevenagel condensation for the synthesis of pyrazole‒rhodanine hybrids (9a‒n).
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Table 3. Inhibitory efficacy of pyrazole‒rhodanine hybrids (9a‒n) against aldehyde and aldose reductase enzymes.
ALR1
ALR2
Compound
R1
R2
IC50 ± SEM (µM)/%inhibition
19.76%
9a
3-Br
3-Br
3-Br
3-Br
3-Br
3-Br
3-Br
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
―
H
2.93 ± 0.77
12.93%
9b
4-OMe
4-Me
4-NO2
4-F
22.68 ± 3.27
1.22 ± 0.67
38.52%
9c
39.38%
9d
16.45%
9e
22.03 ± 2.11
66.13 ± 5.67
40.92%
5.56 ± 1.66
8.08 ± 2.69
42.67%
9f
4-Br
2-OH
H
9g
9h
1.72 ± 0.08
11.07%
7.54 ± 2.67
4.24 ± 1.32
32.55%
9i
4-OMe
4-Me
4-NO2
4-F
9j
15.93%
9k
21.23%
2.34 ± 0.78
46.12%
9l
7.07%
9m
4-Br
2-OH
―
2.28 ± 0.70
5.99 ± 1.23
57.4 ± 10
―
6.33 ± 2.11
32.93%
9n
Valproic acid
Sorbinila
―
―
―
3.10 ± 0.20
aReported IC50 of 3.42 µM of Sorbinil by Rakowitz et al. 47a and Ali et al.47b
pocket formed by amino acids His110, Trp111, Thr113 and Leu300
Inside the active pocket of ALR1, the oxygen moiety of and thus leading to higher binding affinity.
Large difference in the binding free energy i.e. ‒32 KJmol‒1 (inside
thioxothiazolidin-4-one heterocycle ring of compound 9c forms
ALR2) against ‒10 KJmol‒1 (inside ALR1) of compound 9c strongly
supports the selectivity determined under in-vitro enzyme study.
The putative binding mode of the compound 9c inside ALR1 and
ALR2 is illustrated in Fig 4.
hydrogen bonding interaction with amino acid Arg309. The
compound fails to form any hydrogen bonding interaction with
catalytic amino acids His113, previously reported in case of tolrestat
and fidarestat49 which may lead to its poor binding nature and
lower binding affinity inside ALR1. Pi–alkyl interactions with amino
acid Lys23, Phe125, Ala219 and Pro301 were also observed. The
Hyde assessment50 of compound 9c inside the ALR1 active site
OSIRIS drug properties and toxicity profile
The synthesized pyrazole‒rhodanine hybrid compounds (9a‒n)
were screened computationally using online OSIRIS property
determined maximum binding affinity of ‒10 KJmol‒1
.
Despite the inability of the compound 9c to form any hydrogen explorer52 server and were found to possess an average drug score
bonding interactions with previously reported51 catalytic amino because of their lower water solubility and higher polar surface
acids such as Tyr48, His110 and Trp111, the compound binds to area. The compounds with ‒NO2 substituted groups i.e. 9d and 9k
active pocket of ALR2 with higher binding affinity of ‒32 KJmol‒1
.
were found to show lowest overall drug scores, for compound 9d,
The reason for higher binding affinity of the compound 9c inside the estimated overall drug score was found to be 0.16 because of
ALR2 was determined by Hyde assessment that reveals that the its lowest drug likeness of ‒7.42 while overall drug score of
phenyl group attached to the adjacent pyrazole ring of the compound 9k was found to be 0.19 with drug likeness of ‒5.58.
compound 9c lies in a perfect alignment inside the specificity Highest overall drug score was found to be 0.41 for compound 9n
because of the presence of a hydroxyl group with enhanced water
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solubility. Details of the OSIRIS drug properties are included in Table toxicity on tumorigenic, mutagenic, irritability and reproductive
4. When all the synthesized compounds were assessed for drug toxicity, they were found non-toxic.
Fig.
4.
Putative
binding
mode of
compound 9c (colored cyan) inside active pocket of ALR1 (colored green) and ALR2 (colored brown). The hydrogen bonding interactions, Pi-
Pi interaction and Pi-sulphur interactions are shown in green, pink and yellow colored dashed lines and Pi-charge interaction is shown in
solid yellow lines.
Conclusions
In summary, the present study reports successful design and while compounds 9c and 9k displayed strong inhibitory potential as
synthesis of a new class of aldose reductase inhibitors based on compared to the standard inhibitor (sorbinil) against ALR2. The
pyrazole-rhodanine hybrid skeleton. A diverse range of structural structure-activity relationship analysis revealed existence of
a
variations on the aromatic moiety validated the scope of delicate balance of attached functional groups to the aryl rings
synthesized derivatives. The designed compounds were tested for (attached to pyrazole and rhodanine) for potent inhibitory activity,
their ALR1 and ALR2 inhibitory potency determined through an in whereas molecular modelling investigations identified the putative
vitro biological assay using valproic acid and sorbinil as standard binding modes of the lead inhibitors in the active pocket of the
inhibitors. Several new compounds demonstrated
a strong enzymes. Hence, the identified inhibitors from the tested series of
potential to act as selective and potent inhibitors of both isozymes. novel hybrid compounds (9a‒n) may serve as selective and
Among them, 9a, 9e, 9h, 9m and 9n exhibited better activity as promising leads for the development of ALR1 and ALR2 inhibitors to
compared to the standard inhibitor (valproic acid) against ALR1, treat long term diabetic complications.
Table 4. Drug properties of pyrazole‒rhodanine hybrids (9a‒n) determined by OSIRIS property explorer
Compounds
cLogP
Solubility
Mol. Mass
TPSA
Drug Likeness
Overall Drug Score
9a
9b
9c
9d
9e
9f
4.54
4.47
4.88
3.61
4.64
5.26
4.19
4.16
4.09
4.50
3.23
4.26
‒7.33
‒7.35
‒7.67
‒7.79
‒7.64
‒8.16
‒7.03
‒6.84
‒6.86
‒7.18
‒7.30
‒7.15
517
547
531
562
535
595
533
453
483
467
498
471
95.52
104.7
95.52
141.3
95.52
95.52
115.7
95.52
104.7
95.52
141.3
95.52
2.8
0.31
0.29
0.26
0.16
0.28
0.22
0.32
0.39
0.38
0.35
0.19
0.36
2.78
1.28
‒7.42
2.1
2.74
2.35
4.67
4.63
4.46
‒5.58
3.93
9g
9h
9i
9j
9k
9l
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9m
9n
4.88
3.81
‒7.67
‒6.54
531
469
95.52
115.7
2.79
4.24
0.28
0.41
General procedure for the synthesis of phenyl-3-aryl-1H-pyrazole-
4-carbaldehydes (8a‒g)
To a stirred solution of phenyl hydrazine 5 (12 mmol) in absolute
ethanol (20 mL) was added an appropriate acetophenone 6 (10
mmol) and glacial acetic acid (0.3 mL). The reaction mixture was
stirred at 70 °C. After completion of the reaction (TLC), the excess
solvent was evaporated to yield a crude solid which was filtered,
washed with cold ethanol, dried and recrystallized (ethanol) to yield
the corresponding hydrazone (7).
Experimental section
Substrates and reagents
The acetophenones and glacial acetic acid were purchased form
Sigma-Aldrich (Steinheim, Germany). Anilines and ethylenediamine
were the products of Merck (Germany). The reagents used were of
analytical grade. Ethanol, chloroform and methanol were supplied
by Lab scan (Patuman, Thailand). Dichloromethane and diethyl
ether were the products of Riedel de Haen (seezle, Germany) while
ethyl acetate and acetone were obtained from commercial sources.
All solvents used were either anhydrous or dried.
The appropriate acetophenone phenylhydrazone (1.0 mmol)
was added to a cold solution of dimethylformamide (4 mL) and
phosphorus oxychloride (0.5 g, 3.0 mmol), and the resulting mixture
was heated at 70 °C for 3 h. After cooling to room temperature, the
reaction mixture was poured into crushed ice, neutralized with an
aqueous solution of sodium bicarbonate. The precipitated solid was
Instrumentation
All reactions were carried out using oven-dried glassware. Thin filtered and purified by flash column chromatography (20% EtOAc in
layer chromatography (TLC) was performed on Merck precoated petroleum ether) to afford pyrazole-4-carbaldehydes (8a‒g).45 The
60F254 (0.2 mm) DF-Aluminium sheets. Compounds were visualized spectro-analytical data were consistent with those reported in
by exposure to UV light at 254 nm. Flash column chromatography literature.55‒59
was carried out using silica gel (Fisher Scientific 60 Å particle size
35–70 micron). Melting points were recorded in open capillaries
using Gallenkamp melting point apparatus (MP-D) and are
uncorrected. FTIR spectra were recorded on a Thermoscientific
General procedure for the synthesis of 3-aryl-5-((1-phenyl-3-aryl-
1H-pyrazol-4-yl) methylene)-2-thioxothiazolidin-4-ones (9a–n)
To a stirred solution of 3-(4-substituted phenyl)-2-thioxothiazolidin-
4-one (4a or b) (1.0 mmol) in chloroform/methanol (8:1; 18 mL),
Fourier Transform Infra-Red Spectrophotometer Nicolet 6700 (USA)
glacial acetic acid (2 mmol) was added followed by ethylenediamine
using Attenuated total refraction (ATR) technique. NMR spectra
(0.2 mmol, 1.1 µL). The resulting mixture was stirred for 15 minutes
were acquired on Bruker DQX400 and AV300 spectrometers at
followed by the addition of an appropriate 1-phenyl-3-(4-
room temperature. All chemical shifts are reported in parts per
substituted phenyl)-1H-pyrazole-4-carbaldehyde (8a–g) (1.2 mmol)
million (ppm). For CDCl3, the shifts are referenced to 7.27 ppm for
1H NMR spectroscopy and 77.0 ppm for 13C NMR spectroscopy.
High-resolution mass spectra were recorded on a VG Autospec
spectrometer by chemical ionization or on a Micromass LCT
electrospray ionization mass spectrometer operating at a resolution
of 5000 full width half height. The elemental analysis was
performed on Leco CHNS-932 Elemental Analyzer, Leco Corporation
(USA).
at ambient temperature. After completion of the reaction (TLC; 40%
acetone in petroleum ether), the excess solvent was removed to
afford a bright yellow solid. The crude solid was washed with water,
filtered, dried and recrystallized (chloroform/ethanol) to yield
pyrazole-rhodanines (9a–n).
(Z)-3-(3-Bromophenyl)-5-((1,3-diphenyl-1H-pyrazol-4-
yl)methylene)-2-thioxothiazolidin-4-one (9a): Yellow solid (81%):
m.p 253–256 °C; Rf: 0.72 (30% acetone/n
-hexane); IR (ATR, cm‒1):
General procedure for the synthesis of 3-(4-substituted phenyl)-2-
thioxo-1,3-thiazolidin-4-ones (4a,b)
To a suspension of aniline (1) (0.1 mol) and triethylamine (0.2 mol)
in a 250 mL round bottom flask at 0 °C was dropwise added carbon
disulfide (0.2 mol) dissolved in diethyl ether (50 mL). The reaction
mixture was allowed to stir at the same temperature for 5‒6 h.
3011 (Ar‒H), 1727 (C=O), 1612 (C=N), 1568, 1509 (C=C), 1215 (C=S);
1H NMR (400 MHz, CDCl3) δ 8.16 (1H, s, =C-Hpyrazole), 7.77–7.75 (3H,
m, ArH and =C-Holefinic), 7.74–7.64 (3H, m, ArH), 7.59–7.49 (6H, m,
ArH), 7.47–7.41 (2H, m, ArH), 7.30–7.27 (1H, m, ArH); 13C NMR
(100.6 MHz, CDCl3) δ 191.56 (C=S), 166.70 (C=O), 155.25, 139.09,
135.95, 132.90, 131.62, 131.24, 130.70, 129.77, 129.25, 129.00,
127.95, 127.69, 127.28, 124.84, 122.77, 121.11, 119.67, 116.54;
HRMS (ESI) Exact mass calculated for C25H17BrN3OS2 [M+H]+:
517.99909, found: 517.99910. Analysis calcd: C, 57.92; H, 3.11; N,
8.10; S, 12.37. Found: C, 58.01; H, 3.07; N, 8.16; S, 12.43.
After
complete
precipitation,
the
triethylammonium
dithiocarbamate (2) was filtered in vacuo and washed with diethyl
ether. The solid product was used in the next step without further
purification.
To a stirred solution of sodium chloroacetate (0.1 mol) in H2O
(Z)-3-(3-Bromophenyl)-5-((3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one (9b): Yellow
(35 mL) was slowly added
a solution of triethylammonium
dithiocarbamate (2) (0.1 mol) separately dissolved in water/ethanol
(1:1) and the resulting mixture was allowed to stir at 70 °C. After
completion of the reaction (TLC), the mixture was cooled to room
temperature and cautiously poured into boiling hydrochloric acid
(12N, 40 mL). After 5 minutes, the solution was allowed to cool
slowly to room temperature to precipitate the desired 3-(4-
solid (86%): m.p 283–286 °C; Rf: 0.71 (30% acetone/n-hexane); IR
(ATR, cm‒1): 3010 (Ar-H), 2938, 2803 (CH3), 1725 (C=O), 1610 (C=N),
1565, 1502 (C=C), 1218 (C=S); 1H NMR (400 MHz, CDCl3) δ 8.16 (1H,
s, =C-Hpyrazole), 7.82 (2H, m, ArH), 7.66–7.57 (1H, s, =C-Holefinic), 7.56–
7.51 (2H, m, ArH), 7.48 (1H, t,
J = 1.9 Hz, ArH), 7.45–7.37 (3H, m,
ArH), 7.27 (1H, s, ArH), 7.06–7.02 (2H, m, ArH), 3.74 (3H, s, OCH3);
13C NMR (100.6 MHz, CDCl3) δ 191.77 (C=S), 166.65 (C=O), 154.95,
153.82, 139.05, 132.17, 131.56, 130.44, 130.36, 129.92, 129.80,
127.97, 127.72, 123.59, 123.47, 123.24, 122.46, 121.10, 119.64,
116.59, 112.45, 55.98 (OCH3); HRMS (ESI) Exact mass calculated for
C26H19BrN3O2S2 [M+H]+: 548.00966, found: 548.00964. Analysis
substituted
phenyl)-2-thioxo-1,3-thiazolidin-4-one
derivatives
(4a,b). The precipitated solid was filtered and washed with water
followed by cold ethanol, dried and recrystallized (ethanol).44,45 The
spectro-analytical data was consistent to that reported in
literature.53,54
8 | J. Name., 2012, 00, 1-3
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calcd: C, 56.94; H, 3.31; N, 7.66; S, 11.69. Found: C, 55.01; H, 3.35; solid (68%): m.p 211–213 °C; Rf: 0.34 (30% acetone/
N, 7.72; S, 11.75.
n
-hexane); IR
(ATR, cm‒1): 3433 (OH), 3019 (Ar-H), 1735 (C=O), 1613 (C=N), 1560,
1511 (C=C), 1209 (C=S); 1H NMR (400 MHz, CDCl3): δ 8.20 (1H, s, =C-
Hpyrazole), 7.88 (1H, s, =C-Holefinic), 7.68 (2H, d, = 7.9 Hz, ArH), 7.65–
7.52 (2H, m, ArH, OH), 7.49 (2H, t, = 7.7 Hz, ArH), 7.46–7.41 (2H,
m, ArH), 7.38 (2H, dd, = 7.8, 4.3 Hz, ArH), 7.32–7.25 (1H, m, ArH),
7.23–7.19 (1H, m, ArH), 7.06 (1H, d, = 8.2 Hz, ArH), 6.96 (1H, t,
(Z)-3-(3-Bromophenyl)-5-((1-phenyl-3-(p-tolyl)-1H-pyrazol-4-
J
yl)methylene)-2-thioxothiazolidin-4-one (9c): Yellow solid (83%):
m.p 223–226 °C; Rf: 0.69 (30% acetone/n
-hexane); IR (ATR, cm‒1):
J
J
3017 (Ar-H), 2948, 2813 (CH3), 1725 (C=O), 1615 (C=N), 1564, 1502
(C=C), 1214 (C=S); 1H NMR (400 MHz, CDCl3) δ 8.13 (1H, s, =C-
Hpyrazole), 7.73 (3H, m, ArH, =C-Holefinic), 7.51 (1H, m, ArH), 7.45 (2H,
J
J =
7.7 Hz, ArH); 13C NMR (100.6 MHz, CDCl3): δ 191.27 (C=S), 166.46
(C=O), 155.63, 152.13, 138.24, 135.72, 132.89, 131.50, 130.88,
130.65, 129.88, 129.00, 128.77, 127.90, 127.15, 124.11, 122.72,
122.53, 120.15, 119.40, 117.38, 116.54, 115.58; HRMS (ESI) Exact
mass calculated for C25H17BrN3O2S2 [M+H]+: 533.99401, found:
533.99414. Analysis calcd: C, 56.18; H, 3.02; N, 7.86; S, 12.00.
Found: C, 56.24; H, 3.08; N, 7.93; S, 12.05.
d, J = 7.8 Hz, ArH), 7.46–7.29 (3H, m, ArH), 7.34–7.22 (2H, m, ArH),
7.23–7.09 (3H, m, ArH), 2.35 (3H, s, CH3); 13C NMR (100.6 MHz,
CDCl3) δ 190.54 (C=S), 165.63 (C=O), 154.26, 138.23, 138.04,
134.91, 131.81, 130.57, 129.62, 128.68, 128.65, 127.80, 127.28,
126.80, 126.56, 126.23, 123.99, 121.70, 119.81, 118.57, 115.43,
20.36 (CH3); HRMS (ESI) Exact mass calculated for C26H19BrN3OS2
[M+H]+: 532.01474, found: 532.01467. Analysis calcd: C, 58.65; H, (Z)-5-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-2-thioxo-3-(p-
3.41; N, 7.89; S, 12.04. Found: C, 58.69; H, 3.48; N, 7.85; S, 12.00.
tolyl)thiazolidin-4-one (9h): Yellow solid (83%): m.p 226–229 °C; Rf:
0.76 (30% acetone/
-hexane); IR (ATR, cm‒1): 3009 (Ar-H), 2943,
n
(Z)-3-(3-Bromophenyl)-5-((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-
4-yl)methylene)-2-thioxothiazolidin-4-one (9d): Yellow solid (85%):
2817 (CH3), 1746 (C=O), 1616 (C=N), 1567, 1509 (C=C), 1219 (C=S);
1H NMR (400 MHz, CDCl3): δ 8.16 (1H, s, =C-Hpyrazole), 7.79–7.71 (3H,
m, ArH, =C-Holefinic), 7.66–7.56 (2H, m, ArH), 7.44-7.38 (5H, m, ArH),
7.37–7.27 (1H, m, ArH), 7.26–7.06 (3H, m, ArH), 2.35 (3H, s, CH3);
13C NMR (100.6 MHz, CDCl3): δ 192.25 (C=S), 167.08 (C=O), 155.15,
139.90, 139.13, 132.20, 131.33, 130.30, 129.75, 129.18, 129.00,
128.99, 128.01, 127.87, 127.65, 124.31, 121.60, 119.65, 116.64,
21.43; HRMS (ESI) Exact mass calculated for C26H20N3OS2 [M+H]+:
454.10423, found: 454.10407. Analysis calcd: C, 68.85; H, 4.22; N,
9.26; S, 14.14. Found: C, 68.91; H, 4.28; N, 9.29; S, 14.19.
m.p 282–285 °C; Rf: 0.43 (30% acetone/n
-hexane); IR (ATR, cm‒1):
3009 (Ar-H), 1722 (C=O), 1609 (C=N), 1565, 1512 (C=C), 1213 (C=S);
1H NMR (400 MHz, CDCl3): δ 8.34–8.28 (2H, m, ArH), 8.19 (1H, s, =C-
Hpyrazole), 7.84‒7.79 (2H, m, ArH), 7.78–7.73 (2H, m, ArH), 7.70 (1H,
s, =C-Holefinic), 7.60–7.56 (1H, m, ArH), 7.50 (2H, dd, J = 8.6, 7.3 Hz),
7.41–7.34 (3H, m, ArH), 7.21–7.18 (1H, m, ArH); 13C NMR (100.6
MHz, CDCl3): δ 191.00 (C=S), 166.48 (C=O), 152.27, 148.02, 138.73,
137.63, 135.65, 132.93, 131.47, 130.68, 129.81, 129.53, 128.32,
127.98, 127.11, 124.14, 122.87, 122.74, 122.65, 119.67, 116.72;
HRMS (ESI) Exact mass calculated for C25H16BrN4O3S2 [M+H]+: (Z)-5-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-
562.98417, found: 562.98414. Analysis calcd: C, 53.29; H, 2.68; N, 2-thioxo-3-(p-tolyl)thiazolidin-4-one (9i): Yellow solid (86%): m.p
9.94; S, 11.38. Found: C, 53.35; H, 2.76; N, 9.91; S, 11.42.
243–246 °C; Rf: 0.74 (30% acetone/n
-hexane); IR (ATR, cm‒1): 3012
(Ar-H), 2958, 2823 (CH3), 1716 (C=O), 1609 (C=N), 1563, 1502 (C=C),
1211 (C=S); 1H NMR (300 MHz, CDCl3): δ 8.25 (1H, s, =C-Hpyrazole),
7.86–7.83 (3H, m, ArH, =C-Holefinic), 7.66‒7.61 (2H, m, ArH), 7.58–
(Z)-3-(3-Bromophenyl)-5-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-
4-yl)methylene)-2-thioxothiazolidin-4-one (9e): Yellow solid (80%):
m.p 277–279 °C; Rf: 0.67 (30% acetone/n
-hexane); IR (ATR, cm‒1):
7.53 (2H, m, ArH), 7.44‒7.36 (3H, m, ArH), 7.20 (2H, d, J = 8.1 Hz,
3011 (Ar-H), 1710 (C=O), 1625 (C=N), 1568, 1518 (C=C), 1202 (C=S);
1H NMR (400 MHz, CDCl3): δ 8.15 (1H, s, =C-Hpyrazole), 7.77–7.72 (2 H,
m, ArH), 7.73–7.69 (1H, s, =C-Holefinic), 7.62–7.55 (3H, m, ArH), 7.51–
7.43 (2H, m, ArH), 7.41–7.31 (3H, m, ArH), 7.21–7.10 (4H, m, ArH);
13C NMR (100.6 MHz, CDCl3): δ 191.43 (C=S), 166.68 (C=O), 164.69,
162.21, 154.23, 139.01, 135.89, 132.94, 131.60, 130.84, 130.75,
130.72, 129.80, 128.03, 127.71, 127.25, 124.32, 122.79, 119.66,
116.25, 116.03; HRMS (ESI) Exact mass calculated for
ArH), 3.89 (3H, s, OCH3), 2.45 (3H, s, CH3); 13C NMR (75 MHz, CDCl3):
δ 192.32 (C=S), 167.12 (C=O), 160.44, 155.02, 139.90, 139.16,
132.31, 130.30, 130.27, 129.74, 128.01, 127.78, 127.57, 124.57,
123.77, 121.28, 119.60, 116.49, 114.46, 55.43 (OCH3), 21.42 (CH3);
HRMS (ESI) Exact mass calculated for C27H22N3O2S2 [M+H]+:
484.11479, found: 484.11459. Analysis calcd: C, 67.06; H, 4.38; N,
8.69; S, 13.26. Found: C, 67.11; H, 4.43; N, 8.74; S, 13.31.
C25H16BrFN3OS2 [M+H]+: 535.98967, found: 535.98967. Analysis (Z)-5-((1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)-2-thioxo-3-
calcd: C, 55.97; H, 2.82; N, 7.83; S, 11.95. Found: C, 56.03; H, 2.91; (p-tolyl)thiazolidin-4-one (9j): Yellow solid (85%): m.p 235–239 °C;
N, 7.87; S, 11.99.
Rf: 0.71 (30% acetone/n
-hexane); IR (ATR, cm‒1): 3049 (Ar-H), 2968,
2818 (CH3), 1735 (C=O), 1618 (C=N), 1565, 1512 (C=C), 1208 (C=S);
1H NMR (300 MHz, CDCl3): δ 8.25 (1H, s, =C-Hpyrazole), 7.86–7.83 (3H,
m, ArH, =C-Holefinic), 7.61‒7.53 (4H, m, ArH), 7.44–7.28 (5H, m, ArH),
7.21–7.18 (2H, m, ArH), 2.45 (6H, s, 2 × CH3); 13C NMR (75 MHz,
CDCl3): δ 192.33 (C=S), 167.09 (C=O), 155.25, 139.90, 139.22,
139.16, 132.31, 131.31, 129.74, 129.69, 128.87, 128.42, 128.02,
127.80, 127.60, 124.55, 121.36, 119.63, 119.49, 116.59, 21.43 (CH3);
HRMS (ESI) Exact mass calculated for C27H22N3OS2 [M+H]+:
468.11988, found: 468.11968. Analysis calcd: C, 69.35; H, 4.53; N,
8.99; S, 13.71. Found: C, 69.39; H, 4.58; N, 8.94; S, 13.65.
(Z)-3-(3-Bromophenyl)-5-((3-(4-bromophenyl)-1-phenyl-1H-
pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one (9f): Yellow
solid (78%): m.p 255–259 °C; Rf: 0.65 (30% acetone/n-hexane); IR
(ATR, cm‒1): 3009 (Ar-H), 1718 (C=O), 1625 (C=N), 1548, 1518 (C=C),
1225 (C=S); 1H NMR (400 MHz, CDCl3): δ 8.15 (1H, s, =C-Hpyrazole),
7.78–7.66 (3H, d, J = 7.6Hz, ArH), 7.73 (1H, s, =C-Holefinic)7.58 (3H, m,
ArH), 7.53–7.43 (4H, m, ArH), 7.43–7.31 (3H, m, ArH), 7.17 (1H, m,
ArH); 13C NMR (100.6 MHz, CDCl3): δ 191.38 (C=S), 166.64 (C=O),
153.96, 138.98, 135.87, 132.95, 132.22, 131.60, 130.72, 130.43,
130.22, 129.81, 128.09, 127.79, 127.25, 124.05, 123.72, 122.80,
121.64, 119.68, 116.45; HRMS (ESI) Exact mass calculated for (Z)-5-((3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-
C25H16Br2N3OS2 [M+H]+: 595.90961, found: 595.90941. Analysis thioxo-3-(p-tolyl)thiazolidin-4-one (9k): Yellow solid (84%): m.p
calcd: C, 50.27; H, 2.53; N, 7.03; S, 10.74. Found: C, 50.33; H, 2.57; 252–256 °C; Rf: 0.54 (30% acetone/n
-hexane); IR (ATR, cm‒1): 3005
N, 7.09; S, 10.79.
(Ar-H), 2928, 2822 (CH3), 1703 (C=O), 1617 (C=N), 1555, 1512 (C=C),
1223 (C=S); 1H NMR (300 MHz, CDCl3): δ 8.40 (2H, d,
= 6.9 Hz,
ArH), 8.29 (1H, s, =C-Hpyrazole), 7.92 (2H, d, = 6.9 Hz, ArH), 7.85 (2H,
d, = 7.5 Hz, ArH), 7.77 (1H, s, =C-Holefinic), 7.62–7.56 (2H, m, ArH),
J
(Z)-3-(3-Bromophenyl)-5-((3-(2-hydroxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one (9g): Yellow
J
J
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7.49–7.47 (1H, m, ArH), 7.39 (2H, d, J = 8.1 Hz, ArH), 7.18 (2H, m, The enzyme aldehyde reductase was extracted from freshly
ArH), 2.46 (3H, s, CH3); 13C NMR (75 MHz, CDCl3): 191.82 (C=S), slaughtered bovine kidneys (obtained from the local slaughter
166.98 (C=O), 152.28, 148.07, 140.09, 138.87, 137.83, 132.08, house) which were dissected carefully through the cortex area and
131.28, 130.38, 129.90, 129.62, 128.36, 127.94, 124.23, 123.22, the dissected tissue was dissolved in 3 times its volume of 10 mM
122.46, 119.74, 116.93, 21.43 (CH3); HRMS (ESI) Exact mass sodium phosphate buffer containing 2.0 mM ETDA dipotassium salt,
calculated for C26H19N4O3S2 [M+H]+: 499.08931, found: 499.08926. 2.5 mM β-mercaptoethanol and 0.25 M sucrose at 7.2 pH for
Analysis calcd: C, 62.63; H, 3.64; N, 11.24; S, 12.86. Found: C, 62.67; homogenization step. The homogenate was then further
H, 3.69; N, 11.29; S, 12.82.
centrifuged at 12000 rpm for 30 min at 4 °C. The precipitates were
discarded to remove the remaining insoluble lipids. It was further
processed with 40% ammonium sulphate saturation in order to
isolate the required ALR1 enzyme, followed by centrifugation at
12000 rpm for 30 min at 4 °C. The supernatant was again subjected
to 50% and 75% ammonium sulphate saturation after removal of
the precipitates followed by centrifugation at 12000 rpm for 30 min
at 4 °C, thus resulting in the extraction of ALR1 enzyme pellet. The
supernatant was discarded and the pellet was redissolved in the
second buffer containing sodium phosphate 10 mM at pH 7.2, 2.5
mM β-mercaptoethanol and 2.0 mM ETDA dipotassium salt. The
suspension was dialyzed overnight in the dialysis membrane in the
same buffer. The solution in the dialysis membrane contained the
extracted ALR1 and was aliquoted and stored at –80 °C until used.60
(Z)-5-((3-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-
thioxo-3-(p-tolyl)thiazolidin-4-one (9l): Yellow solid (91%): m.p
247–250 °C; Rf: 0.64 (30% acetone/n
-hexane); IR (ATR, cm‒1): 3020
(Ar-H), 2918, 2833 (CH3), 1724 (C=O), 1600 (C=N), 1549, 1512 (C=C),
1219 (C=S); 1H NMR (400 MHz, CDCl3): δ 8.13 (1H, s, =C-Hpyrazole),
7.75–7.73 (3H, m, ArH, =C-Holefinic), 7.51 (1H, m, ArH), 7.45 (2H, d, J =
7.8 ArH), 7.46‒7.29 (3H, m, ArH), 7.34‒7,22 (2H, m, ArH), 7.23‒7.16
(3H, m, ArH), 2.35 (3H, s, CH3); 13C NMR (100.6 MHz, CDCl3): δ
190.54 (C=S), 165.62 (C=O), 154.26, 138.23, 138.04, 134.91, 131.81,
130.57, 129.62, 128.68, 128.65, 127.80, 127.28, 126.80, 126.56,
126.23, 123.99, 121.70, 119.81, 118.57, 115.43, 20.36 (CH3); HRMS
(ESI) Exact mass calculated for C26H19FN3OS2 [M+H]+: 472.09481,
found: 472.09477. Analysis calcd: C, 66.22; H, 3.85; N, 8.91; S,
13.60. Found: C, 66.26; H, 3.89; N, 8.85; S, 13.63.
Isolation and purification of aldose reductase (ALR2)
(Z)-5-((3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-
thioxo-3-(p-tolyl)thiazolidin-4-one (9m): Yellow solid (90%): m.p
The enzyme ALR2 was isolated from the calf lenses removed from
the eyes of freshly slaughtered animals in the local slaughter house.
Lenses (100‒200g) were homogenized in 3 volumes of 10 mM
sodium phosphate buffer (pH 7.2), 2.0 mM EDTA dipotassium salt
and 2.5 mM β-mercaptoethanol for 20 min. Homogenate was
centrifuged at 10,000 rpm for 15 minutes at 4 °C to remove
insoluble material. Precipitated material containing lipids was
discarded. Supernatant layer was separated and ammonium
sulphate salt was added to make the saturation up to 30%. It was
centrifuged at 10,000 rpm for 15 min and the precipitate again was
discarded. Pure ALR2 was precipitated by addition of powdered
ammonium sulfate to 80% saturation. After centrifugation,
supernatant was discarded and the precipitated enzyme was
redissolved in 50 mM NaCl and dialyzed over night against 4 litres
50 mM NaCl. The volume of the suspension was recorded and the
sample was dialyzed overnight against 50 mM NaCI (double
replacement of dialysis solution). After dialysis, the volume of the
sample was recorded, treated with liquid nitrogen and samples
stored in 1 mL aliquots in the eppendorf tubes at ‒80 °C for the
determination of the total protein, enzyme activity and inhibition
studies.60
269–272 °C; Rf: 0.59 (30% acetone/n
-hexane); IR (ATR, cm‒1): 3007
(Ar-H), 2958, 2825 (CH3), 1728 (C=O), 1677 (C=N), 1564, 1508 (C=C),
1219 (C=S); 1H NMR (300MHz, CDCl3): δ 8.35 (1H, s, =C-Hpyrazole),
7.82 (2H, m, ArH), 7.77 (1H, s, =C-Holefinic), 7.68–7.64 (2H, m, ArH),
7.60‒7.53 (4H, m, ArH), 7.46‒7.36 (3H, m, ArH), 7.29 (1H, t, J = 1.3
Hz, ArH), 7.20‒7.17 (2H, m, ArH), 2.46 (3H, s, CH3); 13C NMR (75
MHz, CDCl3): δ 192.09 (C=S), 167.03 (C=O), 153.87, 139.97, 139.02,
132.20, 130.43, 130.34, 129.80, 128.02, 127.98, 127.76, 123.64,
123.54, 122.12, 119.66, 116.55, 21.43 (CH3); HRMS (ESI) Exact mass
calculated for C26H19BrN3OS2 [M+H]+: 532.01474, found: 532.01470.
Analysis calcd: C, 58.65; H, 3.41; N, 7.89; S, 12.04. Found: C, 58.68;
H, 3.46; N, 7.93; S, 12.01.
(Z)-5-((3-(2-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-
2-thioxo-3-(p-tolyl)thiazolidin-4-one (9n): Yellow solid (66%): m.p
224–228 °C; Rf: 0.42 (30% acetone/n
-hexane); IR (ATR, cm‒1): 3428
(OH), 3010 (Ar-H), 2948, 2813 (CH3), 1725 (C=O), 1610 (C=N), 1545,
1512 (C=C), 1219 (C=S); 1H NMR (300 MHz, CDCl3): δ 9.99 (1H, s,
OH), 8.88 (1H, s, =C-Hpyrazole), 7.98–7.92 (2H, m, ArH), 7.68 (1H, s,
=C-Holefinic), 7.67‒7.65 (2H, m, ArH), 7.61‒7.52 (7H, m, ArH), 7.46‒
7.35 (3H, m, ArH), 2.38 (3H, s, CH3); 13C NMR (75 MHz, CDCl3): δ
193.97 (C=S), 167.12 (C=O), 139.57, 139.15, 135.28, 133.06, 131.59,
130.28, 130.20, 130.09, 129.49, 129.26, 128.88, 128.12, 123.29,
122.61, 122.48, 119.97, 119.70, 119.17, 116.16, 21.29 (CH3); HRMS
(ESI) Exact mass calculated for C26H20N3O2S2 [M+H]+: 470.09914,
found: 470.09919. Analysis calcd: C, 66.50; H, 4.08; N, 8.95; S,
13.66. Found: C, 66.56; H, 4.13; N, 8.98; S, 13.62.
Determination of aldose reductase inhibitory activity
UV spectrophotometer was used at 340 nm in order to determine
the activity of aldehyde reductase by measuring the NADPH
consumption. Each well of the 96-well plate contains exactly 200 µL
of assay mixture containing phosphate buffer 100 mM at pH 6.2
with 20 µL of 10 mM test compound followed by addition of 70 µL
of enzyme and 40 µL of substrate. The mixture was incubated at 37
°C for 5 min and for the enzymatic reaction to run properly 0.1 mM
NADPH (50 µL) as a cofactor was added and reading was taken at
340 nm. The mixture was incubated again at 37 °C for 10 min and
reading was taken at the respective UV range in ELIZA plate reader.
As positive and negative control, 20 µL of 10 mM valproic acid and
buffer solution, respectively, were used.60 The enzymatic reaction
was run in triplicate with a final volume of 200 µL in each well.
Absorbance was noted and results were analysed via version 5
GraphPad prism software and IC50 values calculated. The total
percentage inhibition was calculated by the formula:
Enzymatic activity
All chemicals required in the enzyme extraction procedure were of
high analytical grade. For measurement of enzymatic reaction ELIZA
(Bio-Tek ELx800TM Instrument, Inc. USA) microplate reader of 340
nm UV range and 96 well-plates was used for the sample analysis.
Micropipettes from Gilson were used for sample loading. Sodium D-
glucoronate and D,L-glyceraldehyde were used as substrates along
with a cofactor i.e. NADPH (nicotinamide adenine dinucleotide
phosphate) from Sigma Aldrich (London, UK).
Isolation and purification of aldehyde reductase (ALR1)
10 | J. Name., 2012, 00, 1-3
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%Inhibition = [100 − (Absorbancetestwell/Absorbancecontrol)] × 100
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Molecular Docking
6
7
To carry out molecular docking study of compound 9c, the
corresponding structure was drawn using ACD/ChemSketch61 and
was 3D optimized. Crystal structure of porcine aldehyde reductase
PDB ID 3FX4 and crystal structure of human aldose reductase PDB
ID 1US0 was downloaded from RCSB Protein Databank.62 Using
‘Load or Prepare receptor’ utility of LeadIT from BioSolveIT GmbH,
Germany, the enzymes crystal structure was loaded and prepared.
The co-crystallized cofactor inside the receptor structure was
selected as part of the docking study. The cofactor in case of ALR1
was nicotinamide adenine dinucleotide phosphate (NADP), while in
case of ALR2, the cofactor was dihydro nicotinamide adenine
dinucleotide phosphate (NADPH). Using Protoss63 utility of the
software water handling, protonation state and tautomer state of
the active site residues was determined and used for preparing
receptor for docking.
8
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8
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{[(4-bromo-2-fluorophenyl)methyl]carbamothioyl}-5-
fluorophenoxy)acetic acid ).
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corresponding compound was performed after successfully docking
the co-crystallized reference ligands. The top most 30 docking poses
of the compound was retained for further assessment. The final
putative binding mode of the compound was selected after careful
visual and Hyde assessment.50
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5
Acknowledgements
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H. Andleeb is thankful to the Higher Education Commission of
Pakistan for the financial support under Indigenous 5000 Ph.D
Fellowship program. The University of Oxford (UK) is gratefully
acknowledged for analysis of some of the synthesized
compounds.
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12 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6RA14531K
Identification of novel pyrazole‒rhodanine hybrid scaffolds as potent inhibitors of
aldose reductase: design, synthesis, biological evaluation and molecular docking
analysis
Hina Andleeb,a Yildiz Tehseen,b Syed Jawad Ali Shah,b Imtiaz Khan,a Jamshed Iqbalb*and Shahid Hameeda*
For TOC use only:
A series of novel pyrazole-rhodanine derivatives was designed, synthesized, and biologically evaluated for their potential
inhibitory effect on both aldehyde reductase (ALR1) and aldose reductase (ALR2).