SAR studies of differently functionalized chalcones based hydrazones and their cyclized derivatives as inhibitors of mammalian cathepsin B and cathepsin H

Article abstract of DOI:10.1016/j.bmc.2014.05.037

Cathepsins have emerged as potential drug targets for melanoma therapy and engrossed attention of researchers for development and evaluation of cysteine cathepsin inhibitors as cancer therapeutics. In this direction, we have designed, synthesized, and ass

Full text of DOI:10.1016/j.bmc.2014.05.037

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
SAR studies of differently functionalized chalcones based hydrazones
and their cyclized derivatives as inhibitors of mammalian cathepsin
B and cathepsin H
⇑
Neera Raghav , Mamta Singh
Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Cathepsins have emerged as potential drug targets for melanoma therapy and engrossed attention of
researchers for development and evaluation of cysteine cathepsin inhibitors as cancer therapeutics. In
this direction, we have designed, synthesized, and assayed in vitro a small library of 30 low molecular
weight functionalized analogs of chalcone hydrazones for evaluating structure–activity relationship
aspects and inhibitory potency against cathepsin B and H. The maximum inhibitory effect was exerted
by chalcone hydrazones, which are open chain analogues followed by their cyclized derivatives, pyrazo-
lines and pyrazoles. All the synthesized compounds were established as reversible inhibitors of these
enzymes. Cathepsin B was selectively inhibited by the compounds in each series. Compounds 1d, 2d
and 4d were recognized as most potent inhibitors of cathepsin B in this study with Ki values of
Received 22 March 2014
Revised 14 May 2014
Accepted 15 May 2014
Available online xxxx
Keywords:
Cathepsin B and cathepsin H
Reversible inhibitors
Chalcone hydrazones
Pyrazolines and pyrazoles
0.042 lM, 0.053 lM and 0.131 lM whereas 1b (Ki = 1.111 lM), 2b (Ki = 1.174 lM) and 4b (Ki = 1.562 lM)
inhibited cathepsin H activity effectively. And, preeminent cathepsin B inhibitors were –NO2 functional-
ized however, –Cl substituted moieties were the most persuasive inhibitors for cathepsin H among all the
designed compounds. Molecular docking studies performed using iGemdock provided valuable insights.
Ó 2014 Published by Elsevier Ltd.
1. Introduction
been reported as cysteine protease inhibitors (Fig. 1) for example,
chalcones (1 and 2) as cathepsin B inhibitor,12–14 hydrazone (3)
Cathepsin B and H are up regulated in a variety of malignant
tumors and play a significant role in cancer cell invasion and
migration.1–6 Cysteine cathepsin inhibition has resulted in a signif-
icant decrease in tumor invasiveness. These result encouraged the
development and enduring evaluation of cysteine cathepsin
inhibitors as cancer therapeutics.7–11 Cathepsin B and H are unique
biomolecular targets for anticancer drug development, and are
especially significant due to the important role they play in cancer
metastasis.
Cathepsin inhibitors range from endogenous peptide inhibitors
to low molecular weight natural and synthetic inhibitors. However,
with peptidyl inhibitors gastric instability and immunological
problems are associated; therefore, envisage the importance of
low molecular weight inhibitors which can be easily synthesized.
We, here, give a complete spectra of inhibitory potency of structur-
ally related differently functionalized chalcones based hydrazones,
pyrazolines and pyrazoles against cathepsin B and H which may be
interconvertible in vivo as the reactions involved are common;
cyclization and oxidation. Some compounds of these classes have
as parasitic cysteine protease inhibitor,15 hydrazone (4) as cathep-
sin S inhibitor,16 1,2,4-oxadiazole-N-acylhydrazone17 (5) and pyr-
azoline analogues (6) as cysteine protease cruzain inhibitor18 and
pyrazole-based (7–10) cathepsin S inhibitors.19–22
In a recent study, some acyl hydrazides (11)23 and bischalcones
and their derivatives (12 and 13)24 as inhibitors to cathepsin B and
H have been reported. In addition, chalcones,25–28 hydrazones,29–31
pyrazolines32–34 and pyrazoles35–37 moieties have also been
explored as key functional groups in a variety of anticancer agents.
Encouraged from our previous work where we found that some
class of compounds inhibit endogenous protease activity38–43 at a
pH where most of the activity is attributed to cysteine proteases
and also that such compounds can lead to the identification of
some potent inhibitors of cathepsin B and H,23,24 we here report
SAR studies of some differently functionalized chalcones based
hydrazones and their cyclized derivatives as inhibitors of mamma-
lian cathepsin B and cathepsin H. In chalconephenyl hydrazones
two pharmacologically active moieties are present which can act
in synergism with each other and their cyclized derivatives having
diversity of substituents are reported due to their broad utility as
anticancer drugs as well as cysteine protease inhibitor potentiality.
The compounds can also exert anti-inflammatory effect. In this regard
⇑
Corresponding author. Tel.: +91 9896918277.
E-mail address: nraghav.chem@gmail.com (N. Raghav).
http://dx.doi.org/10.1016/j.bmc.2014.05.037
0968-0896/Ó 2014 Published by Elsevier Ltd.
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 1. Representative small molecular weight inhibitors of cysteine protease.
it may be worth mentioning here that lysosomes have also been eval-
uated as agents of inflammation in bacterial endotoxicity, rheuma-
toid arthritis and polyarthritis,44–46 therefore lysosomal enzymes
are also important targets along with Cyclo-oxygenases47 while con-
sidering the development of new anti-inflammatory compounds.
Inspired from our previous work where we have established
that among differently positioned substituents, p-positioned sub-
stituents inhibit endogenous protease activity to maximum
extent,39,40 the designed compounds in the present study contain
p-positioned substituents only. The present study may provide
new therapeutic opportunities in conditions caused by imbalanced
activities of cathepsin B and cathepsin H.
Feinchemikalien AG, Switzerland. Sephadex G-100, CM-Sephadex
C-50 and DEAE-Sephadex A-50 were obtained from Pharmacia Fine
Chemicals, Uppsala, Sweden. The protein sample was concentrated
using Amicon stirred cells with YM 10 membrane under nitrogen
pressure of 4–5 psi. The source of enzyme was fresh goat liver
obtained from local slaughter house.
2.2. Methods
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, cmꢀ1) were recorded on a Perkin–Elmer
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 aluminium 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.
2. Materials and methods
2.1. Materials
All the chemicals were of analytical grade. Fast Garnet GBC
(o-aminoazotoluene diazonium salt,
a-N-benzoyl-D, L-arginine-2-
naphthylamide (BANA) and Leu-bNA were purchased from Bachem
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
2.2.1. Synthesis of various compounds
2.2.1.1. General procedure for the synthesis of chalconehydraz-
2.2.1.5. General procedure for the synthesis of 1,3,5-trip-
henylpyrazole. Pyrazoles were synthesized by the addition
one.
Hydrazones were synthesized by adding calculated
of IBD (1.5 mmol) to a stirred solution of substituted pyrazolines
(l.0 mmol) in dichloromethane (30 ml) at room temperature. The
reaction mixture was stirred for 5–6 h. Excess of dichloromethane
was distilled off on a water bath and the residual mass was
recrystallized from an appropriate solvent. Spectral analysis of
the synthesized hydrazones is consistent with the proposed struc-
tures and with those reported.43
quantity of phenylhydrazine to a saturated acetic acid solution of
the substituted chalcone with efficient stirring at room tempera-
ture. When the product was crystallized out, it was immediately
collected on a gravity filter, and washed with several portions of
warm methyl alcohol. Spectral analysis of the synthesized hydra-
zones is consistent with the proposed structures and with those
reported.48
2.2.2. Enzymatic studies
2.2.1.2. General procedure for the synthesis of 1,3,5-triphenyl
2.2.2.1. Purification of cathepsin B and H.
All the purifica-
pyrazolines.
A mixture of chalcone (0.01 mol) and phen-
tion steps were carried out at 4 °C. Cathepsin B and H were iso-
lated, separated and purified from goat liver using the previously
reported procedure23,24 including the steps of acetone powder
preparation, homogenization in cold 0.1 M sodium acetate buffer
pH 5.5 containing 0.2 M NaCl and 1 mM EDTA, acid-autolysis at
pH 4ꢁ0 and 30–80% ammonium sulfate fractionation. Fractionation
of proteases based on molecular weight on Sephadex G-100 col-
umn chromatography and finally ion-exchange chromatographies
on CM-Sephadex C-50 and DEAE-Sephadex A-50. The specific
activities of the cathepsin B and H were ꢂ10.38 nmol/min/mg
and ꢂ22.56 nmol/min/mg, respectively.
ylhydrazine (0.02 mol) was refluxed for 6 h in absolute ethanol
(50 ml). The solution was left for cooling at room temperature
and the solid formed was filtered off, washed with water, dried
and crystallized from absolute ethanol. Spectral analysis of the
synthesized hydrazones is consistent with the proposed structures
and with those reported.42
2.2.1.3. General procedure for the synthesis of N-acetyl-3,5-
diphenyl pyrazolines.
A mixture of chalcone (0.01 mol),
hydrazine hydrate (0.02 mol) in acetic acid (40 ml) was refluxed
for 3 h then poured into ice cold water. The precipitate was sepa-
rated by filtration, washed free of acid and crystallized from meth-
anol to afford 2-pyrazolines, dried and recrystallized from ethanol.
Spectral analysis of the synthesized hydrazones is consistent with
the proposed structures and with those reported.42
2.2.2.2. Enzyme assays.
were first activated in presence of thiol activators at pH 6.0
and pH 7.0, respectively. Activated enzyme solution (100 l)
The purified cathepsin B and H
l
was equilibrated with 0.1 M phosphate buffer at respective
pHs containing 1 mM EDTA separately for 10 min at 37 °C. Then,
2.2.1.4. General procedure for the synthesis of 3,5-diphenyl
20 ll of stock solution (1 mM prepared in DMSO) of different
pyrazolines.
A mixture of chalcone (0.01 mol) and hydrazine
compounds under study were added separately to the activated
hydrate (0.02 mol) was heated at reflux for 6 h in absolute ethanol
(50 ml). The solution was left to cool at room temperature and the
solid formed was filtered off, washed with water, dried and crystal-
lized from absolute ethanol. Spectral analysis of the synthesized
hydrazones is consistent with the proposed structures and with
those reported.42
enzyme assay mixtures to effect final concentrations as
2 ꢃ 10ꢀ5 M in 1 ml assay. After 30 min, 25
ll of 100 mM sub-
strate stock solution was added to start the reaction. Cathepsins
B and H activities were then analyzed using BANA and leu-bNA
as substrates, respectively. The released b-naphthylamine was
quantitated colorimetrically at 620 nm by the use of fast garnet
Table 1
Effect of chalcone hydrazones (1), 1,3,5-triphenyl-2-pyrazolines (2), N-acetyl-3,5-diphenyl pyrazolines (3), 3,5-diphenyl-2-pyrazolines (4) and 1,3,5-triphenyl-2-pyrazoles (5) on
the activities of cathepsin B and cathepsin H
Code
(1)
(2)
(3)
(4)
(5)
Mean SMDI
Mean SMDI
Mean SMDI
Mean SMDI
Mean SMDI
BII
M)IV
HIII
BII
M)IV
HIII
BII
M)IV
HIII
BII
M)IV
HIII
BII
M)IV
HIII
(
l
(l
M)IV
(
l
(l
M)IV
(
l
(l
M)IV
(
l
(
l
M)IV
(
l
(l
M)IV
a
b
c
d
e
f
4.86 0.21
(10)
2.82 0.03
(10)
2.88 0.02
(10)
0.494 0.14
(1.0)
2.64 0.01
(10)
2.49 0.28
(10)
2.00 0.03
(10)
0.925 0.003
(10)
1.48 0.04
(10)
1.11 0.07
(10)
1.52 0.05
(10)
1.0 0.09
(10)
5.48 0.13
(10)
4.01 0.08
(10)
4.29 0.05
(10)
0.778 0.16
(1.0)
1.41 0.09
(10)
1.30 0.09
(10)
2.22 0.04
(10)
1.01 0.02
(10)
1.55 0.05
(10)
1.46 0.04
(10)
1.89 0.07
(10)
1.15 0.03
(10)
2.11 0.26
(10)
5.15 0.06
(10)
5.01 0.06
(10)
0.846 0.42
(10)
5.45 0.11
(10)
5.28 0.11
(10)
2.33 0.07
(10)
1.82 0.06
(10)
2.17 0.09
(10)
2.62 0.01
(10)
2.85 0.08
(10)
3.38 0.04
(10)
5.47 0.10
(10)
5.20 0.24
(10)
5.19 0.03
(10)
1.51 0.02
(1.0)
5.61 0.14
(10)
5.59 0.03
(10)
2.28 0.07
(10)
1.14 0.03
(10)
2.13 0.14
(10)
1.48 0.06
(10)
2.41 0.15
(10)
2.58 0.04
(10)
5.59 0.17
(10)
4.75 0.32
(10)
5.52 0.17
(10)
1.69 0.01
(10)
3.12 0.15
(10)
2.32 0.32
(10)
3.26 0.29
(10)
1.29 0.07
(10)
1.81 0.06
(10)
1.43 0.33
(10)
2.47 0.08
(10)
1.56 0.24
(10)
First figure in each block represents the enzyme activity in presence of compounds and the second figure in parenthesis represents the concentration of compounds at which
the activity was measured.
Value of mean SMD of control is 5.65 0.0276 for cathepsin B and 3.70 0.0387 for cathepsin H.
Value of mean SMD of leupeptin is 0.067 0.0012 (1
Value of mean SMD of Leu-CH2-Cl is 5.03 0.024 (10
l
l
M) for cathepsin B and 0.241 0.015 (10
l
M) for cathepsin H.
M) for cathepsin B and 1.78 0.016 (10
lM) for cathepsin H.
The specific activity of the cathepsin B and cathepsin H were ꢂ10.38 nmol/min/mg and ꢂ22.56 nmol/min/mg, respectively. The % residual activity is calculated wrt control
where no compound was added but an equivalent amount of solvent was present.
I
Represents mean SMD of the experiment conducted in triplicate and is calculated as activity in nmol/min/ml in enzyme preparation.
Enzyme assays were conducted using BANA as substrate for cathepsin B.
II
III
Enzyme assays were conducted using Leu-bNA as substrate for cathepsin H.
In parenthesis represents concentration of compounds in lM.
IV
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
GBC dye using established procedures.23,24 The experi-
ments were performed in triplicate for each concentration and
averaged before further calculations. In control experiments,
the equivalent amount of DMSO was added and percent resid-
ual activities were calculated with reference to control
(Table 1).
3. Pharmacology
3.1. Enzyme inhibition studies
Cathepsins B and H activities were first estimated at 2 ꢃ 10ꢀ5
M
concentration of each compound. The compounds which showed
Figure 2. Effect of varying concentrations of chalcone hydrazones (I), 1,3,5-triphenyl-2-pyrazolines (II), N-acetyl-3,5-diphenyl pyrazolines (III), 3,5-diphenyl-2-pyrazolines
(IV) and 1,3,5-triphenyl-2-pyrazoles (V) at pH 6.0 on cathepsin B activity in presence of 2.5 mM concentration of BANA. Results are the mean of the experiment conducted in
triplicates taking different concentrations of compounds. Activities are expressed as percent of control which contains equivalent amount of solvent and Lineweaver–Burk
plot for cathepsin B activity on varying concentrations of BANA in presence of minimum concentration of chalcone hydrazones (VI), 1,3,5-triphenyl-2-pyrazolines (VII),
N-acetyl-3,5-diphenyl pyrazolines (VIII), 3,5-diphenyl-2-pyrazolines (IX) and 1,3,5-triphenyl-2-pyrazoles (X) as reported in Table 1 below at pH 6.0.
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
complete inhibition at 2 ꢃ 10ꢀ5 M concentration were further
studied for their inhibitory effect at lower concentrations by
adding appropriate amount of individual compounds in the
reaction mixture separately to effect the final concentration of
each compound as 0.10 ꢃ 10ꢀ5, 0.25 ꢃ 10ꢀ5 M, 0.50 ꢃ 10ꢀ5 M,
0.75 ꢃ 10ꢀ5 M, 1.0 ꢃ 10ꢀ5 M and 1.5 ꢃ 10ꢀ5 M (Figs. 2 and 3).
interactions in the active site of the enzyme was analyzed. For
these studies, small molecular weight ligands were prepared and
enzyme structure active site was retrieved from the Protein Data
Bank. The structures of cathepsin B and H were retrieved from Pro-
tein Data Bank (http://www.rcsb.org/) as cav2IPP B_PYS.pdb49 and
cav8PCH H_NAG.pdb,50 respectively. The structures of ligands
were prepared in Marvin sketch, minimized and were saved as
MDL Mol File. After loading the prepared ligands and the binding
site, docking was run by setting the GA parameters at drug screen-
ing. Fitness is the total energy of a predicted pose in the binding
site. The docked poses of most inhibitory compounds in the three
respective series with the active site of cathepsin B and the amino
acyl binding site of cathepsin H are shown in Figures 4 and 5. A
total decrease in energy resulting due to H-bonds and Van der
Waals interaction between the legends are provided as
Supplementary data for cathepsin B and H, respectively.
3.2. Enzyme kinetic studies
After establishing the inhibitory action of synthesized compounds
on cathepsin B and H, experiments were designed to evaluate the
type of inhibition and to determine their Ki values. For that, enzyme
activity was evaluated at six different substrate concentrations
(25 ꢃ 10ꢀ5 M,
20 ꢃ 10ꢀ5 M,
15 ꢃ 10ꢀ5 M,
10 ꢃ 10ꢀ5 M,
5.0 ꢃ 10ꢀ5 M, 3.0 ꢃ 10ꢀ5 M, 2.5 ꢃ 10ꢀ5 M and 2.0 ꢃ 10ꢀ5 M) in pres-
ence and absence of a fixed concentration of inhibitor. The enzyme
concentration was kept constant in all the experiments as detailed
previously. The results are presented in Figures 2 and 3. The val-
ues represent mean SMD of at least three individual experiments.
The Ki values of compounds were calculated using the Lineweaver–
4. Results and discussion
Chalconephenyl hydrazones and their cyclized derivatives, pyr-
azolines and pyrazoles have a wide potential role as antitumor
agents and anti-inflammatory agents. Different pyrazolines (14a,
14b and 14c) have been reported to possess significantly high cyto-
toxic activity against MCF-7, HCT 116 cell lines.51 Anticancer activ-
ity of some pyrazolines (15) has also been reported by Manna
et al.52 Pyrazoles and pyrazolines (16) possessed antiproliferative
activity in SK-OV-3, HT-29, and HeLa human cancer cells lines.53
0
Burk equation Km = Km (1 + [I]/Ki) for competitive inhibition and
0
Vmax = Vmax (1 + [I]/Ki) for non-competitive inhibition (Table 2).
3.3. Drug modeling studies
All docking studies were performed using iGemdock. Individ-
ual binding poses of each compound was assessed and their
Fig. 2 (continued)
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3,5-Diaryl-pyrazoline (17a) and its N1-acetylated derivatives (17b)
showed potent and selective activity in the NCI 60 human cancer
cell lines panel.54
acetic acid.42 1,3,5-Triaryl-2-pyrazoles (5) were synthesized by
oxidation of substituted pyrazolines in presence of dichlorometh-
ane and Iodo Benzene Diacetate (IBD) at room temperature.43
Simple pyrazolines and pyrazoles having potential anticancer
activities ignited a thought to evaluate these designed molecules
as inhibitors to cysteine proteases, cathepsin B and H as elevated
levels of these cathepsins have been found in various cancerous
conditions.7–11 Therefore, some chalcone based hydrazones and
their cyclized derivatives were synthesized to screen their effect
on cathepsin B and H. Preliminary studies of these synthesized
compounds on the inhibition of endogenous protein substrates
were also conducted.42,43 It has been evaluated that the designed
pyrazolines and pyrazoles inhibited proteolytic activity on endog-
enous protein substrates appreciably at pH 5.0. In some cases
ꢂ100% inhibition is achieved at 1 mM concentration. Since most
of the proteolytic activity is attributed to cysteine proteases at this
pH, it was thought to explore their inhibition on cathepsin B and H,
two important cysteine proteases which are the key factors in the
pathogenesis of cancer invasion, osteoporosis, arthritis, etc. The
study can be helpful in providing a case history for relating the
enzyme inhibition studies with pharmacological significance of
pyrazolines and pyrazoles.
The synthesized compounds were characterized with the help of
melting points, 1H NMR and 13C NMR data.
4.2. Pharmacological evaluation
Cysteine protease inhibitors may be suitable as drugs for treat-
ment of tumors, inflammatory diseases, pathological processes
which involve increased degradation of bone, cartilage or muscle,
infections with viruses, and bacterial and protozoa infections.
Analysis of the effect of designed compounds on cathepsin B and
cathepsin H with target-directed chemical specificity suggests an
optimistic future for their use as cysteine protease inhibitors in
various therapies such as in cancer, inflammation, etc.
Some of the compounds exhibited inhibition comparable to ref-
erence peptidic inhibitors, for example, leupeptin for cathepsin B
and Leu-CH2-Cl for cathepsin H. Leupeptin having exact comple-
mentarities with the enzyme active site can bind to it more effec-
tively causing more inhibition but use of peptides as drugs have
immunological and stability problems. In this regard, the invention
of new cathepsin inhibitors with consistent quality, potency and
stability still poses a considerable challenge in diseased conditions
where cathepsins are druggable targets such as in cancers, rheu-
matoid arthritis and other important diseases. With the back-
ground of previously reported associated biological activities
possessed by these compounds in literature, the compounds were
screened to evaluate their inhibitory potency on cathepsin B and H.
A complete spectrum of variety of compounds in structurally
related series has been reported here. When these analogs were
screened against cathepsin B and H in enzyme assays, several
promising inhibitors were identified.
4.1. Chemistry
These analogs were prepared by chemical synthesis as detailed
in Scheme 1. Chalcone phenyl hydrazones (1) were synthesized by
the reaction of chalcones with phenyl hydrazine in saturated acetic
acid solution.48 Chalcones were also allowed to react with phenyl
hydrazine or hydrazine hydrate in hot acetic acid to afford substi-
tuted 1,3,5-triaryl-2-pyrazolines (2) or substituted N-acetyl-3,
5-diaryl-2-pyrazolines (3) in high yields and 3,5-diphenyl pyrazo-
lines (4) were obtained in presence of alcohol and in absence of
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
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Figure 3. Effect of varying concentrations of chalcone hydrazones (I), 1,3,5-triphenyl-2-pyrazolines (II), N-acetyl-3,5-diphenyl pyrazolines (III), 3,5-diphenyl-2-pyrazolines
(IV) and 1,3,5-triphenyl-2-pyrazoles (V) at pH 7.0 on cathepsin H activity in presence of 2.5 mM concentration of Leu-bNA. Results are the mean of the experiment conducted
in triplicates taking different concentrations of compounds. Activities are expressed as percent of control which contains equivalent amount of solvent and Lineweaver–Burk
plot for cathepsin H activity on varying concentrations of Leu-bNA in presence of minimum concentration of chalcone hydrazones (VI), 1,3,5-triphenyl-2-pyrazolines (VII), N-
acetyl-3,5-diphenyl pyrazolines (VIII), 3,5-diphenyl-2-pyrazolines (IX) and 1,3,5-triphenyl-2-pyrazoles (X) as reported in Table 1 at pH 7.0.
4.2.1. Cathepsin B inhibition studies
pounds were most inhibitory to cathepsin B, for example, 4-nitroch-
alcone phenyl hydrazone (1d), 5-(-40-nitrophenyl)-1,3-diphenyl-2-
pyrazoline (2d), N-acetyl 5-(-40-nitrophenyl)-3-phenyl-2-pyrazoline
The activities of cathepsin B was estimated at varying concentra-
tions of synthesized structural analogs. The plots of % residual activ-
ities versus the concentrations of different compounds are presented
in Figure 2; I–V. Among the designed analogs, nitro substituted com-
(3d),
5-(-40-nitrophenyl)-3-phenyl-2-pyrazoline
(4d)
and
5-(-40-nitrophenyl)-1,3-diphenyl-2-pyrazole (5d) showed IC50
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Fig. 3 (continued)
values of ꢂ0.45
lM, ꢂ0.50
lM, ꢂ5.9
l
M, ꢂ0.74
lM and ꢂ5.68
lM,
as competitive inhibitors whereas the results of N-acetyl-3,5-
diphenyl pyrazolines (3) are consistent with noncompetitive inhi-
bition. The Km values for cathepsin B and cathepsin H were found
respectively.
4.2.2. Cathepsin H inhibition studies
to be 400 lM and 500 lM, respectively whereas the 1/Vmax values
Similarly, the activities of cathepsin H were estimated at varying
concentrations of synthesized compounds (Fig. 3; I–V). In case of
cathepsin H, chloro substituted compounds in each series such as
4-chlorochalcone phenyl hydrazone (1b), 5-(-40-chlorophenyl)-1,3-
diphenyl-2-pyrazoline (2b), N-acetyl 5-(-40-chlorophenyl)-3-phenyl-
2-pyrazoline (3b), 5-(-40-chlorophenyl)-3-phenyl-2-pyrazoline (4b)
and 5-(-40-chlorophenyl)-1,3-diphenyl-2-pyrazole (5b) were most
were found to be 0.120 nmol/min/ml and 0.170 nmol/min/ml,
respectively. The mode of inhibition was investigated, and Ki
values obtained are tabulated in Table 2.
4.2.3. SAR studies
The potential of inhibition shown by the synthesized com-
pounds is related with the presence of electron donating or with-
drawing substituents on benzene ring as well as with the type of
nucleus present in the molecule which indicates the effect of struc-
tural features on inhibitory potential on the activity of cathepsin B
and cathepsin H.
inhibitory and demonstrated IC50 values of ꢂ5.7
lM, ꢂ6.25
lM,
ꢂ9.0 M, ꢂ7.0 M and ꢂ7.1 M, respectively.
l
l
l
Once, the inhibitory potency of individual compounds was
established, experiments were designed to find out the type of
inhibition and determination of their Ki values. The enzyme activ-
ities were assayed in presence and absence of a fixed concentration
of compounds at different substrate concentrations (Figs. 2; VI–X
and 3; VI–X). It was found that in general chalcone hydrazones
were shown to be a potent and selective inhibitor followed by their
cyclized derivatives, pyrazolines and pyrazoles series.
Among the synthesized compounds, nitro substitution, that is,
1d, 2d and 4d demonstrated an impressive Ki value of 0.042
0.053 and 0.131 M, respectively against cathepsin B.
However, chloro substituted compounds 1b, 2b and 4b were best
inhibitors with the Ki values of 1.111 M, 1.174 M and 1.562 M,
lM,
l
M
l
l
l
l
respectively (Fig. 6) for cathepsin H. The inhibition achieved in
these cases is of nanomolar and sub-micromolar range for cathep-
sin B and cathepsin H, respectively. The compounds are better
inhibitors for cathepsin B than cathepsin H. In our previous study
also we have demonstrated that some acyl hydrazides23 and
bischalcones based quinazoline-2(1H)-ones and quinazoline-
2(1H)-thione derivatives24 showed better inhibition to cathepsin
The Ki values of compounds were calculated using the Linewe-
0
aver–Burk equation Km = Km (1 + [I]/Ki) for competitive inhibition
0
and Vmax = Vmax (1 + [I]/Ki) for non-competitive inhibition. From
the figures, it is clear that chalcone phenyl hydrazones (1), 1,3,5-
triphenyl-2-pyrazolines (2), substituted 3,5-diphenyl-2-pyrazo-
lines (4) and substituted triphenyl-2-pyrazoles (5) were identified
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
Table 2
Ki values and type of inhibition exerted by chalcone hydrazones (1), 1,3,5-triphenyl-2-pyrazolines (2), N-acetyl-3,5-diphenyl pyrazolines (3), 3,5-diphenyl-2-pyrazolines (4) and
1,3,5-triphenyl-2-pyrazoles (5) on the activities of cathepsin B and cathepsin H
Ar
Series
(3)
(1)
M)
(2)
M)
(4)
M)
(5)
KiI
(lM)
KiI
(
l
KiI
(
l
KiI
(
lM)
KiI
(
l
BII
HIII
BII
HIII
BII
HIII
BII
HIII
BII
HIII
a;
12.74, C
3.28, C
4.810, C
1.111, C
2.658, C
1.905, C
90.90, C
10, C
5.388, C
1.174, C
3.158, C
2.907, C
17.67, NC
17.54, NC
40,C
6.024,C
1.562,C
5.376,C
3.333,C
80, C
18.59, C
2.346, C
3.80, C
H
b;
108.70, NC
67.11, NC
11.41, NC
10.01, NC
12.66, NC
24.32, NC
38.09,C
14.81,C
0.131,C
17.80,C
52.60,C
1.311,C
Cl
c;
3.883, C
0.042, C
12.74, C
0.053, C
Br
d;
2.579, C
O2N
e;
3.15, C
2.906, C
1.695, C
1.365, C
1.311, C
4.184, C
2.119, C
1250, NC
35.46, NC
140.8, NC
114.3,C
74.07,C
8.2,C
7.843,C
4.706,C
7.396, C
3.33, C
H3C
f;
2.888, C
149.25, NC
9.804,C
H3CO
C = competitive, NC = non-competitive.
I
Represents mean SMD of the experiment conducted in triplicate and is calculated as activity in nmol/min/ml in enzyme preparation 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 Lineweaver–Burk plots and then the Ki values were calculated
using Lineweaver–Burk equations for competitive and non-competitive inhibition depending upon the results.
II
Enzyme assays were conducted using BANA as substrate for cathepsin B.
III
Enzyme assays were conducted using Leu-bNA as substrate for cathepsin H.
(I)
(II)
(III)
(IV)
Figure 4. Docking results (I), (II), (III) and (IV) showing the alignment of most inhibitory compounds 1d, 2d, 4d and BANA in the active site of cathepsin B (cav2IPP B_PYS.pdb).
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10
N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
(I)
(II)
(IV)
(III)
Figure 5. Docking results (I), (II), (III) and (IV) showing the alignment of most inhibitory compounds 1b, 2b, 4b and Leu-bNA in the active site of cathepsin H (cav8PCH
H_NAG.pdb).
B when compared with H, giving a clue that active site of cathepsin
B is more exposed to these compounds. Involvement of these cys-
teine proteases in inflammatory conditions also indicates the
potential use of these compounds as anti-inflammatory agents. In
addition, enhanced expression of cathepsin B and H in various
tumor conditions, signify the role of these moieties as a therapeutic
agent against cancer metastasis.
A detailed analysis of Table 2 further extends the structure
activity relationship based on the field effect and resonance effect.
It is clear from the study that cathepsin B is better inhibited by the
presence of more electron withdrawing substituent (–NO2) as
compared to cathepsin H. However, in rest of the synthesized com-
pounds having substituents –Cl, –Br and –OCH3 where overall nat-
ure of substituents is electron releasing as a result of field and
resonance effect, are better inhibitors to cathepsin H as compared
to cathepsin B. Chloro- and bromo-substituted compounds show
electron withdrawing field effect with a more dominating electron
releasing resonance effect. The unsubstituted compounds (–H) in
each series also resulted in preferential inhibition to cathepsin H.
The most potent inhibitor in our current study features nitro sub-
stitution pattern in the ring for cathepsin B and chloro substitution
for cathepsin H, thus extending the SAR with functional group
diversity at these positions.
Among the different categories, chalcone phenyl hydra-
zones were most inhibitory. The divergence and inhibition
achieved by these compounds validates the significance of this
study.
Some of the compounds exhibited inhibition comparable to
reference peptidic inhibitors, for example, leupeptin for cathepsin
B and Leu-CH2-Cl for cathepsin H. As reported in literature,
leupeptin being a potential peptide inhibitor of cathepsin B,55
inhibited the goat brain cathepsin B competitively with Ki value
of 0.0125
was reported to be 0.007
cathepsin H was reported to be 9.2
l
M56 whereas Ki value for human liver cathepsin B57
M. However, Ki value for human liver
M.58
l
l
Our results obtained are comparable with earlier results reported
for brain cathepsin B56 and cathepsin H.59
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
Scheme 1. Synthesis of hydrazones and their cyclized derivatives.
The proposed mechanism of inhibition for chalcone hydrazone
is shown in Figure 7. The most likely mechanism of inactivation
involves an attack of the cysteine-29 thiolate of cathepsin B to
the carbonAnitrogen double-bond of the azomethine group result-
ing in a covalent or transient covalent bond. The tetrahedral
intermediate formed can result in reactivation of active site after
some time. The hypothesis is supported by preliminary studies of
cysteine proteases on endogenous protein substrates,42,43 where
the enzyme inhibition has been found to be reversed when the
reaction time was increased. The diversity in inhibition obtained
by differently substituted compounds indicates that specific
binding interactions of designed compounds affect the inhibitory
properties of these compounds.
propose that these compounds also interact with the active site
cysteine-SH (Cys-29) group as supported by the docking studies
(Figs. 4 and 5). The reversible inhibition in the present study is
supported by kinetic studies of cathepsin B.
4.3. Molecular docking experiment
The empirical scoring function presented as Etotal of iGemdock
is the estimated sum total of Van der Waal, H-bonding and
electrostatic interaction between enzyme and the ligand.
On the basis of the interaction data of docking experiments, it
was observed that phenyl hydrazones (1a–1f), phenyl pyrazolines
(2a–2f) and their subsequent pyrazoles (5a–5f) and acetyl pyrazo-
lines (3a–3f) and diphenyl pyrazolines (4a–4f) showed a lesser
interaction than the reference leupeptin, a peptidyl inhibitor.
Decrease in total energy for leupeptin-cathepsin B has come out
be ꢀ114.86 of which the contribution of Van der Waal interactions
A similar mechanism has been proposed for inhibition of
cathepsin L by benzophenone functionalized thiosemicarbazones60
where the active site is regenerated. Leupeptin is also reported
reversible inhibitor of cathepsin B.61,62 Similar to leupeptin, we
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12
N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 6. Chemical structures of some representative compounds as potent inhibitors.
Figure 7. Proposed mechanism for inhibition of cathepsin B.
are more with a score of ꢀ79.2743 as compared to H-bonds with a
score of ꢀ35.5857. Leupeptin-cathepsin B binding energy is due to
peptide protein interaction. Leupeptin is peptidyl in nature and
therefore being a flexible molecule binds effectively with the
enzyme active site resulting in higher binding energy. iGemdock
provide algorithms for flexible docking approach for both ligands
and proteins63 therefore flexible ligands like leupeptin will show
a larger decrease in total energy as compared to the molecules
under study because these are smaller in structure and provide
lesser surface area compared to leupeptin. Therefore, the binding
energy of title compounds is less than leupeptin. But, the in vitro
analysis reveals that the compounds show comparable inhibition
to this reference inhibitor. The compound 1d, found to be most
inhibitory to cathepsin B show a total decrease in energy as
ꢀ93.2753 of which ꢀ73.5028 is assigned to Van der Waal interac-
tions whereas ꢀ19 is of H-bond. The proposed mechanism is
shown in Figure 7. The results clearly indicate the significance of
the in vitro inhibition studies.
However in cathepsin H, the decrease in total energy for the ref-
erence inhibitor leu-CH2Cl was less as compared to all the designed
compounds. Here, it can be seen that though leu-CH2Cl is specific
inhibitor for cathepsin H,59,64 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
ꢀ61.1812 of which ꢀ44.8751 is the Van der Waal interaction and
ꢀ16.3061 is due to hydrogen bonds. All the designed compounds
have been found to show more decrease in ligand–cathepsin H
interaction energy than Leu-CH2Cl–cathepsin H. The compound
1b, found to be most inhibitory to cathepsin H, showed a total
decrease in energy as ꢀ93.7752 of which ꢀ79.7752 is owed to
Van der Waal interactions while ꢀ14 is related to H-bond.
In silico studies have been used as a supporting tool for enzyme
inhibition studies. The in silico predictable behavior of enzyme–
ligand interaction can give an idea about the interaction between
these two but cannot substitute the in-solution studies.
4b and Leu-bNA for cathepsin H. The compounds are presented as
pink fine line structures. The side chain amino acid residues inter-
acting with the active site are shown in bold and detail. However,
rest of the backbone of the enzyme is ribbon shaped. The residues
of enzymes interacting via hydrogen bond are displayed in green
and those exhibiting Van der Waal interaction are in shown in grey
color. The figures were saved after applying the consensus scoring
criteria which improved the enrichment in virtual screening.65 As
we can see that most of the amino acids residues which interact
with the substrate and the reference inhibitors also interact with
the designed compounds. This further adds to the in vitro studies
carried out that the compounds competes with the substrate and
inhibit the enzyme in a competitive manner.
5. Conclusion
We have evaluated structurally related five series of 30 com-
pounds for their inhibitory activity against cathepsin B and H
and have identified some synthetic non-peptidyl inhibitors for
cathepsin B and H. The compounds were potent inhibitors to
cathepsin B compared to cathepsin H. In general, the inhibitory
potency of compounds was related to the electro negativity prop-
erty of substituent. Through enzyme kinetics, it was revealed that
chalcone phenyl hydrazones, phenyl pyrazolines, diphenyl pyrazo-
lines and pyrazoles inhibit cathepsin B and cathepsin H in a com-
petitive manner whereas cathepsin B and cathepsin H were
inhibited in a non-competitive manner by acetyl pyrazolines,
which were found to be least inhibitory. The results presented in
this study also signify the importance of in vitro, solution studies
in addition to in silico studies.
Acknowledgements
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.
Figures 4 and 5 represents the docking poses of the most inhib-
itory compounds 1d, 2d, 4d and BANA for cathepsin B and 1b, 2b,
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N. Raghav, M. Singh / Bioorg. Med. Chem. xxx (2014) xxx–xxx
24. Raghav, N.; Singh, M. Eur. J. Pharm. Sci. 2014, 54, 28.
13
Supplementary data
25. Modzelewska, A.; Pettit, C.; Achanta, G.; Davidson, N. E.; Huang, P.; Khan, S. R.
Bioorg. Med. Chem. 2006, 14, 3491.
Supplementary data (synthesis and characterization of com-
pounds and Tables related to docking studies showing decrease
in different energies of cathepsin B and H in presence of synthe-
sized compounds) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.05.037.
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