Synthesis, docking, and in vitro studies of some substituted bischalcones on acid and alkaline phosphatases

Article abstract of DOI:10.1007/s00044-013-0780-4

A series of bischalcones was synthesized and screened for their effect on acid and alkaline phosphatases. In addition, molecular modeling and docking of these compounds into alkaline phosphatase using iGemdock was performed in order to predict the affinity and orientation of the designed compounds at the active site and was compared with the established inhibitor levamisole. The iGemdock docking fitness resulted in decrease in total energy (ranging from -75.50 to -100.84) for all the synthesized compounds which were less than levamisole (-50.69) revealing higher binding with the enzyme. The compounds were synthesized by Clasien-Schimdt condensation and their effect was observed on the activity of acid and alkaline phosphatases. The results showed that synthesized bischalcones were inhibitory to alkaline phosphatases, whereas an activating effect was observed on the activity of acid phosphatase. The type of inhibition and K_i values of bischalcones on alkaline phosphatase were also determined. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0780-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1781–1788
DOI 10.1007/s00044-013-0780-4
ORIGINAL RESEARCH
Synthesis, docking, and in vitro studies of some substituted
bischalcones on acid and alkaline phosphatases
Mamta Singh Neera Raghav
•
Received: 16 January 2013 / Accepted: 31 August 2013 / Published online: 13 September 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of bischalcones was synthesized and
screened for their effect on acid and alkaline phosphatases.
In addition, molecular modeling and docking of these
compounds into alkaline phosphatase using iGemdock was
performed in order to predict the affinity and orientation of
the designed compounds at the active site and was com-
pared with the established inhibitor levamisole. The
iGemdock docking fitness resulted in decrease in total
energy (ranging from -75.50 to -100.84) for all the
synthesized compounds which were less than levamisole
antimicrobial (Asiri and Khan, 2011; Husain et al., 2011,
2012, 2013; Nagaraj and Reddy, 2007), anticancer (Mod-
zelewska et al., 2006), antimalarial (Ram et al., 2000;
Srivastava et al., 2008), radioprotective and antiviral agents
(Raj et al., 2012a), in-vivo peritoneal antiangiogenesis, and
in vitro antiproliferative agents (Raj et al., 2012b). Acid
and Alkaline phosphatase are enzymes found in all living
tissues, and are concerned with the removal of the phos-
phate from protein and other molecules. These enzymes are
used as diagnostic tools in various diseased conditions.
Their altered concentration in various pathological disor-
ders necessitates the evaluation of various classes of nat-
ural and synthetic compounds on the activities of these
physiologically important enzymes. Acid phosphatases
(EC 3.1.3.2) catalyze the nonspecific hydrolysis of phos-
phate monoesters and oxygen exchange from water into
inorganic phosphate in an acidic environment (Hollander
and Boyer 1971). These enzymes are widely distributed in
mammalian serum (Nakanishi et al., 2000), plants (Yam
et al., 1971), and in microorganisms (Nakanishi et al.,
2000). The highest levels of acid phosphatase are found in
the circulation of prostate cancer patients, bone diseases,
diseases of blood cells, or lysosomal storage diseases, such
as Gaucher’s disease (Nair and Johnson, 2008) etc. Alka-
line phosphatases (ALP, EC 3.1.3.1) hydrolyze various
monophosphate esters at a high pH optimum (McComb
et al., 1979). These enzymes, which are widely distributed
in nature, exist as membrane-bound glycoproteins in higher
organisms. These enzymes consist of a group of isoen-
zymes that are encoded by at least four gene loci: tissue
nonspecific, intestinal, placental, and germ cell ALP
(
-50.69) revealing higher binding with the enzyme. The
compounds were synthesized by Clasien–Schimdt con-
densation and their effect was observed on the activity of
acid and alkaline phosphatases. The results showed that
synthesized bischalcones were inhibitory to alkaline
phosphatases, whereas an activating effect was observed on
the activity of acid phosphatase. The type of inhibition and
K values of bischalcones on alkaline phosphatase were
i
also determined.
Keywords Acid phosphatase ꢀ Alkaline phosphatase ꢀ
Bischalcones ꢀ p-Nitrophenyl phosphatase
Introduction
The pharmaceutical importance of bischalcone derivatives
lies in the fact that they can be effectively utilized as
ameliorative (Sarojini et al., 2011), nitric oxide production
inhibitors, and cytotoxic agents (Reddy et al., 2012),
(
Weiss et al., 1986, 1988; Millan 1986, 1988). Elevated
M. Singh ꢀ N. Raghav (&)
alkaline phosphatase levels have been used as diagnostic
tool in a number of diseases, including those associated with
liver, bile duct, and bone. Elevated alkaline phosphatase
Department of Chemistry, Kurukshetra University,
Kurukshetra 132119, India
e-mail: nraghav.chem@gmail.com
123

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Med Chem Res (2014) 23:1781–1788
levels are found in cholecystitis, cholestasis, cholangitis,
cirrhosis, hepatitis, fatty liver, liver tumor, liver metastasis,
drug intoxication, Paget’s disease, osteosarcoma, bone
metastasis, prostatic cancer, renal osteodystrophy, fractured
bone, multiple myeloma associated with fractures, osteo-
malacia, and others such as policythemiavera, myelofibrosis,
seminoma (Gennari et al., 2005; Lange et al.,1982; Schiele
et al., 1998). We are involved in the synthesis of different
class of compounds and their evaluation as phosphatase
inhibitors. In the present work, we report the synthesis of
various bischalcones and their effect has been investigated
on the activity of acid and alkaline phosphatase of mam-
malian origin.
Assay for acid phosphatase Enzyme homogenate having
specific activity 42.2 n moles/min/mg was incubated with
0.1 M acetate buffer pH 5.3. Enzyme activities were
measured using p-nitrophenyl phosphate as substrate
(Plummer 1987).
Determination of acid phosphatase activity in presence of
synthesized bischalcones Enzyme homogenate (50 ll)
was incubated with 0.1 M acetate buffer pH 5.3 containing
1 mM final concentration of compounds (1a–1i). The
solution of all compounds was prepared in DMSO. After
half an hour, the residual enzyme activities were measured
using p-nitrophenyl phosphate as substrate. The concen-
tration of liberated p-nitrophenol was determined from the
absorbance at 410 nm. The results were compared with the
controls run along with the experiments. The reaction was
terminated by the addition of 100 ll of 0.1 M NaOH.
Table 1 represent the percentage residual activity left in
solution after the interaction of acid phosphatase with the
individual compound for 30 min.
Experimental
Materials and methods
General remarks
Melting points were determined in open capillary tubes and
are uncorrected. All the chemicals and solvents used were
Alkaline phosphatase
-
of laboratory grade. IR spectra (KBr, cm ) were recorded
1
Isolation Fresh goat liver purchased from local slaughter
house was washed with cold isotonic saline solution and
was disintegrated in a mixer-cum-blender and 10 %
homogenate was prepared in 0.1 M glycine–NaOH buffer
pH 10.5 containing 0.2 M NaCl for alkaline phosphatase
activity. The homogenate was centrifuged at 10,000 rpm
for 10 min at 4 °C to obtain a clear solution which was
further used as enzyme source.
1
on a Horizon 300 MHz spectrometer. H NMR spectra was
recorded on Brucker 300 MHz NMR spectrometer
(
chemical shifts in d ppm) using TMS as an internal
standard. The purity of the compounds was ascertained by
thin layer chromatography on aluminium plates per coated
with silica gel G (Merck) in various solvent systems using
iodine vapors as detecting agent or by irradiation with
ultraviolet lights (254 nm). ELISA plate reader was used
for measuring absorbance in the visible range. Refrigerated
ultracentrifuge Remi C-24BL was used for centrifugation
purpose under cold conditions.
Assay for liver alkaline phosphatase Enzyme homoge-
nate having specific activity 41.7 n moles/min/mg was
incubated with 0.1 M glycine–NaOH buffer pH 10.5.
Enzyme activities were measured using p-nitrophenyl
phosphate as substrate (Sahney and Singh, 2001).
Phosphatase activity
Acid phosphatase
Determination of alkaline phosphatase activity in presence
of synthesized bischalcones Enzyme homogenate
(150 ll) was incubated with 0.1 M glycine–NaOH buffer
pH 10.5 containing 1 mM concentration of compounds
(1a–1i). After half an hour, the residual enzyme activities
were measured using p-nitrophenyl phosphate as substrate.
The concentration of liberated p-nitrophenol was deter-
mined from the absorbance at 410 nm. The results were
compared with the controls run along with the experiments.
The reaction was terminated by the addition of 100 ll ml
of 0.1 M NaOH. Table 1 represents the percentage residual
activity left in solution after the interaction of alkaline
phosphatase with the individual compound for 30 min.
Isolation Fresh goat liver purchased from local slaughter
house was washed with cold isotonic saline solution and
was cut into small pieces and disintegrated in a mixer-cum-
blender with chilled acetone. The crude extract was filtered
through Buchner funnel and washed 2–3 times with 10
volumes of chilled acetone. A stream of N gas was flushed
2
through it and dried over concentrated H SO and stored
2
4
over anhydrous CaCl . 10 % homogenate was prepared in
2
0
.1 M acetate buffer pH 5.3 containing 0.2 M NaCl for
acid phosphatase activity. The homogenate was centrifuged
at 10,000 rpm for 10 min at 4 °C to obtain a clear super-
natant solution which was further used as enzyme source.
1
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Med Chem Res (2014) 23:1781–1788
1783
Table 1 Percentage residual activities of acid and alkaline phosphatases in presence of bischalcones
S. no.
Compound name
Acid phosphatase
Alkaline phosphatase
M.D. ± S.M.D.
Percentage
residual
activity
M.D. ± S.M.D.
Percentage
residual
activity
Control
0.609 ± 0.0095
0.855 ± 0.0145
0.784 ± 0.0085
0.771 ± 0.0104
0.905 ± 0.0120
0.963 ± 0.0134
0.809 ± 0.0118
0.788 ± 0.0147
0.688 ± 0.0088
100
0.600 ± 0.102
0.347 ± 0.0390
0.277 ± 0.0062
0.261 ± 0.0023
0.279 ± 0.0106
0.190 ± 0.0032
0.156 ± 0.0012
0.185 ± 0.0034
0.241 ± 0.0072
100
0
1.
2.
3.
4.
5.
6.
7.
8.
2,6-Bis(4 -fluoro benzylidene) cyclohexanone (1a)
0
140.39 ± 2.38
128.73 ± 1.39
126.60 ± 1.71
148.60 ± 1.97
158.13 ± 2.20
132.84 ± 1.94
129.39 ± 2.41
112.97 ± 1.44
57.83 ± 6.50
46.16 ± 1.03
43.50 ± 0.38
46.00 ± 1.77
31.67 ± 0.53
26.00 ± 0.20
30.83 ± 0.57
40.17 ± 1.20
2,6-Bis(4 -chloro benzylidene) cyclohexanone (1b)
0
2,6-Bis(4 -bromo benzylidene) cyclohexanone (1c)
0
2,6-Bis(4 -methyl benzylidene) cyclohexanone (1d)
0
2,6-Bis(4 -methoxy benzylidene) cyclohexanone (1e)
0
2,6-Bis(4 -nitro benzylidene) cyclohexanone (1f)
0
2,6-Bis(3 -phenylallylidene) cyclohexanone (1g)
0
2,6-Bis(4 - (dimethyl amino) benzylidene)cyclo
hexanone (1h)
9
1
.
2,6-Di benzylidene cyclo hexanone (1i)
Levamisole
0.734 ± 0.0103
120.52 ± 1.69
0.247 ± 0.0069
0.528 ± 0.0075
41.17 ± 1.15
88.00 ± 1.25
0.
The results are the mean and S.M.D. of the experiment conducted in triplicate and is calculated as phosphatase activity in nmol/min/ml enzyme
homogenate. The enzyme activity was determined at 1 mM concentration of each compound and % residual activity is calculated w.r.t. control
where no compound was added but an equivalent amount of solvent was present. The specific activities of the acid phosphatase and alkaline
phosphatase were 42.2 and 41.7 nmol/min/mg, respectively
0
General procedure for the synthesis of bischalcones Mix-
2,6-Bis(4 -bromo benzylidene) cyclohexanone (1c) Yield
-
1
ture of cyclohexanone (0.01 mol) and aromatic aldehyde (0.
78.25 %; m.p. 162–164 °C; IR (KBr, cm ): 1,659([C=O
str), 1,489–1,574(-C=C- str), 825(-C–Br str); H-NMR
1
0
2 mol) in methanol (25 ml) was cooled for 10–15 °C in ice
bath. To this cold solution 40 % sodium hydroxide (5 ml)
was added drop wise, with continuous stirring for 30 min
using magnetic stirrer and was then left overnight. The
reaction mixture was poured into crushed ice and acidified
carefully using dilute hydrochloric acid. The solid obtained
was filtered, washed with ice-cold water, dried and recrys-
tallized from ethanol to give compounds.
(300 MHz, CDCl , d ppm): 1.71–1.73(s, 2H, [CH ), 2.
3
2
85–2.92(s, 4H, [CH ), 7.47–7.65(m,8H, arom. H), 7.56(s,
2
1
3
2H, =CH-); C-NMR (300 MHz, CDCl , d ppm): 190.05,
3
164.34, 161.02, 135.87, 135.74, 132.30, 132.19, 132.09,
115.67, 115.38, 77.43, 77.01, 76.58, 28.35, 22.91.
0
2,6-Bis(4 -methyl benzylidene) cyclohexanone (1d) Yield
-
1
8
3.00 %; m.p. 166–168 °C; IR (KBr, cm ): 1,659([C=O
0
2
,6-Bis(4 -fluoro benzylidene) cyclohexanone (1a) Yield
-
str), 1,412–1,597(-C=C- str), 2,916 (-C–H- str);
1
1
8
4.76 %; m.p. 152–154 °C; IR (KBr, cm ): 1,651([C=O
str), 1,489–1,612(-C=C- str), 648(-C–F str); H-NMR
H-NMR (300 MHz, CDCl , d ppm): 1.77–1.85(s, 2H,
3
1
[CH ), 2.40(s, 3H, -CH ), 2.92–2.96 (s, 4H, [CH ), 7.
2
3
2
1
22–7.41(m, 8H, arom. H), 7.79(s, 2H, =CH-); C-NMR
3
(
300 MHz, CDCl , d ppm): 1.78–1.82(s, 2H, [CH ), 2.
3
2
9
2
1
0
0–2.93(s, 4H,[CH ), 6.94–7.71(m, 8H, arom. H), 7.99(s,
(300 MHz, CDCl , d ppm): 190.41, 138.80, 136.87, 135.
51, 133.23, 130.48, 129.14, 77.45, 77.03, 76.60, 28.54, 23.
2
3
1
3
H, =CH-); C-NMR (300 MHz, CDCl , d ppm): 189.85,
3
36.54, 135.83, 134.75, 131.79, 131.65, 122.94, 77.43, 77.
04, 21.40.
0, 76.58, 28.37, 22.80.
2
,6-Bis(4-methoxy benzylidene) cyclohexanone (1e) Yield
-1
0
2
,6-Bis(4 -chloro benzylidene) cyclohexanone (1b) Yield
-
74.54 %; m.p. 156–158 °C; IR (KBr, cm ): 1,659([C=O
str), 1,489–1,574 (-C= C- str), 2,932(-C–H- str),
1
8
6.57 %; m.p. 148-150 °C; IR (KBr, cm ): 1,666([C=O
str), 1,427–1,605(-C=C- str), 774(-C–Cl str); H-NMR
1
1
1,250(-C-OCH3 str); H-NMR (300 MHz, CDCl , d
3
(
300 MHz, CDCl , d ppm): 1.69–1.73(s, 2H, [CH ), 2.
ppm): 1.74–1.76(s, 2H, [CH ), 2.46(3H, s, –COCH ), 2.
3
2
2
3
8
2
1
0
5–2.88(s, 4H,[CH ), 7.49–7.59(m, 8H, arom. H), 7.55(s,
88–2.92(s, 4H,[CH ), 7.32–7.55(m, 8H, arom. H), 7.65(s,
2
2
1
3
13
H, =CH-); C-NMR (300 MHz, CDCl , d ppm): 189.85,
2H, =CH-); C-NMR (300 MHz, CDCl , d ppm): 190.41,
3
3
36.43, 135.77, 134.62, 134.32, 131.57, 128.69, 77.44, 77.
138.79, 136.87, 135.51, 133.24, 130.47, 129.13, 77.44, 77.
1, 76.59, 28.38, 22.82.
02, 76.59, 28.53, 23.04, 21.39.
123

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Med Chem Res (2014) 23:1781–1788
0
2
,6-Bis(4 -nitro benzylidene) cyclohexanone (1f) Yield
-
and absence of inhibiting bischalcones at varing concen-
tration of substrate. The assays were performed as descri-
1
8
8.26 %; m.p. 200–202 °C; IR (KBr, cm ): 1,674([C=O
str), 1,435–1,589(-C=C- str), 1,342, 1,512 (-C-NO
1
bed earlier and the K values were calculated by plotting
i
2
str); H-NMR (300 MHz, CDCl , d ppm): 1.75–1.77(s, 2H,
Lineweaver–Burk plot.
3
[
CH ), 2.93–2.99(s, 4H, [CH ), 7.79–7.89(m, 8H, arom.
2
2
13
H), 7.68(s, 2H, =CH-); C-NMR (300 MHz, CDCl , d
3
ppm): 190.85, 147.40, 142.13, 138.66, 134.88, 130.79, 123.
Drug modeling studies
6
8, 77.42, 77.00, 76.58, 28.35, 22.54.
All docking studies were performed using iGemdock. For
these studies small molecular weight ligands were prepared
and enzyme structure active site was retrieved from the
Protein Data Bank. The structures were prepared in Chem
Draw 3D and were saved as MDL Mol File. The structure
of alkaline phosphatase was retrieved from Protein Data
Bank as cav1B8 J alkvan_SVA.pdb file (Holtz et al., 1999;
Murphy et al., 1997; Kim and Wyckoff, 1991). After
loading the prepared ligands and the binding site Docking
was started at Standard Docking Accuracy Settings.
0
2
,6-Bis(3 -phenyl allylidene) cyclohexanone (1g) Yield
1
-
7
1.34 %; m.p. 182–184 °C; IR (KBr, cm ): 1,651([C=O
str), 1,450–1,612(-C=C-str);
1
H-NMR (300 MHz,
CDCl , d ppm): 1.77-1.81(s, 2H,[CH ), 2.81–2.84(s, 4H,
CH ), 7.30–7.66(m, 10H, arom. H), 7.09(dd, 2H), 7.
1
1(dd, 2H), 7.30(s, 2H, =CH-); C-NMR (300 MHz,
3
2
[
2
2
3
CDCl , d ppm): 189.85, 140.76, 136.80, 136.29, 135.40,
28.68, 123.69, 121.17, 77.42, 77.00, 76.58, 26.65, 22.09.
3
1
0
2
,6-Bis(4 -(dimethyl amino)benzylidene)cyclohexanone (1h)
-
1
Yield 72.45 %; m.p. 240–242 °C; IR (KBr, cm ):
,659([C=O str), 1,427–1,574(-C=C- str), 2,916(-C–H-
1
Results and discussion
1
str); H-NMR (300 MHz, CDCl , d ppm): 1.70–1.76(s, 2H,
3
[
CH ), 2.88–2.92(s, 4H, [CH ), 7.31–7.55(m, 8H, arom.
The synthesis of bischalcones was achieved through base
catalyzed Claisen–Schmidt reaction (Scheme 1) between
cyclohexanone and p-substituted aryl aldehydes. The syn-
thesis of compounds was confirmed with the help of their
2
2
1
3
H), 7.63(s, 2H, =CH-); C-NMR (300 MHz, CDCl , d
ppm): 190.02, 150.36, 136.98, 132.39, 124.21, 111.64, 77.
3
5
2
1, 77.29, 77.08, 76.66, 40.63, 40.35, 40.12, 39.79, 28.70,
3.17.
1
physical data, IR, and H NMR spectra.
Bishalcones showed a characteristic IR absorption peak
-
at v 1,690–1,650 cm indicating the presence of a con-
1
2
,6-Bis benzylidene cyclohexanone (1i) Yield 83.58 %;
-
1
m.p. 116–118 °C; IR (KBr, cm ): 1,659([C=O str),
jugated carbonyl group ([C=O) as well as an olfenic
1
-1
1
1
,489–1,605(-C=C- str); H-NMR (300 MHz, CDCl , d
C=C band in the region 1,400–1,605 cm . In H NMR
the characteristic peak of 2H near d7.50–d7.99 was
observed.
3
ppm): 1.70–1.76(s, 2H,[CH ), 2.88–2.92(s, 4H,[CH ), 7.
2
2
1
1–7.55(m, 10H, arom. H), 7.63(s, 2H, =CH-); C-NMR
3
3
(
300 MHz, CDCl , d ppm): 190.38, 136.94, 136.21, 136.
1, 130.37, 128.58, 128.39, 77.45, 77.05, 76.61, 28.47,
3.04.
Table 1 and Fig. 1 show the effect of bischalcones on
the activity of acid phosphatase, it can be observed that
bischalcones showed an activating effect on the enzyme. It
can be the inference that bischalcones can not be used as
inhibitors for the conditions related to elevated acid phos-
phatase levels, and therefore such moieties are of no use in
the treatment of diseases where elevated acid phosphatase
3
0
2
Determination of K values and type of inhibition of alka-
i
line phosphatase activity in presence of bischalcones The
alkaline phosphatase activity was estimated in presence
O
O
Ar'
Ar'
NaOH
+
Ar'-CHO
Here, Ar' = p-F-C H , p-Cl-C H , p-Br-C H , p-CH -C H , p-OCH -C H , p-NO -C H , C H -CH=CH,
6
4
6
4
6
4
3
6
4
3
6
4
2
6
4
6 5
p-N(CH ) -C H , H
3
2
6 4
Scheme 1 Synthesis of bischalcones
1
23

Med Chem Res (2014) 23:1781–1788
1785
Fig. 1 Effect of substituted bis-
benzylidenecyclohexanones
(1a–1i) on Acid and alkaline
phosphatase activity
1
1
60
40
120
00
1
8
6
4
2
0
0
0
0
0
Acid phosphatase
Alkaline phosphatase
Substituted bis-benzylidenecyclohexanones
level is observed but at the same time, the compounds can
be beneficial at low cellular acid phosphatase levels which
have been reported in prostate cancer cells (Veeramani
et al., 2005).
phosphatase (Van Belle, 1976). It is also used as an ant-
helmentic agent. It has been reported by Kelleher et al.
(1996) that the inhibitors of alkaline phosphatase can be
successfully used in the treatment of neurological disor-
ders, where the inhibitors were administered to reduce
degeneration of cells and reduces peripheral neuropathy.
The compound and its derivatives have been successfully
used in the treatment of Alzheimer’s disease. The present
study can lead to the discovery of novel class of alkaline
phosphatase inhibitors.
From the results, it can also be observed that bischal-
cones exerted an inhibitory effect on alkaline phosphatase
(
Table 1; Fig. 1). The inhibition has been found between
*
40 and 75 % in different bischalcones.
Table 2 represents the data of in vitro inhibition and
docking studies of bischalcones on alkaline phosphatase
activities. This gives an understanding of the inhibition
caused by the target compounds on structural basis, where
the designed compounds were docked on one of the crystal
structures of alkaline phosphatase available through RCSB
Protein Data Bank (PDB entry cav1B8 J alkvan_SVA.pdb)
The binding of levamisole with the binding site of the
enzyme shows a slight overlap with the p-nitrophenyl-
phosphate binding with the enzyme active site (Fig. 2).
0
The lowest energy has been shown in case of 2,6-bis(4 -
nitro benzylidene)cyclohexanone and out of this total
energy stabilization -78.0976 is due to van der Waal
interactions and -24.3751 is the contribution of H-bonds.
The p-nitrophenyl group of 1f and substrate are perfectly
aligned.
(
Holtz et al., 1999; Murphy et al., 1997; Kim and Wyck-
off, 1991). The decrease in total energy of bischalcone–
alkaline phosphatase complex (in the range of approxi-
mately -75.5084 to -100.845) has been found to be less
than the levamisole–enzyme complex (approximately
The results clearly indicate a higher binding affinity of
designed compounds over the existing inhibitor of enzyme
-
59.6946),
a
well reported inhibitor of alkaline
Table 2 Docking and in vitro inhibition studies of alkaline phosphatase in presence of bischalcones
S. no. Name of ligand
Total energy VDW
H bond
K
i
value (mM) Type of inhibition
Competitive
0
1
2
3
4
5
6
7
8
9
1
1
.
2,6-Bis(4 -fluoro benzylidene)cyclohexanone (1a)
0
-76.5245
–82.5661
–81.4958
–75.5744
–88.0326
–100.845
–75.5084
-71.3748 -5.14966 1.09
.
2,6-Bis(4 -chloro benzylidene)cyclohexanone (1b)
0
–77.591
–4.97508 0.670
Competitive
Competitive
Non-competitive
Competitive
Competitive
Non-competitive
Competitive
Competitive
Competitive
–
.
2,6-Bis(4 -bromo benzylidene)cyclohexanone (1c)
0
–75.1658 –6.33004 0.565
–69.9312 –5.64319 0.845
–76.3711 –11.6615 0.389
–78.0976 –24.3751 0.250
–68.2076 –7.30077 0.441
–84.0486 –9.85157 0.470
–67.1408 –7.93812 0.470
.
2,6-Bis(4 -methyl benzylidene)cyclohexanone (1d)
0
.
2,6-Bis(4 -methoxy benzylidene)cyclohexanone (1e)
0
.
2,6-Bis(4 -nitro benzylidene)cyclohexanone (1f)
0
.
2,6-Bis(3 -phenylallylidene)cyclohexanone (1g)
0
.
2,6-Bis(4 -(dimethyl amino)benzylidene)cyclohexanone (1h) –93.9001
.
2,6-Di-benzylidene-cyclo-hexanone (1i)
Levamisole
–75.0789
–59.6946
–114.225
0.
1.
–49.5207 –10.1739
–48.2566 –58.6114
4
–
p-Nitrophenylphosphate
The results presented are one of the docking studies carried out using iGemdock at standard docking accuracy settings. The K
i
values and the type
i
of inhibition were determined at varying substrate concentrations in absence and presence of a fixed concentration of inhibitor and the K values
were calculated with the help of line-weaver Burk plot
123

1
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Med Chem Res (2014) 23:1781–1788
but at the same time it is less than the substrate. The affinity
with the substrate p-nitrophenylphosphate has been shown
to be the highest and the total energy stabilization is
absence of compounds to determine the K value and to
i
establish the type of inhibition (Nelson and Cox, 2008). It
was found that most of the compounds inhibited the
0
-
114.225, of which -48.2566 is the contribution of van
enzyme in a competitive manner except that of 2,6-bis(4 -
0
der Waal interactions and -58.6114 is the stabilization
resulting due to H-bonds.
methylbenzylidene) cyclohexanone and 2,6-bis(3 -phenyl-
allylidene)cyclohexanone, where the inhibition has been
found to be of non-competitive type. In a more detailed
study (Figs. 3, 4, 5) of the docking results it can be
observed that all the bischalcones except these two interact
at the binding site of p-nitrophenylphosphate. From Figs. 4
and 5 it is clear that two compounds, i.e., 1d and 1g are
showing interactions at a site other than the binding of
p-nitrophenyl phosphate.
It was found that in vitro studies correlated well with the
0
results of the docking studies. The 2,6-bis(4 -nitro ben-
zylidene)cyclohexanone was found to most inhibitory to
alkaline phosphatase showing an inhibition of *26 % at
1
mM concentration (Table 1; Fig. 1) and the compound
resulted in the highest binding energy –100.845 into the
active site of the enzyme (Table 2). In most of the bis-
chalcones docking results showing more stabilization were
found to be more inhibitory and those showing less sta-
bilization were less inhibitory. The effect of synthesized
compounds was further studied on alkaline phosphatase
at varying concentration of substrates in presence and
Fig. 4 Binding of bischalcones into the binding site of alkaline
phosphatase. Two compounds 1d and 1g are showing the binding at a
site other than the binding site
Fig. 2 Binding of levamisole and p-nitrophenyl phosphate into the
binding site of alkaline phosphatase
Fig. 5 Another view of binding of bischalcones into the binding site
of alkaline phosphatase. Two compounds 1d and 1g are showing the
binding at a site other than the binding site
0
Fig. 3 Binding of 2,6-bis(4 -nitro benzylidene)cyclohexanone 1f,
into the binding site of alkaline phosphatase
1
23

Med Chem Res (2014) 23:1781–1788
Conclusion
1787
McComb RB, Bowers GN Jr, Posen S (1979) Alkaline phosphatase.
Plenum Press, New York
Millan JL (1986) Molecular cloning and sequence analysis of
human placental alkaline phosphatase. J Biol Chem 261(7):
3112–3115
The compounds under study have been synthesized success-
1
fully as characterized by the IR and H NMR spectral data.
From the enzymatic studies, it was concluded that the com-
pounds show a differential effect on acid and alkaline phos-
phatases of liver. The acid phosphatase activity was enhanced
in presence of these compounds, whereas the activity of
alkaline phosphatase was inhibited. All the bischalcones
showed a greater affinity with alkaline phosphatase than lev-
amisole, an established inhibitor of alkaline phosphatase. 2,6-
Millan JL (1988) T Seminoma-derived Nagao isozyme is encoded by
a germ-cell alkaline phosphatase gene. Proc Natl Acad Sci USA
8
5:3024–3028
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SR (2006) Anticancer activities of novel chalcone and bis-
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0
bis(4 -nitrobenzylidene)cyclohexanone was found to be most
inhibitory to alkaline phosphatase The compounds inhibited
the enzyme activity in a competitive manner except that of
0
0
2
,6-bis(4 -methylbenzylidene)cyclohexanone and 2,6-bis(3 -
phenylallylidene)cyclohexanone, where the inhibition has
been found to be of non-competitive type.
Nelson DL, Cox MM (2008) Lehninger principles of biochemistry,
5
Acknowledgments One of the authors, Mamta Singh is thankful to
CSIR New Delhi, India for award of SRF and also to Kurukshetra
University, Kurukshetra for providing necessary research laboratory
facilities.
th edn. W H Freeman, New York
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BEK, Raghavendra R (2012a) Studies on radioprotective and
antiviral activities of some bischalcone derivatives. Med Chem
Res 21:2671
Conflict of interest The authors have declared no conflict of
interest.
Raj CGD, Sarojini BK, Ramakrishna MK, Ramesh SR, Manjunatha H
(
2012b) In vivo peritoneal antiangiogenesis and in vitro anti-
proliferative properties of some bischalcone derivatives. Med
Chem Res 21:453
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