Development of novel pyrazolone derivatives as inhibitors of aldose reductase: An eco-friendly one-pot synthesis, experimental screening and in silico analysis

Article abstract of DOI:10.1016/j.bioorg.2014.02.002

Aldose reductase is the key enzyme of polypol pathway leading to accumulation of sorbitol. Sorbitol does not diffuse across the cell membranes easily and therefore accumulates within the cell, causing osmotic damage which leads to retinopathy (cataractoge

Full text of DOI:10.1016/j.bioorg.2014.02.002

Bioorganic Chemistry 53 (2014) 67–74
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Development of novel pyrazolone derivatives as inhibitors of aldose
reductase: An eco-friendly one-pot synthesis, experimental screening
and in silico analysis
Aparna Kadam a, Bhaskar Dawane a, Manisha Pawar a, Harshala Shegokar b, Kapil Patil b, Rohan Meshram b,
Rajesh Gacche b,
⇑
a School of Chemical Sciences, Swami Ramanand Teerth Marathwada University, Nanded (MS), India
b School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded (MS), India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 November 2013
Available online 19 February 2014
Aldose reductase is the key enzyme of polypol pathway leading to accumulation of sorbitol. Sorbitol does
not diffuse across the cell membranes easily and therefore accumulates within the cell, causing osmotic
damage which leads to retinopathy (cataractogenesis), neuropathy and other diabetic complications.
Currently, aldose reductase inhibitors like epalrestat, ranirestat and fidarestat are used for the ameliora-
tion of diabetic complications. However, such drugs are effective in patients having good glycemic control
and less severe diabetic complications. In present study we have designed novel pyrazolone derivative
and performed eco-friendly synthesis approach and tested the synthesized compounds as potential
inhibitors of aldose reductase activity. Additional in silico analysis in current study indicates presence
of highly conserved chemical environment in active site of goat lens aldose reductase. The reported data
is expected to be useful for developing novel pyrazolone derivatives as lead compounds in the manage-
ment of diabetic complications.
Keywords:
Goat lens aldose reductase
Pyrazolone derivatives
Carbothioamide derivatives
Polyethylene glycol (PEG-400)
Paired potential analysis
Virtual screening
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
to be developed. Thus, research on AR inhibition currently domi-
nates the area of anti-diabetic therapeutics regimen.
In recent past, diabetes was believed to be associated with
overindulgent life-style and thus was thought as major health
concern in industrialized countries. Recent reports have revealed
that this disease has now engulfed entire globe, specifically India
is suspected to have around 15 million people suffering from this
disease [1,2]. The pathogenesis of the diabetic complications en-
tails multifaceted aspects and hence many mechanisms are pro-
posed for its origin; however, one of the major proposed theories
for development of the diabetic complications involves the polyol
pathway [3]. According to a study involving polymorphic markers
of the human aldose reductase (AR) gene, a very strong association
is observed between expression of AR gene and susceptibility to
develop diabetic complications. These observations point towards
the key role of AR in development of diabetes in human [4–6]. De-
spite of several recent reports describing in vitro and experimental
animal models involving AR inhibition and significant protection
against diabetic complications [7–9], a successful drug is still yet
Compounds containing pyrazolone as parent scaffold are known
to have wide varieties of therapeutic applications. These com-
pounds are used as starting material for the synthesis of various
biologically active compounds [10]. Pyrazolone derivatives are an
important class of heterocyclic compounds that occur in many
drugs and synthetic products [11,12], and thus exhibit a wide
spectrum of biological properties including anti-microbial,
anti-bacterial, anti-fungal, anti-oxidant, anti-gout activities [13].
Pyrazole and its synthetic analogs have been reported to possess
industrial, agricultural and some biological application. These com-
pounds show remarkable anti-tubercular [14], analgesic [15], anti-
inflammatory [16], anti-neoplastic [17] and anti-tumor activity
[18]. A very recent research describing anti-diabetic activity of pyr-
azolone containing compounds [19], promoted us to investigate
the possibility of AR inhibitory activity of pyrazolone derivatives.
In present work we describe efficient green synthesis of some
novel pyrazolone derivatives followed by their in vitro screening
and subsequent in silico analysis. The pyrazolone derivatives were
synthesized by an eco-friendly approach using PEG-400 as a green
solvent and bleaching earth clay (pH12.5) as a catalyst. Bleaching
earth clay is easily available in India with very low cost and hence
⇑
Corresponding author. Fax: +91 2462 259461.
E-mail address: rngacche@rediffmail.com (R. Gacche).
http://dx.doi.org/10.1016/j.bioorg.2014.02.002
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

68
A. Kadam et al. / Bioorganic Chemistry 53 (2014) 67–74
are actively utilized on industrial scale for refining of vegetable oils
(British Patent GB-1077557, 1970) fats, greases and used as cata-
lyst in reactions (US Patent 4970295, 1990). The formed com-
pounds were confirmed by spectroscopic methods.
The resulting compounds via the schemes with both series
(1a-e) and (2a-e) described above are represented in supplemen-
tary Fig. 1.
2.1.3. Spectroscopic data of selected compounds
3-Amino-4-(4I-chloro benzylidene)-1H-pyrazol-5(4H)-one (1a). IR
(KBr):3363(ANH)cmÀ1
,
3063(ACH@)cmÀ1
,
3009(ArAH)cmÀ1
,
2. Material and methods
1705(>C@O)cmÀ1
,
1543(C@N)cmÀ1
, 1234(ANANAC of pyra-
zole)cmÀ1, 600–800(CACl)cmÀ1
,
1H NMR (DMSO-d6): d4.7 (s, 2H,
Melting points were uncorrected and determined in an open
capillary tube. IR spectras were recorded on FTIR Schimadzu
spectrometer. 1H NMR spectras were obtained in DMSO-d6 on
avance 300 MHz spectrometer using TMS as an internal standard.
The mass spectra were documented on EI-Shimadzu-GC–MS spec-
trometer.
NH2), d6.7(s, 1H, @CH), d7.6(d, 2H, ArAH), d7.9(d, 2H, ArAH),
d8.7(s, 1H, ANH), EIMS(m/z): 221.47(M+).
3-Amino-4-(4I-fluoro benzylidene)-1H-pyrazol-5(4H)-one (1b). IR
(KBr):3420(ANH)cmÀ1
,
3090(ACH@)cmÀ1
,
2928(ArAH)cmÀ1
,
1629(>C@O)cmÀ1
, , 1225 (ANANAC of pyra-
1600(C@N)cmÀ1
zole)cmÀ1 1H NMR (DMSO-d6): d4.8(s, 2H, NH2), d6.8(s, 1H,
.
2.1. Synthesis of pyrazolone derivatives
@CH), d7.1(d, 2H, ArAH), d7.8(d, 2H, ArAH), d8.6(s, 1H, NH), EIMS
(m/z): 205.35(M+).
2.1.1. General procedure for the synthesis of 3-amino-4-(4I-substituted
benzylidene)-1H-pyrazol-5(4H)-one derivatives
3-Amino-4-[3-(4I-methoxyphenyl)-1-phenyl-1H-pyrazol-5-yl)methy-
An equimolar mixture of substituted benzaldehyde/heteroalde-
hyde (1 mmol), ethylcyanoacetate (1 mmol), was heated in PEG-
400(20 ml) at 40–50 °C for 4–5 h. After completion of reaction
(monitored by TLC), add 5–10 ml Hydrazine hydrate 99% with
continuous heating. After the completion of reaction, the crude
mixture was worked up in ice-cold water (100 ml). The product
was separated by using filtration. The filtrate was evaporated to re-
move water and PEG leaving behind and obtained corresponding
products (1a-e). The purity of the synthesized compound was
checked by TLC on microscopic slide with silica gel layer. The struc-
tures of compounds were assigned on the basis of spectral data
such as IR, NMR and MASS.
lene]-1H-pyrazol-5(4H)-one (1d). IR (KBr): 3440 (ANH)cmÀ1
,
,
3071(ACH@)cmÀ1
1593(C@N)cmÀ1
,
2921(ArAH)cmÀ1
,
1670(>C@O)cmÀ1
,
1213(ANANAC of pyrazole)cmÀ1
.
1H NMR
(DMSO-d6): d3.9(s, 3H, AOCH3), d4.5(s, 2H, NH2), d6.2(s, 1H,
@CH), d7.2–8.0(m, 10H, ArAH and ACH of pyrazole), d8.6(s, 1H,
NH).
3-Amino-4-(4I-fluro benzylidene)-4,5-dihydro-5-oxopyrazole-1-car-
bothioamide (2b). IR (KBr): 3302(ANH)cmÀ1, 3011(ACH@)cmÀ1
,
,
2933(ArAH)cmÀ1
,
1654(>C@O)cmÀ1
,
1609(C@N)cmÀ1
1230(ANANAC of pyrazole)cmÀ1
,
1184(C@S)cmÀ1
.
1H NMR
(DMSO-d6): d4.2(s, 2H, ANH2), d6.5(s, 1H, ACH@), d7.0–7.9(m,
S
4H, ArAH), d9.8(s, 2H,
). 13C NMR (CDCl3): d189.42,
H2N
2.1.2. General procedure for the synthesis of 3-amino-4-(4I-substitued
benzylidene)-4,5-dihydro-5-oxopyrazole-1-carbothioamide
derivatives
d166.42, d163.08, d160.96, d130.71, d130.60, d130.50, d130.46,
d129.93, d116.29, d116.00.
EIMS (M/z): 263(M+).
An equimolar mixture of substituted benzaldehyde/heteroalde-
hyde (1 mmol), ethylcyanoacetate (1 mmol) and thiosemicarba-
zide (1 mmol) was heated in PEG-400 (20 ml) at 50–60 °C for
4–5 h. After completion of reaction (monitored by TLC), the crude
mixture was worked up in ice-cold water (100 ml). The product
was separated by filtration. The filtrate was evaporated to remove
water and PEG leaving behind and obtained corresponding prod-
ucts (2a-e). The purity of the synthesized compounds was checked
by TLC on microscopic slide with silica gel layer. The structures of
compounds were assigned on the basis of spectral data such as IR,
NMR and MASS. Both the above schemes are represented in Fig. 1.
2.2. Biological activity
AR activity was checked by goat lens aldose reductase inhibi-
tion assay. The AR inhibitory activity was measured according to
the method described earlier by Suryanarayana et al. [20]. The
reaction cocktail contained 1 ml of 1 M potassium phosphate buf-
fer (pH 6.2), 0.4 mM lithium sulfate and 5
10 M DL-glyceraldehydes, 0.1 M NADPH, and crude enzyme. The
reaction mixture was incubated at 37 °C for 10 min. The enzymatic
lM 2-mercaptoethanol,
l
l
Fig. 1. Scheme for preparation of derivatives from 1a–e and 2a–e.

A. Kadam et al. / Bioorganic Chemistry 53 (2014) 67–74
69
reaction was initiated by addition of NADPH and activity was mea-
sured by recording the decrease in absorbance at 340 nm. Various
concentrations of selected compounds were added to assay mix-
ture and appropriate blanks were prepared for correction. AR activ-
ity in the absence of inhibitor was considered as 100% for the
determination of IC50 values, the concentration of individual com-
pounds required to achieve 50% AR inhibition were calculated.
in PyMol. Two dimensional schematic representation of inhibitor
with residues from active site were generated using Ligplot+
[29,30].
3. Results and discussion
3.1. Chemical synthesis
Quercetin (IC50, 3.12 lM/ml) was used as a standard AR inhibitor.
We have realized that processes for synthesizing chemical
products are highly inefficient and it has been estimated that for
every kilogram of fine chemical and pharmaceutical products pro-
duced, 5–100 times that amount of chemical waste is generated
[31]. The prime component of waste in the majority chemical pro-
ductions is related with use of solvent, hence as part of green
chemistry efforts, various cleaner solvents have been proposed as
safer substitution [32]. It has now been proved that solvent PEG-
400 is not only an environmentally benign reaction solvent but
also non-toxic, inexpensive, water soluble (which facilitates its re-
moval from the reaction product) and is highly reusable [33,34].
Hence we have utilized this green solvent in conjugation with
bleaching earth clay that is used as a catalyst which is also fully
recyclable and can be collected from the reaction by simple
filtration and thus are essential requirements for the principles of
green chemistry. Considering an ever increase concern towards
the development of new environmental friendly synthesis
procedures for the production of biologically active compounds,
here we reported an efficient method for the synthesis of
2.3. Docking investigation
2.3.1. Ligand preparation
2D structures of all compounds were drawn in ChemDraw 8.0
(CambridgeSoft, Cambridge, MA, USA) and their SMILES were ob-
tained. The 3D conformers of these compounds were then gener-
ated in SDF format using FROG2 server [21]. Autodock 4.0 [22,23]
implemented in python prescription 0.8 (PyRx) was used in dock-
ing analysis. The ligand molecules in sdf format were imported in
PyRx environment via OpenBabel utility and were subject to en-
ergy minimization using UFF forcefield [24–26], Conjugate gradi-
ent optimization algorithms was applied for over 200 steps while
molecules were updated for every 1 step.
2.3.2. Receptor preparation
Structural model of AR in complex with zopolrestat with PDB ID
2DUX was downloaded from protein data bank [27]. This mono-
meric structural model of AR was co-crystallized with solvent mol-
ecules, inhibitor zopolrestat along with its essential prosthetic
group nicotinamide adenine dinucleotide phosphate (NADP).
Receptor molecule was prepared in traditional pdbqt format after
assigning charges to the receptor coordinate file by using ‘Make
macromolecule’ command from Autodock menu of PyRx.
3-amino-4-(4I-substituted
benzylidene)-1H-pyrazol-5(4H)-one
and 3-amino-4-(4I-substitued benzylidene)-4,5-dihydro-5-oxopy-
razole-1-carbothioamide by simultaneous one pot condensation
of substituted aldehyde/heteroaldehyde, ethylcyano acetate,
hydrazine hydrate (99%) and thiosemicarbazide by using PEG-
400 and bleaching earth clay(pH12.5).
2.3.3. Testing validity of AutoDock 4.2 and virtual screening
The reaction time, yield and melting point of pyrazolone deriv-
atives are described in Table 1 and Table 2. The reactions proceed
rapidly and completed within 4–5 h. The newly synthesized
compounds 1a-e and 2a-e were established on the basis of
spectroscopic data. IR spectra of pyrazolone and pyrazole-1-carbo-
thioamide derivatives showed characteristic peak in between 3400
The validity of a docking system can be checked by docking the
experimentally verified pose back into the receptor. This procedure
follows the rationale that a good docking engine should replicate
the experimental binding modes of the ligand. After docking, root
mean square deviation (RMSD) value of the predicted pose to
experimentally verified pose is calculated. RMSD value indicates
the measure of spatial similarity between two structures. If the
RMSD value is found to be less than certain value (typically
<2.00 angstrom), the prediction of binding mode is considered as
successful. Auto-grid program was utilized to obtain grid file. The
affinity grid of 50 Â 50 Â 50 points was set using spacing of
0.375 angstrom in order to encompass entire active site. The
lamarckian genetic algorithm was used for the conformational
search. Each lamarckian job was set to have 10 runs. The initial
population was restricted to 150 structures; while the maximum
number of energy evaluation and generation were set to 27,000.
Single top individual was allowed to survive to next generation,
rate of gene mutation was set to 0.02 and rate of crossover was
set to 0.8; the rest of parameters were set to default values. The fi-
nal structures were clustered and according to native autodock
scoring function. RMSD value of 0.43 angstrom was obtained from
the re-docking experiment of zopolrestat. This value indicates that
predicted binding mode is nearly identical to the X-ray crystallog-
raphy conformer (supplementary Fig. 2). Same settings were used
to dock selected pyrazolone derivatives. The top ranked conforma-
tions of each ligand were selected.
Table 1
Table showing data for synthesis of 3-amino-4-(4I-substituted benzylidene)-1H-
pyrazol-5(4H)-one derivatives catalyzed by bleaching earth clay (pH 12.5) in
PEG-400.
Compound code
1a
(Aromatic/heterocyclic aldehydes)
Time (h)
3.0
Cl
CHO
1b
1c
1d
4.0
3.5
3.0
F
CHO
H3CO
CHO
H3CO
N
CHO
CHO
N
1e
4.5
Cl
2.3.4. Post virtual screening analysis (paired potential analysis)
Further analysis of docked conformers were carried out using
on-line program DSX-ONLINE v0.88, knowledge-based DSX pair
potentials that are based on the Drug Score potential [28]. Results
obtained from docking and those from DSX server were visualized
N
N

70
A. Kadam et al. / Bioorganic Chemistry 53 (2014) 67–74
Table 2
compounds produced in scheme 1, first three compounds, 1a, 1b
and 1c showed a little inhibitory activity (IC50 values 16.70 M,
18.80 M and 17.60 M respectively). This might have occurred
Table showing data for synthesis of 3-amino-4-(4I-substitued benzylidene)-4,5-
dihydro-5-oxopyrazole-1-carbothioamide derivatives catalyzed by bleaching earth
clay (pH 12.5) in PEG-400.
l
l
l
due to direct attachment of hydrophobic chloro, fluoro and meth-
oxy benzylidene moieties respectively to pyrazolone. Their lower
potency towards AR inhibition is further explained using paired
potential analysis in upcoming sections.
Compound code
2a
(Aromatic/heterocyclic aldehydes)
Time (h)
4.0
Cl
But when this substituted benzylidene group was replaced by
3-(4I-methoxyphenyl)-1-phenyl-1H-pyrazole group yielded com-
pound 1d. These substitutions resulted in its sudden improved per-
formance (two fold improvement in IC50) as shown in Table 3. In
compound 1e, 3-(4I-methoxyphenyl)-1-phenyl-1H-pyrazole was
found to be placed at first position, this feature can be attributed
to increased performance as compared to first three compounds
CHO
2b
2c
2d
4.5
3.5
4.5
F
CHO
H3CO
CHO
(IC50 value 9.20 lM) indicating importance of proper positioning
of pyrazole group in inhibition.
H3CO
N
All compounds in second series contained carbothioamide
group. First three compounds of second series (2a, 2b and 2c) still
performed poor owing to direct connection of substituent chloro,
fluoro and methoxy benzylidene fragments to pyrazolone, how-
ever it is to be noted that existence of carbothioamide group
slightly improved inhibitory effect of these compounds (IC50 val-
CHO
N
2e
4.0
Cl
N
CHO
N
ues 15.50 lM, 11.90 lM and 12.80 lM as compared to that of com-
pound 1a, 1b and 1c described in previous paragraph) This
observation is inductive of the fact that hydrophobic moieties like
phenyl group directly attached with pyrazolone adversely affects
inhibitory properties of AR. By virtue of replacement of phenyl sub-
stituents from previous compounds (1a, 1b, 1c, 2a, 2b and 2c) with
3-(4I-methoxyphenyl)-1-phenyl-1H-pyrazole and 3-(4I-chloro-
phenyl)-1-phenyl-1H-pyrazole (compound 1d, 2d and 1e, 2e);
inhibitory potential significantly improved (for example IC50 of
and 3500 cmÀ1 due to ANH2. The peak at 1650–1690 cmÀ1 is due
to >C@O stretch vibrations. The peak was observed at 1200–
1235 cmÀ1 due to (ANANAC of pyrazole ring), while the peak
was observed at 1270–1190 cmÀ1 due to C@S stretching. Beside
these bands 680–800 cmÀ1 due to CACl stretching whenever pres-
ent in the compound. 1H NMR spectra of compounds were studied
in CDCl3 and DMSO showed characteristic peak at d 3.9 ppm of
AOCH3, peak at d6.6 ppm of ACH@CH, d7–8 ppm of Aromatic pro-
tons, the characteristic peak was observed at d8.6–9.0 ppm of –NH
of pyrazole ring. The peak was observed at d4.6 ppm of ANH2. The
8.10
all the synthesized derivatives was observed in compound 2e (IC50
of 6.30 M). This performance can be attributed to synergistic
lM in case of compound 2d). The best inhibition value among
l
effect produced due to simultaneous presence of carbothioamide
group and 3-(4I-methoxyphenyl)-1-phenyl-1H-pyrazole and
3-(4I-chlorophenyl)-1-phenyl-1H-pyrazole substituent at first
position.
S
peak was observed at d9.8 ppm of
. The mass spectra of
H2N
3.3. In silico evaluations
these compounds were showed molecular ion peak corresponding
to their molecular formula.
Binding cleft of target AR is approximately 10.84 angstrom deep
containing NADP and Zopolrestat and possess huge volume of
3505.36 Å3, since it is known to accommodate prosthetic group
as well as substrate (or inhibitor) simultaneously, as observed from
its binary complex structures [35]. This big cleft can be
conceptually imagined to be formed by two main sub pockets. A
hydrophobic pocket P1 is formed by residues 112-PTGF-115,
303-C, 309-YPFHE-313 and represented by cyan color. Pocket P1
3.2. Biological evaluation
All the compounds produced by both the schemes were evalu-
ated for their AR inhibitory activity in terms of IC50 values. The re-
sults of in vitro AR activity are presented in Table 3. Out of 5
Table 3
Aldose reductase inhibition results of selected pyrazolone and carbothioamide derivatives.
Compound code
Mol. formula
Mol. Wt.
M.P. (°C)
Yield%
Solubility
Aldose reductase activity IC50 (lM)
1a
1b
1c
1d
1e
2a
2b
2c
C
C
C
C
10H8N3OCl
10H8N3OF
11H11N3O2
20H17N5O2
221
205
217
359
363
280
264
264
418
422
–
178
185
182
240
248
189
184
210
256
248
–
83.33
82.41
75.47
81.78
69.45
75.94
85.10
82.25
71.42
74.62
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
–
16.70
18.80
17.60
7.70a
9.20a
12.50
11.90
12.80
8.10a
6.30a
3.12b
C19H14N5OCl
C11H9N4OSCl
C11H9N4OSF
C12H12N4O2S
C21H18N6O2
2d
2e
Quercetin
C20H15N6OSCl
–
a
Compounds showing promising IC50 values.
Standard compound.
b

A. Kadam et al. / Bioorganic Chemistry 53 (2014) 67–74
71
is separated by residue 111-W via formation of separating wall
(yellow color) that leads to pocket P2 which is lined by residues
110-H, 159-SN-160, 183-Q, 209-Y, 260-I and represented by
orange color. Pocket P2 is in continuum with cavity that holds
NADP (supplementary Fig. 3). Experimentally verified structure
of AR in complex with zopolrestat illustrates vital interaction of
trifluoromethyl moiety attached via benzothiazole scaffold of
inhibitor with residues from pocket P1, while phthalazinone ring
protrudes towards pocket P2 as seen in supplementary Fig. 3
[36,37].
It is reported that AR prefers highly hydrophobic compounds
owing to its lipophilic active site [35,38]; the first three inhibitors
of first series (1a, 1b and 1c) contained hydrophobic chloro, fluoro
and methoxy benzylidene moieties attached to pyrazolone.
Comparatively reduced inhibitory activity of these compounds
can be effectively explained by its in silico docking and paired
potential analysis.
Paired potential analysis of docked conformers enables to
scrutinize the per-atom score contribution of inhibitor in active
site of structural model in terms of ‘good’ and ‘bad’ potentials.
These paired potential represent favorable and unfavorable inter-
actions respectively. Favorably interacting atoms from inhibitor
are depicted to be surrounded by blue spheres; while the sizes of
the spheres correspond to the values of the contributing per-atom
scores (Figs. 2 and 3). In simpler words, larger the sphere, more
favorable is the interaction. If the inhibitor is not structurally com-
petent with active site of enzyme, it makes unfavorable contacts
with surrounding residues. Such unfavorable contacts are depicted
as ‘bad contacts’ in paired potential analysis and are represented as
red lines (refer Fig. 2 in case of compound 1a and 1b). Analyzing
Fig. 2 it becomes evident that chloro and fluoro benzylidene moie-
ties of compound 1a and 1b have shown to interact favorably in ac-
tive site since these are hydrophobic moieties and preferred by
hydrophobic enzyme active site [35,38], while amino pyrazolone
containing region of same compounds makes numerous bad
contacts (indicated by red lines) signifying unacceptable mode of
binding. These unfavorable contacts thus results in lower binding
affinity as correlated in docking calculations (refer Table 4), bind-
ing energies À6.89 kcal/mol and À6.31 kcal/mol for 1a and 1b
respectively). These facts might result in lower potency of com-
pound 1a and 1b in AR inhibition. Similar results of binding affinity
as well as paired potential analysis was obtained for compound 1c
(figure not shown).
Literature survey indicate that carbothioamide moiety in many
compounds is proved to be responsible for diverse biological activ-
ities including antibacterial, anti fungal [39,40], anti proliferative
[41] as well as anti tumor [42,43], hence its presence in existing
pyrazolone derivatives (compounds 2a, 2b and 2c) can be hypoth-
esized to increase potency of AR inhibition activity. This assump-
tion was verified to be convincing, since compound containing
carbothioamide group in this study are found to increased inhibi-
tion to a little extent (IC 50 values improved to 12.50, 11.90 and
12.80 lM for compound 2a, 2b and 2c respectively). This improve-
ment can be attributed to the fact that placement of this group
seems to reorient the pyrazolone moiety, hence avoid the bad con-
tacts and enable it to maintain favorable interaction in active site
as observed from result of paired potential analysis. Fig. 2 also
point out complete elimination of bad contacts and proper orienta-
tion of pyrazolone moiety to attain complimentary interactions
with residues from active site. This fact is again supported by
improvement in binding energies obtained from docking analysis
for compounds 2a, 2b and 2c (À7.56, À7.06, À7.28 kcal/mol
respectively).
Structural analysis of docking poses of previous compounds (1a,
1b, 1c, 2a, 2b and 2c) revealed the fact that due to small size of all
the above compounds, they may not occupy both the binding pock-
ets. Better results are observed for compounds that are absolutely
complemented to active site area (i.e. compounds 1d, 1e, 2d, 2e).
These compounds possess optimally placed hydrophobic moieties
attached to parent pyrazolone via methylene bridges. These
structural features can be suspected for striking improvement in
observed AR inhibition. These structural peculiarities might result
in improvement by approximately 3 folds (IC 50 6.30
lM in case of
inhibitor 2e as compared to initial 18.80 M for compound 1b).
l
Fig. 2. Figure showing paired potential analysis results for compound 1a, 1b, 2a and 2b (CPK colored sticks). Atoms surrounded by blue sphere indicate paired potential per-
atom score while red lines surrounding amino pyrazole moiety (compound 1a and 1b) represent bad contacts with residues in active site (residues in active site are not shown
here for sake of clarity). For compound 2a and 2b, note improvement in per atom score of amino pyrazole moiety resulted due to introduction of carbothioamide group
(highlighted by circle).

72
A. Kadam et al. / Bioorganic Chemistry 53 (2014) 67–74
Fig. 3. Figure showing paired potential analysis results for compound 1d, 1e, 2d and 2e (CPK colored sticks). Atoms surrounded by blue sphere indicate paired potential
per-atom score. Note significant improvement in per atom score depicted as large blue spheres.
Table 4
Molecular docking results of selected pyrazolone and
carbothioamide derivatives.
3.4.2. Structural analysis of docking results for compound 1e
Amino-pyrazole moiety of compound 1e is found to be involved
Name of ligand
Binding energy
in polar contacts with side chains of residues in active site. N3
atom of amino pyrazole appears to form hydrogen bond with O
atom of Tyr 48 side chain (Fig. 4c). In a study involving inhibition
of AR by citrate and cacodylate as inhibitors revealed critical role
of polar interaction involving Tyr 48 as observed in present study
[44]. SG sulfur atom of thiol group from Cys 298 forms a salt bridge
with keto oxygen of amino pyrazole portion of compound1e
(Fig. 4c). Inhibitor’s interaction observed in present study with
Cys 298 is experimentally verified to be essential for inhibition of
AR [45,46]. Chloro-phenyl part of inhibitor occupies P1 hydropho-
bic pocket lined by residues Cys 303, Phe 122, Leu 300, and Trp 79;
while P2 pocket is occupied by amino-pyrazole moiety imparting
total complementary with that of active site (Fig. 4d).
Zopolrestat
À10.59a
À6.89
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
À6.31
À6.56
À8.64b
À9.32b
À7.56
À7.06
À7.28
À9.35b
À10.58b
a
Experimentally crystallized inhibitor, used as ref-
erence structure to validate accuracy of autodock
algorithm.
b
Compounds showing best binding affinity towards
aldose reductase.
3.4.3. Structural analysis of docking results for compound 2d
Terminal anisole group of compound 2d seems to occupy P1
pocket, simultaneously making an ionic contact via its O2 oxygen
atom with OG1 atom of Thr 113 side chain (Fig. 5a and b). Such
ionic contacts via Thr 113 are experimentally verified to be critical
in inhibition of AR for inhibitors like fidarestat (SNK-860) and
minalrestat (WAY-509) [47]. Benzyl group of ligand completely
occupies P2 pocket. N6 atom of carbothioamide component of
compound 2d seems to be hydrogen bonded with backbone oxy-
gen of Val 47 thus stabilizing active site (Fig. 5a and b).
These structural features seems to bring about exact complemen-
tary of compounds to active site resulting in proper placement of
hydrophobic moieties in respective binding pockets. These facts
are evident from structural analysis of docking results as well as
from paired potential analysis (Fig. 3).
3.4. Structural analysis of docking results
3.4.4. Structural analysis of docking results for compound 2e
3.4.1. Structural analysis of docking results for compound 1d
Methoxy benzylidene moiety of compound 1d is found to be
stack on heterocyclic aromatic side chain of Trp 111. Such stacking
interaction with Trp 111 is found to be one of basic force stabilizing
experimentally observed inhibitors [36]. Moreover, N1 atom of
central pyrazole ring seems to make ionic contact with NE1 atom
of Trp 111 (Fig. 4a). More than half of the van der Waals contacts
between inhibitor and residues of active site are formed between
carbon atoms, demonstrating that enzyme–inhibitor interaction
are highly hydrophobic in nature (Fig. 4a and b).
Compound 2e look like to be implicated in lot of polar interac-
tions with residues lining pocket P2 (Fig. 5c and d). Salt bridge be-
tween conserved Cys 298 and keto oxygen of amino-pyrazole
portion is also observed in case of compound 2e. S atom of carbo-
thioamide group from compound 2e forms a bridge of hydrogen
bonds connecting residues Ser 159 and Asn 160. Another series
of hydrogen bonds appear to stabilize inhibitor involves N3 and
N6 atoms of compound 2e that hydrogen bonds with NE2 atom
from His 110, OH atom from Tyr 48 and OE1 atom of Gln 183
(Fig. 5c). Chlorobenzyl group seems to occupy P1 pocket

A. Kadam et al. / Bioorganic Chemistry 53 (2014) 67–74
73
Fig. 4. Interaction of compound 1d and 1e with residues in active site of aldose reductase (a and c respectively). (b and d) represents perfect placement of two hydrophobic
moieties of compound 1d and 1e in pocket P1 and P2 of aldose reductase active site.
Fig. 5. (a) Interaction of compound 2d and 2e with residues in active site of aldose reductase (a) and (c) respectively). (b) and (d) Represents perfect placement of two
hydrophobic moieties of compound 2d and 2e in pocket P1 and P2 of aldose reductase active site.
(Fig. 5d). Hydrogen bonding network as observed in this study is
known to be very essential in terms of AR inhibition and has been
confirmed experimentally [48,49].
analysis carried out in this study indicate that mere hydrophobic-
ity in ligands is not sufficient for inhibition of AR. For optimal AR
inhibition, excellent complementarily between active site and li-
gand structure is essentially required. Strategic placement of
hydrophobic groups over pyrazole moiety may play a key role in
achieving best inhibitory activity. Docking results on AR from this
study is expected to provide springboard for initiating further de-
tailed investigations of these compounds focusing on effective AR
inhibition.
4. Conclusion
Here we report synthesis of novel pyrazolone and pyrazole car-
bothioamide derivatives. in vitro testing and subsequent in silico

74
A. Kadam et al. / Bioorganic Chemistry 53 (2014) 67–74
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Authors are thankful to Hon’ble Vice Chancellor, SRTM
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and DST-SERB, New Delhi, India for financial assistance (File No.
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2014.02.
002.
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