SAR studies of o-hydroxychalcones and their cyclized analogs and study them as novel inhibitors of cathepsin B and cathepsin H

Article abstract of DOI:10.1016/j.ejps.2014.04.006

Cathepsins have emerged as a potential target for anti-cancer drug development. In the present study, we have synthesized three structurally related series of flavanoids i.e., 2′-hydroxychalcones, flavanones and flavones and assayed in vitro to study their inhibitory potency against cathepsin B and H, promising drug candidate for cancer therapy. Enzyme kinetics studies were carried out in presence of these compounds after preliminary proteolytic studies on endogenous protein substrates. SAR studies suggested that open chain flavanoids were better inhibitors as compared to their cyclized analogs. The most potent inhibitors among the three series were nitro substituted compounds 1g, 2g and 3g with K_i values of ～6.18 × 10^-8 M, 4.8 × 10^-7 M and 7.85 × 10^-7 M for cathepsin B and K_i values of ～2.8 × 10^-7 M, 31.8 × 10^-6 M and 33.7 × 10 ^-6 M for cathepsin H, respectively. The relationship between chalcone, flavanones and flavone structures interpreted by docking studies on cathepsin B and H also provided useful insights.

Full text of DOI:10.1016/j.ejps.2014.04.006

PHASCI 2993
30 April 2014
No. of Pages 9, Model 5G
European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx
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Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
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SAR studies of o-hydroxychalcones and their cyclized analogs and study
them as novel inhibitors of cathepsin B and cathepsin H
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S. Garg, N. Raghav
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Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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Cathepsins have emerged as a potential target for anti-cancer drug development. In the present study, we
have synthesized three structurally related series of flavanoids i.e., 20-hydroxychalcones, flavanones and
flavones and assayed in vitro to study their inhibitory potency against cathepsin B and H, promising drug
candidate for cancer therapy. Enzyme kinetics studies were carried out in presence of these compounds
after preliminary proteolytic studies on endogenous protein substrates. SAR studies suggested that open
chain flavanoids were better inhibitors as compared to their cyclized analogs. The most potent inhibitors
among the three series were nitro substituted compounds 1g, 2g and 3g with Ki values of ꢀ6.18 ꢁ 10ꢂ8 M,
4.8 ꢁ 10ꢂ7 M and 7.85 ꢁ 10ꢂ7 M for cathepsin B and Ki values of ꢀ2.8 ꢁ 10ꢂ7 M, 31.8 ꢁ 10ꢂ6 M and
33.7 ꢁ 10ꢂ6 M for cathepsin H, respectively. The relationship between chalcone, flavanones and flavone
structures interpreted by docking studies on cathepsin B and H also provided useful insights.
Ó 2014 Elsevier B.V. All rights reserved.
Received 17 December 2013
Received in revised form 20 March 2014
Accepted 7 April 2014
Available online xxxx
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Keywords:
20-Hydroxychalcone
Flavanone
Flavone
Cathepsin B inhibitors
Cathepsin H inhibitors
Endogenous proteolysis
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1. Introduction
The work on the identification of small molecular weight com-
pounds as inhibitors of cysteine proteases in the past decade has
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Recently, several studies have reported close association
between cancer extension and cathepsin expression (Gocheva
and Joyce, 2007; Jedeszko and Sloane, 2004; Turk et al., 2004). Pro-
tease inhibitors have considerable potential utility for therapeutic
intervention in a variety of disease states such as inflammation,
osteoporosis, microbial infections and cancer cell invasion
(Kerrow and James, 1996; Mort and Buttle, 1997; Frlan and
Gobec, 2006; Jedinak and Maliar, 2005; Lim et al., 2004). Lysosomal
cysteine proteinases are involved in intracellular protein degrada-
tion and inflammatory conditions. Increased levels of cathepsin B
[EC 3.4.22.1] and cathepsin H [EC 3.4.22.16] with simultaneously
diminished amount of cysteine protease inhibitors have been ver-
ified in a variety of tumors and therefore, approve their contribu-
tion towards invasion and metastasis. The cathepsin family of
lysosomal enzymes is also of special interest because of their
involvement in bone resorption, joint inflammation, destruction
in rheumatoid arthritis and extracellular matrix degradation.
Based on the aforementioned characteristic features, cathepsin B
and H has become the focus of intensive investigation in hope to
understand its precise role in severe diseases like cancer, angiogen-
esis, rheumatoid arthritis and especially its potential for use as a
target for chemoprevention and anticancer therapy.
been in focus. Recently, different chalcones have been identified
as cathepsin B inhibitors (Kim et al., 2013; Caracelli et al., 2012).
Chalcones are known as precursors for flavonoids, and show varied
biological activities, such as anticancer (Heyerick et al., 2012;
Zhang et al., 2012), antioxidant (Zheng et al., 2012; Kohli et al.,
2012), antiinflammatory (Shin et al., 2013) and antimicrobial prop-
erties (Tran et al., 2012a,b). The chalcone has been studied for its
inhibition of prostaglandin E2 production, inducing the biosynthe-
sis of glutathione and antibacterial activities against methicillin-
resistant staphylococcus aureus (Tran et al., 2009, 2012a,b;
Kachadourian et al., 2012). Flavanones and flavones have been
reported to have significant potential to cure, treat and prevent
tumor, senescence and cancer (Ramos, 2007). Flavanones have
been a potential source in the search for lead compounds and bio-
logically active components and have been the focus of much
researches and development in the last 30 years (Halsteen, 1983;
Silberberg et al., 2006). 2-Hydroxyflavanone markedly inhibited
the invasion, motility and cell-matrix adhesion of A549 cells
(Yung et al., 2007). Lavandulyl flavanones showed potent b-secre-
tase inhibitory activity which was strongly implicated in the cause
of Alzheimer’s disease (Shi et al., 2010). Naringin has been shown
to play a role in preventing the development of cardiovascular dis-
ease (Sung et al., 2008). As such, the identification of inhibitors of
cathepsin B and H would provide valuable tools to explore them as
potential drug-candidates in treatment of cancer, arthritis,
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Corresponding author. Tel.: +91 9896918277.
E-mail address: nraghav.chem@gmail.com (N. Raghav).
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http://dx.doi.org/10.1016/j.ejps.2014.04.006
0928-0987/Ó 2014 Elsevier B.V. All rights reserved.
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Please cite this article in press as: Garg, S., Raghav, N. SAR studies of o-hydroxychalcones and their cyclized analogs and study them as novel inhibitors of
cathepsin B and cathepsin H. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.006

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inflammation and other tissue degeneration processes. We have
identified different classes of compounds as inhibitors to endoge-
nous proteolysis at a pH where cysteine proteases are active
(Raghav et al., 2010a,b, 2011, 2012; Raghav and Singh, 2013).
Recently we have reported bischalcones, their derivatives
(Raghav and Singh, 2014a) and acylhydrazides, triazoles (Raghav
and Singh, 2014b) as novel inhibitors of cathepsin B & H. To further
achieve this endeavor, we here present the synthesis of some 20-
hydroxychalcones and their corresponding flavanone and flavone,
followed by their evaluation as cysteine protease inhibitors and
inhibitory studies on cathepsin B and cathepsin H.
tionation, molecular sieve chromatography on Sephadex G-100
column chromatography and finally ion-exchange chromatogra-
phies on CM-Sephadex C-50 and DEAE Sephadex A-50 column.
The specific activities of the cathepsin B and cathepsin H were
ꢀ11.15 nmol/min/mg and ꢀ22.91 nmol/min/mg, respectively. The
specific activity was calculated by the following equation.
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Specific activity ¼ total activity units=total protein
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2.2.4. Effect of 2-hydroxychalcones, flavanones and flavones on the
activity of cathepsin B and cathepsin H
The activities of cathepsin B were estimated at varying concen-
trations of synthesized 20-hydroxychalcones, flavanones and flav-
ones (Fig. 2a–c), separately. First of all, enzyme was equilibrated
in 0.1 M phosphate buffer of pH 6.0 at 37 °C. The stock solutions
of compounds were prepared in DMSO. Appropriate amount of
stock solutions of individual compounds and corresponding
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2. Materials and methods
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All the chemicals (analytical grade) and biochemicals, Fast Gar-
net GBC (o-aminoazotoluene diazonium salt, substrate a-N-ben-
zoyl-D,L-arginine-2-naphthylamide (BANA) and Leu-bNA were
purchased either from Sigma Chemical Co., USA or from Bachem
Feinchemikalien AG, Switzerland. The protein sample was concen-
trated using Amicon stirred cells with YM 10 membrane under
nitrogen pressure of 4–5 psi. The source of enzyme, fresh goat liver,
was obtained from local slaughter house.
amount of DMSO (in total 20 ll) was added in the reaction mixture
to effect different concentrations of each compound as indicated in
Fig. 2a–f, separately. After incubation time of 30 min residual
enzyme activity was estimated by the usual enzyme assay
(Kamboj et al., 1990) at pH 6.0 using a-N-benzoyl-D,L-arginine-2-
naphthylamide (BANA) as substrate. The experiments were per-
formed in triplicate for each concentration and averaged before
further calculations. The % activity in each case has been calculated
with respect to the control where no compound was added but an
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2.1. General procedure
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Melting points were determined in open capillary tubes and are
uncorrected. All the chemicals and solvents used were of labora-
tory grade. IR spectra (KBr, cmꢂ1) were recorded on a PerkinElmer
spectrometer. 1H NMR spectra was recorded on Bruker 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 aluminum plates percoated 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.
equivalent amount of DMSO (20 ll) was present. The results are
presented in Table 1. Similar experiments were designed to evalu-
ate the effect of varying concentrations of synthesized 2-hydroxy-
chalcones, flavanones and flavones separately on cathepsin H using
LeubNA as substrate at pH 7.0 (Fig. 2d–f) (Raghav et al., 1995).
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2.2.5. Kinetic studies of synthesized compounds on cathepsin B and
cathepsin H
After establishing the inhibitory action of synthesized com-
pounds on cathepsin B, experiments were designed to evaluate
the type of inhibition and to determine the Ki value of these com-
pounds on respective enzymes. For that, enzyme activities were
evaluated at different substrate concentrations in presence and
absence of a fixed concentration of inhibitor. The enzyme concen-
tration was kept constant in all the experiments. Line-weaver Burk
plot were drawn between 1/S and 1/V in presence and absence of
different series of compounds on cathepsin B (Fig. 3a–c) and
cathepsin H (Fig. 3d–f). And the Ki values were calculated using
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2.2. Proteolytic studies
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2.2.1. Preparation of liver homogenate
Goat liver, purchased fresh from the local slaughter house was
washed with cold isotonic saline solution. The tissue was then
homogenized in 0.1 M acetate buffer pH 5.5 containing 0.2 M NaCl
in a mixer-cum-blender to obtain 10% (w/v) homogenate and was
stored at 4 °C till further use.
0
the line-weaver Burk equation for competitive inhibition Km = -
Km(1 + I/Ki).
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2.2.2. Assay for proteolytic activity
The proteolytic activity was estimated at pH 5.0, 37 °C using
0.1 M acetate buffer as the incubation medium (Kamboj et al.,
1992). The homogenate prepared above was incubated with the
buffer at 37 °C for 3 h and 24 h, separately. The reaction was
stopped by the addition of TCA and the resulting solution was cen-
trifuged to precipitate proteins. The acid soluble proteins were
quantitated in the supernatant using Bradford method (Bradford,
1976). The experiments were conducted in triplicate and the
results are presented in Table 1 (Fig. 1).
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2.3. Drug modeling studies
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All docking studies were performed using iGemdock. For these
studies, small molecular weight ligands and enzyme active site
structure is required. The structure of cathepsin B was retrieved
from Protein Data Bank (http://www.rcsb.org/) as cav2IPP
B_PYS.pdb (Huber et al., 2013). The structures were prepared in
Marvin sketch minimized and were saved as MDL Mol File. The
prepared ligands and the binding site were loaded in the iGemdock
software and docking was started by setting the GA-parameters at
drug screening setting. The results presented in Table 2 pertain to
the interaction data. Fitness is the total energy of a predicted pose
in the binding site. The empirical scoring function of iGemdock is
the sum total of Van der Waal, H-bonding and electrostatic energy.
The docked poses of the ligands in the active site of cathepsin B
along with the substrate BANA and different compounds are
shown in Fig. 4.
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2.2.3. Purification of goat brain cathepsin B and cathepsin H
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All the purification steps were carried out at 4 °C. Cathepsin B
and H were isolated, separated and purified from goat liver using
the following procedure (Kamboj et al., 1990; Raghav et al.,
1995) including acetone powder preparation, homogenization in
cold 0.1 M acetate buffer pH 5.5 containing 0.2 M NaCl and 1 mM
EDTA, acid-autolysis at pH 4.0 and 30–80% ammonium sulfate frac-
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147 Q5
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Please cite this article in press as: Garg, S., Raghav, N. SAR studies of o-hydroxychalcones and their cyclized analogs and study them as novel inhibitors of
cathepsin B and cathepsin H. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.006

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Table 1
Enzyme inhibition studies in presence of substituted 20-hydroxychalcones, flavanones and flavones.
(Protease activity)
S. no. Compound name
Enzyme inhibition studies
Cathepsin B
% Residual activity at 10ꢂ4
M
Cathepsin H
concentration of compounds
3 h incubation 24 h incubation % Residual activity Ki
% Residual activity Ki
at (Z) ꢁ 10ꢂ5 M
concentration
of compounds
(10ꢂ8 M) at (Z) ꢁ 10ꢂ4
M
(10ꢂ7 M)
concentration
of compounds
Control
1-(-2-Hydroxyphenyl)-3-phenylprop-2-en-1-one (1a)
1-(-2-Hydroxyphenyl)-3-(-4-methylphenyl)prop-2-
en-1-one (1b)
100 8.99
6.23 0.429
9.3 0.69
100 0.404
57.57 5.25
96.9 9.69
100 1.01
34.26 3.22(0.1)
63.29 6.27(0.1)
100 0.675
62.43 4.720(0.1)
91.62 2.21(0.1)
1.
2.
21.17
501
58.7
528
3.
4.
5.
6.
7.
8.
9.
1-(-2-Hydroxyphenyl)-3-(-4-methoxyphenyl)prop-2-
en-1-one (1c)
1-(-2-Hydroxyphenyl)-3-(-3-chlorophenyl)prop-2-en-
1-one (1d)
1-(-2-Hydroxyphenyl)-3-(-4-chlorophenyl)prop-2-en-
1-one (1e)
1-(-2-Hydroxyphenyl)-3-(-3-nitrophenyl)prop-2-en-
1-one (1f)
1-(-2-Hydroxyphenyl)-3-(-4-nitrophenyl)prop-2-en-
1-one (1g)
1-(-2-Hydroxyphenyl)-3-(-2-methoxyphenyl)prop-2-
en-1-one (1h)
1-(-2-Hydroxyphenyl)-3-(-3-methoxyphenyl)prop-2-
en-1-one (1i)
9.1 0.901
5.11 0.509
4.29 0.421
2.65 0.265
0.63 0.063
7.97 0.707
7.07 0.623
94.14 8.28
42.42 0.424
57.74 5.74
44.44 4.29
8.88 0.707
34.34 0.34
30.49 0.94
48.31 4.77(0.1)
23.22 2.24(0.1)
21.71 2.15(0.1)
16.09 1.360(0.1)
15.33 1.54(0.1)
35.33 2.54(0.1)
36.72 3.59(0.1)
131
18.04
17
88.37 8.81(0.1)
59.72 5.75(0.1)
46.31 4.64(0.1)
45.13 4.491(0.1)
45.00 3.760(0.1)
78.11 7.080(0.1)
82.59 7.37(0.1)
272
46.5
41
8.21
6.18
2.22
2.5
29
28
84.5
137.3
10.
11.
12.
2,3-Dihydro-2-phenylchromen-4-one (2a)
2,3-Dihydro-2-(-4-methylphenyl)chromen-4-one (2b)
2,3-Dihydro-2-(-4-methoxyphenyl)chromen-4-one
(2c)
18.81 1.84
26.99 2.65
26.58 2.59
24.84 3.12
51.51 0.867
29.09 1.86
46.27 4.517(1.0)
86.62 8.592(1.0)
75.84 7.574(1.0)
195
2137
950.5
79.45 7.54(1.0)
89.46 8.81(1.0)
80.72 6.24(1.0)
875
3906
3546
13.
14.
15.
16.
17.
2,3-Dihydro-2-(-3-chlorophenyl)chromen-4-one (2d)
2,3-Dihydro-2-(-4-chlorophenyl)chromen-4-one (2e)
2,3-Dihydro-2-(-3-nitrophenyl)chromen-4-one (2f)
2,3-Dihydro-2-(-4-nitrophenyl)chromen-4-one (2g)
2,3-Dihydro-2-(-2-methoxyphenyl)chromen-4-one
(2h)
18.73 1.16
17.89 1.178
14.14 1.08
10.02 1.075
19.55 1.955
11.19 1.93
43.19 0.877
61.01 1.85
38.3 1.84
34.64 3.08(1.0)
25.84 0.480(1.0)
20.09 2.008(1.0)
16.09 1.603(1.0)
46.62 4.599(1.0)
175
121
108
48
57.08 5.75(1.0)
43.40 4.35(1.0)
24.35 2.43(1.0)
43.40 4.35(1.0)
70.70 7.00(1.0)
595
432
337
318
875
31.31 0.867
211
18.
2,3-Dihydro-2-(-3-methoxyphenyl)chromen-4-one
(2i)
21.88 2.08
35.15 1.86
56.09 5.601(1.0)
479
79.45 7.9(1.0)
2493
19.
20.
21.
22.
23.
24.
25.
26.
27.
2-Phenyl-4H-chromen-4-one (3a)
34.56 3.39
60.44 5.316
53.16 4.29
31.28 3.1
30.26 3.026
29.24 2.92
28.83 2.86
45.19 4.51
51.73 4.29
31.90 2.638
72.59 7.034
70.34 5.535
26.38 2.525
25.25 2.06
20.6 1.767
17.67 1.767
35.40 3.131
55.35 5.35
39.88 4.10(1.0)
63.92 6.35(1.0)
53.32 5.13(1.0)
33.47 3.09(1.0)
31.14 2.99(1.0)
21.70 2.071(1.0)
10.48 1.039(1.0)
49.43 4.76(1.0)
51.47 5.097(1.0)
201.1
5010
1314
190.7
146
118.00
78.5
222.5
511.7
74.29 6.64(1.0)
88.67 8.143(1.0)
87.18 8.71(1.0)
64.86 6.480(1.0)
54.29 4.930(1.0)
50.97 4.930(1.0)
33.85 3.143(1.0)
79.02 7.900(1.0)
84.97 8.49(1.0)
875
2-(-4-Methylphenyl)-4H-chromen-4-one (3b)
2-(-4-Methoxyphenyl)-4H-chromen-4-one (3c)
2-(-3-Chlorophenyl)-4H-chromen-4-one (3d)
2-(-4-Chlorophenyl)-4H-chromen-4-one (3e)
2-(-3-Nitroyphenyl)-4H-chromen-4-one (3f)
2-(-4-Nitroyphenyl)-4H-chromen-4-one (3g)
2-(-2-Methoxyphenyl)-4H-chromen-4-one (3h)
2-(-3-Methoxyphenyl)-4H-chromen-4-one (3i)
5966.5
4854.3
766
595
432
337
1090.5
3322.2
The TCA soluble peptides were estimated at 630 nm using Bradford method (Bradford, 1976) and the results are the mean and S.M.D. of the experiment conducted in
triplicate and is calculated as % residual protease activity in 0.1% liver homogenate w.r.t. control where no compound was added but an equivalent amount of solvent was
present. Cathepsin B and cathepsin H activities were calculated using BANA and Leu-bNA as substrates at pH 6.0 and 7.0, respectively. The enzyme activity was determined at
minimum inhibitory concentration of each compound given in parenthesis. The specific activitiy of the Cathepsin B and cathepsin H were ꢀ11.15 nmol/min/mg and
ꢀ22.91 nmol/min/mg, respectively. In order to determine Ki values, experiments were conducted in triplicates in presence and absence of a fixed concentration of different
compound, separately. The results were then plotted between 1/V and 1/S to obtain line-weaver Burk plots and all the compounds were established as competitive inhibitors.
The Ki values were calculated using line-weaver Burk equations for competitive inhibition.
Fig. 1. Effect of substituted 20-hydroxychalcone, flavanone and flavones on the endogenous proteolytic activity for 3 h and 24 h reaction. The data in each bar represents the %
residual activity in presence of individual compound w.r.t. control taken as 100.
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Please cite this article in press as: Garg, S., Raghav, N. SAR studies of o-hydroxychalcones and their cyclized analogs and study them as novel inhibitors of
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Fig. 2. Effect of varying concentration of 20-hydroxychalcone (1a–1i) (a), flavanones (2a–2i) (b), flavones (3a–3i) (c) at pH 6.0 on cathepsin B activity. Effect of varying
concentration of 20-hydroxychalcone (1a–1i) (d), flavanones (2a–2i) (e), flavones(3a–3i) (f) at pH 7.0 on cathepsin H activity. Results are mean of experiment conducted in
triplicate. Activities are % of control which contain equivalent amount of solvent.
20-hydroxychalcone (1a–1i) in CDCl3 shows signals at d 12.4,
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3. Result and discussion
7.60–7.67 and 7.46–7.59 ppm assignable to 20-OH and CH@CH pro-
20-Hydroxychalcones and flavanones were synthesized under
ecofriendly, solvent free conditions (Scheme 1). The products were
tons, respectively. In flavanones (2a–2i), a characteristic ABX
pattern is observed: H appear as double doublets at d 2.79–3.10,
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220
3.25–3.67 and 5.50–5.80 ppm with (Jab = 16.2–18.0 Hz, Jax
=
obtained in pure form in about 15–30 min in 80–90% yield. The
structure elucidation of compounds was based on the spectral data
(IR, 13C NMR). All the synthesized compounds show a characteris-
tic IR absorption peak. The IR spectra showed mainly stretching
bands at 3100–3000, 1630–1600, and 1600–1450 cmꢂ1 assigned
to (OH), (C@O) and aromatic (C@C), in 20-hydroxychalcone respec-
tively. However in flavanones, the ˜C@O peak observed at
1697–1657 cmꢂ1 showing a shift from 20-hydroxychalcone due to
lesser extension of conjugation. The 1H NMR spectrum of
4.5–7.2 Hz, Jbx = 10.8–12.3 Hz). In 1H NMR spectrum of flavones
(3a–3i), @CH proton appears as singlet at d 3.4 ppm.
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229
3.1. Effect of 2-hydroxychalcones and their cyclized analogs flavanones
and flavones on in vitro endogenous proteolysis in liver homogenate
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Table 1 presents the results of proteolytic studies conducted at
pH 5.0 on endogenous protein substrate in presence of different
Q1
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Fig. 3. Line-weaver Burk plot for cathepsin B at varying concentrations of BANA in presence of 1 ꢁ 10ꢂ6 M concentration of 20-hydroxychalcone (a), 1 ꢁ 10ꢂ5 M concentration
of flavanones (b), 1 ꢁ 10ꢂ5 M concentration of flavones (c), respectively at pH 6.0 using 100
ll of enzyme having specific activity ꢀ11.15 units/mg. The Km value for control
have been found to be 3.64 ꢁ 10ꢂ4 M. Similarly line-weaver Burk plot for cathepsin H on varying concentrations of leu-bNA in presence of 1 ꢁ 10ꢂ5 M concentration of
20-hydroxychalcone (d), 1 ꢁ 10ꢂ4 M concentration of flavanones (e), 1 ꢁ 10ꢂ4 M concentration of flavones (f), respectively at pH 7.0 using 100
ll of enzyme having specific
activity ꢀ22.91 units/mg. The Km value for control have been found to be 5.34 ꢁ 10ꢂ4 M. The Ki values as calculated from this graph are presented in Table 1.
compounds separately at 10ꢂ4 M. It can be observed that proteo-
lytic activity is inhibited appreciably in presence of these com-
pounds. Further it was found that the inhibition was more at
3.0 h and less at 24.0 h. suggesting that inhibition caused by the
compounds is of reversible type because initial inhibition caused
by the compounds is reversed when incubated for a long time.
The compounds bearing nitro group led to a dramatic decrease in
proteolytic activity and showed higher inhibition than those of
the corresponding compounds with methyl or methoxy group at
the same position indicating that activity decreases in presence
of electron withdrawing groups whereas it is less affected by the
presence electron donating groups. A similar trend has previously
been reported for semicarbazones (Raghav and Singh, 2013), aryl-
hydrazones (Raghav et al., 2012) and phenylhydrazones (Raghav
et al., 2010a). An important finding in the study was that 20-
hydroxychalcones were better inhibitor to endogenous proteolysis
followed by flavanones and flavones in that order. This is due to
presence of a–b unsaturated carbonyl moiety in 20-hydroxychal-
cones which are more prone to attack by nucleophilic groups pres-
ent at the active site of proteolytic enzymes and lead to michael
addition as compared to flavanones and flavones. Further 1,3-pro-
pene backbone is more flexible than the corresponding cyclized
derivatives. After establishing the inhibition of endogenous prote-
olytic activity in presence of flavanoids at pH 5.0, where most of
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Table 2
Docking studies showing decrease in different energies of cathepsin B & H in presence of different 2-hydroxychalcones, flavanones and flavones.
Enzyme
Compound
Cathepsin B
Total energy
Cathepsin H
Total energy
VDW
HBond
Electronic
VDW
HBond
Electronic
BANA
Leupeptin
Leu-bNA
LeuCH2Cl
1a
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
3d
3e
3f
3g
3h
3i
ꢂ129.82
ꢂ112.59
–
ꢂ93.43
ꢂ81.66
–
ꢂ34.084
ꢂ30.935
–
ꢂ2.311
–
–
–
–
–
–
–
–
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
–
–
0
0
0
0
0
ꢂ76.88
ꢂ59.32
ꢂ72.76
ꢂ73.47
ꢂ71.99
ꢂ73.11
ꢂ69.31
ꢂ89.51
ꢂ85.26
ꢂ76.71
ꢂ75.82
ꢂ68.80
ꢂ66.12
ꢂ70.68
ꢂ66.04
ꢂ65.74
ꢂ85.83
ꢂ80.49
ꢂ74.49
ꢂ74.42
ꢂ70.45
ꢂ72.32
ꢂ76.46
ꢂ74.40
ꢂ72.18
ꢂ86.31
ꢂ85.52
ꢂ76.29
ꢂ73.65
ꢂ60.91
ꢂ46.50
ꢂ66.76
ꢂ64.97
ꢂ62.53
ꢂ59.57
ꢂ57.78
ꢂ60.53
ꢂ62.33
ꢂ73.31
ꢂ61.33
ꢂ59.30
ꢂ59.12
ꢂ61.23
ꢂ60.36
ꢂ58.74
ꢂ63.33
ꢂ58.99
ꢂ65.00
ꢂ61.56
ꢂ61.07
ꢂ62.82
ꢂ63.90
ꢂ64.99
ꢂ62.71
ꢂ59.18
ꢂ63.02
ꢂ66.80
ꢂ70.15
ꢂ15.97
ꢂ12.82
ꢂ6
–
–
–
ꢂ77.94
ꢂ78.68
ꢂ84.04
ꢂ79.59
ꢂ80.12
ꢂ96.11
ꢂ86.44
ꢂ85.52
ꢂ82.17
ꢂ74.53
ꢂ75.48
ꢂ76.96
ꢂ72.50
ꢂ75.14
ꢂ85.90
ꢂ77.12
ꢂ77.73
ꢂ76.90
ꢂ71.41
ꢂ71.69
ꢂ75.00
ꢂ74.06
ꢂ71.25
ꢂ88.13
ꢂ77.20
ꢂ81.43
ꢂ77.91
ꢂ64.62
ꢂ65.94
ꢂ66.94
ꢂ65.83
ꢂ65.87
ꢂ69.86
ꢂ68.32
ꢂ65.97
ꢂ62.34
ꢂ65.05
ꢂ66.01
ꢂ67.46
ꢂ63.00
ꢂ65.64
ꢂ60.82
ꢂ66.32
ꢂ68.96
ꢂ64.101
ꢂ59.46
ꢂ59.94
ꢂ65.50
ꢂ62.26
ꢂ60.12
ꢂ64.78
ꢂ67.708
ꢂ63.49
ꢂ62.04
ꢂ13.31
ꢂ12.74
ꢂ17.09
ꢂ13.76
ꢂ14.24
ꢂ26.90
ꢂ18.37
ꢂ19.54
ꢂ19.83
ꢂ9.476
ꢂ9.472
ꢂ9.5
ꢂ8.5
ꢂ9.457
ꢂ13.54
ꢂ11.53
ꢂ28.97
ꢂ22.92
ꢂ3.398
ꢂ14.48
ꢂ9.5
0.660
0.255
0
0
0
0
0
0
ꢂ7
ꢂ9.45
ꢂ5.675
ꢂ7
ꢂ9.5
ꢂ9.5
0
ꢂ25.75
ꢂ9.5
0.674
ꢂ1.293
ꢂ22.5
ꢂ21.49
ꢂ9.488
ꢂ12.85
ꢂ9.381
ꢂ9.5
ꢂ8.770
ꢂ12.80
ꢂ11.94
ꢂ11.75
ꢂ9.5
0
0
0
0
0
0
0
ꢂ12.56
ꢂ9.40
ꢂ9.47
ꢂ26.88
ꢂ22.5
ꢂ9.489
ꢂ3.5
0
0
0
ꢂ11.80
ꢂ11.12
ꢂ23.78
ꢂ9.5
0.444
ꢂ0.238
0
0
0
0
0
0
ꢂ17.93
ꢂ15.86
The results are one of the docking experiments run using iGemdock under drug screening settings. The ligands were loaded as MDL mol file. The active site was extracted
from the structure of cathepsin B and cathepsin H were retrieved from Protein Data Bank (http://www.rcsb.org/) as cav2IPP B_PYS.pdb (Huber et al., 2013), and (cav8PCH
H_NAG.pdb) (Guncar et al., 1998), respectively.
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the proteolytic activity is attributed to cysteine proteases (Raghav
et al., 2010b). It was thought proper to study the effect of synthe-
sized compounds on purified cathepsin B and cathepsin H.
3.3. Enzyme kinetic studies
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Afterv establishing the inhibitory action of synthesized com-
pounds on cathepsin
B and cathepsin H, experiments were
3.2. Effect of 20-hydroxychalcones, flavanones, flavones on the activity
of cathepsin B and cathepsin H
designed to evaluate the type of inhibition and to determine
the Ki value of these compounds on cathepsin B and cathepsin
H. For that, enzyme activities were evaluated at different sub-
strate concentrations in presence and absence of a fixed concen-
tration of different compounds. The enzyme concentration was
kept constant in all the experiments. Line-weaver Burk plots
were drawn in 1/S and 1/V in presence and absence of inhibitor
for cathepsin B (Fig. 3a–c) and for cathepsin H (Fig. 3d–f) sepa-
rately. It was found that the plots of 1/V and 1/S were straight
lines intersecting at the Y-axis and showed that value of V
remained constant in all the compounds whereas the value of
K changes with each compound. These studies suggested a com-
petitive type of inhibition exhibited by these compounds. Using
the line-weaver Burk equation for competitive inhibition the
Km values were calculated, which has been presented in Table
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The activities of cathepsin B and cathepsin H was estimated
at varying concentrations of title compounds (Fig. 2a–f), respec-
tively. The figures show the relationship between the enzyme
activity and concentration of different flavanoids. The plots of %
residual activities versus the concentrations of different
compounds gave a relationship where increased compound con-
centration caused more inhibition. Among the various compounds
tested, 1-(-2hydroxyphenyl)-3-(-4-nitrophenyl)prop-2-en-1-one
(1g) was found to be most inhibitory to cathepsin B and cathepsin
H activity. Similar trend was observed in flavanones and flavones.
It has been found that cathepsin B and cathepsin H activity is
inhibited and is affected by the substituent present in B ring.
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7
Fig. 4. Docking results showing the alignment of most inhibitory compounds different compounds with BANA in the active site of cathepsin B (cav2IPP B_PYS.pdb) and
LeubNA in the active site of cathepsin H (cav8PCH H_NAG.pdb). Here (a–c) shows alignment of 20-hydroxychalcone (1g), flavanones (2g), flavones (3g) with BANA in the active
site of cathepsin B (cav2IPP B_PYS.pdb) respectively. And (d–f) showing the alignment 1g, 20-hydroxychalcone (1d), flavanone (2g), flavones (3g) with Leu bNA in the active
site of cathepsin H (cav8PCH H_NAG.pdb) respectively.
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1. The Ki value of most inhibiting compound for cathepsin B in
the corresponding series i.e. chalcone, flavanone and flavone
3.4. Molecular docking experiment
has been evaluated as ꢀ6.18 ꢁ 10ꢂ8 M, 4.8 ꢁ 10ꢂ7
M
and
On the basis of the interaction data of docking experiments
that include total energy and individual energy terms, an
indicative of the fitness of a predicted pose in the binding site,
it is suggested that the level of interaction is highest for
20-hydroxychalcone (1a–1i) followed by flavanones (2a–2i) and
flavones (3a–3i) with the active site of cathepsin B (Table 2)
and in case of cathepsin H (Table 2), the order was 20-hydroxy-
chalcone (1a–1i) > flavones (3a–3i) > flavanones (2a–2i). In
cathepsin B, all the compounds showed a lesser interaction than
the peptidyl inhibitor, leupeptin. Decrease in total energy for
leupeptin–cathepsin B has come out be ꢂ122.529 which is equiv-
alent to substrate BANA, ꢂ122.442 of which the contribution of
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7.85 ꢁ 10ꢂ7
M for compounds 1g, 2g and 3g, respectively;
similarly for cathepsin H, these compound showed maximum
inhibition with Ki values ꢀ2.8 ꢁ 10ꢂ7 M, 31.8 ꢁ 10ꢂ6
M and
33.7 ꢁ 10ꢂ6 M respectively. We have demonstrated here that in
general, these compounds showed more inhibition on cathepsin
B activity in comparison to cathepsin H. Analysis of the effect
of these compounds on cathepsin B and cathepsin H by the com-
bination of serendipitous biological selectivity with target-direc-
ted chemical specificity suggests an optimistic future for their
use as cysteine protease inhibitors as therapy in a number of
disease processes.
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Scheme 1. Synthesis of 20-hydroxychalcones, flavanones and flavones.
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Van der Waal interactions are more with a score of ꢂ89.9932 as
compared to H-bonds with a score of 32.5308. This is due to pep-
tide protein interaction. Leupeptin is peptidyl in nature and
therefore binds effectively with the enzyme active site resulting
in higher binding energy. As compared to this the binding energy
of title compounds are less (Table 2). When compared with in the
designed series to evaluate the interaction with cathepsin B, the
results clearly support the in vitro inhibition studies. 20-hydroxy-
chalcones show a higher interaction than flavanones and flavones
(Table 1) in that order. The in silico predictable behavior of
enzyme–ligand interaction can give an idea about the interaction.
Docking methods can provide valuable insight into the binding
mode between the ligand and the enzyme active site thereby
have an important role in the understanding of ligand–enzyme
interactions. From molecular docking experiments, we observed
that all of compounds inhibit the enzyme in a competitive man-
ner and because these compete at the binding site of enzyme
with substrate. However in cathepsin H, the decrease in total
energy for the reference inhibitor Leu-CH2Cl was less as
compared to all the designed compounds. Here, it can be seen
3.5. Structure activity relationship studies
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in vitro inhibition studies of differently functionalized
20-hydroxychalcone (1a–1i), flavanones (2a–2i) and flavones
(3a–3i) on cathepsins B & H clearly demonstrated that the
designed compounds inhibit these enzyme effectively. Among
these different series it has been observed that 20-hydroxychal-
cones have been evaluated as potential inhibitors to these cys-
teine proteases probably due to presence of a–b unsaturated
carbonyl moiety in the molecule. Chalcones have previously been
reported as inhibitors of cathepsin B (Kim et al., 2013; Caracelli
et al., 2012) the ACH2SH moiety present at the active site of
enzymes can interact with the electron deficient center present
in the chalcones inhibiting the enzymes activity. Flavanones and
Flavones have also been found inhibitory to the target enzymes.
Previously, the structure–activity relationship (SAR) and binding
mechanism of three biflavones isolated from Taxodium mucrona-
tum has been reported (Pan et al., 2005) as novel natural inhibi-
tors of human CatB with IC50 values of 1.75, 1.68 and 0.55 l M.
In the present study, substitution pattern in each series was care-
fully altered to acquire a comprehensible correlation between the
electronic environment of ligands and the enzyme active site or
binding site. Among the similarly substituted designed com-
pounds nitro substituent has been evaluated as most inhibitory
to both the enzymes. In a similar study on bischalcones and qui-
nazoline-2(1H)-one and quinazoline-2(1H)-thione derivatives
(Raghav and Singh, 2014a), we have evaluated that nitro
substituted quinazoline-2(1H)-one and quinazoline-2(1H)-thione
derivatives are potential inhibitors to cathepsins B & H. in another
study, carried out in order to identify non-peptidyl inhibitors to
cathepsins B and H. we have reported that 3-phenyl-5-(4-nitro-
phenyl)-4-amino-1,2,4-triazole and 3-phenyl-5-(30-nirophenyl)-
4-amino-1,2,4-triazole (Raghav and Singh, 2014b) inhibited
cathepsins B & H effectively. It can be observed that nitro-subsi-
tution provide an effective interaction for cathepsin B & H. All the
synthesized compounds were evaluated as better inhibitors to
that though Leu-CH2Cl is specific inhibitor for cathepsin
H
(Schwartz and Barrett, 1980). But possess only one amino acid
residue as compared to leupeptin–cathepsin B. therefore, the
leu-CH2Cl-cathepsin H interaction cause a decrease in energy of
ꢂ60.205 of which ꢂ44.643 is the interaction and ꢂ15.9762 is
due to hydrogen bonds. As listed in Table 2, all the designed com-
pounds have been found to show higher decrease in ligand
cathepsin H interaction energy than leu-CH2Cl-cathepsin H. It is
clear in (Fig. 4a–c) showing the docked view of most inhibitory
20-hydroxychalcone, flavanones and flavones in the active site of
cathepsin B, where active site groups Cys-29 and His-199 interact
with all these compounds, (Fig. 4d–f) show the docked view of
most inhibitory 20-hydroxychalcone, flavanones and flavones in
the active site of cathepsin H, all the compounds interact with
amino acyl acceptor site of the enzyme. In order to explore
non-peptidyl novel inhibitors to cathepsin H, such studies are
important where the structurally related compounds showing
potential biological activities are evaluated for their inhibitory
effect on physiologically significant enzyme cathepsin B and
cathepsin H. We here for the first time report comparative study
of the three related class of compounds on clinically significant
enzymes cathepsin B and H.
cathepsin
B as compared with cathepsin H, indicating that
cathepsin B active site is more susceptible to these compounds
as compared to cathepsin H. In the present study, we have
discussed that backbone structure as well as substituent effects
the enzyme activities. The results were consistent when
compared with in- silico docking studies.
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4. Conclusion
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sin
B
activity maximally with Ki values of ꢀ6.18 ꢁ 10ꢂ8 M,
4.8 ꢁ 10ꢂ7 M and 7.85 ꢁ 10ꢂ7 M, respectively. The compounds
were also identified as most potent inhibitors to cathepsin H with
the Ki values of ꢀ2.8 ꢁ 10ꢂ7 M, 31.8 ꢁ 10ꢂ6 M and 33.7 ꢁ 10ꢂ6
M
respectively. All the compounds were evaluated as competitive
inhibitors of enzymes and the results are well documented with
in-silico experiments.
422
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424
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Acknowledgements
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One of the authors, Shweta Garg is thankful to UGC New Delhi,
India for award of JRF and also to Kurukshetra University, Kuruksh-
etra for providing necessary research laboratory facilities.
The authors have declared no conflict of interest.
431
Appendix A. Supplementary material
432
433
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejps.2014.04.006.
434
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Please cite this article in press as: Garg, S., Raghav, N. SAR studies of o-hydroxychalcones and their cyclized analogs and study them as novel inhibitors of
cathepsin B and cathepsin H. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.04.006