SAR studies of differently functionalized 4′-phenylchalcone based compounds as inhibitors of cathepsins B, H and L

Article abstract of DOI:10.1039/c5ra00357a

Conditions related to the elevated levels of cathepsin B [3.4.22.1], cathepsin H [3.4.22.16] and cathepsin L [3.4.22.15] in various cancerous, rheumatoid arthritis and tissue degenerative disorders motivate the design, synthesis and evaluation of compound

Full text of DOI:10.1039/c5ra00357a

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SAR studies of differently functionalized 40-
phenylchalcone based compounds as inhibitors of
Cite this: RSC Adv., 2015, 5, 50440
cathepsins B, H and L†
*
Indu Ravish and Neera Raghav
Conditions related to the elevated levels of cathepsin B [3.4.22.1], cathepsin H [3.4.22.16] and cathepsin L
[3.4.22.15] in various cancerous, rheumatoid arthritis and tissue degenerative disorders motivate the
design, synthesis and evaluation of compounds as cathepsin B, H and L inhibitors. In the present work
we report the synthesis and analysis of 36 different compounds belonging to 4 related series as
cathepsin B, H and L inhibitors. After a preliminary screening of the synthesized compounds as inhibitors
to the hydrolysis of endogenous protein substrates at pH 5.0, their inhibitory effects were evaluated on
cathepsins B, H and L. Kinetic studies were carried out to determine the inhibition constants. It was
found that the compounds belonging to different classes affected the enzyme activity significantly, and
the activity was greatly affected by the presence of substituents. Maximum inhibition was caused by 2b
and 2g in the cases of cathepsin B and cathepsin L, and 4d in the case of cathepsin H, where Ki values of
the order of 10ꢀ8 M, 10ꢀ9 M and 10ꢀ6 M, respectively, were obtained. The results are correlated with the
docking results.
Received 8th January 2015
Accepted 7th April 2015
DOI: 10.1039/c5ra00357a
www.rsc.org/advances
various clinical reports and experimental models.8–11 Increased
levels of these enzymes in the tumor state have been predictive
factors for cancer patients.12,13 In addition, elevated levels of
1. Introduction
Cysteine proteases, possessing a –CH2SH group at the active
site, have become important therapeutic targets for the treat-
ment of a number of diseases. The cysteine proteases of para-
sites, such as falcipain, cruzain and amoebepain1,2 of
Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei
rhodesiense, Toxoplasma gondii and Entamoeba hystolytica3–5 are
some of the important targets in the development of anti-
parasitic agents. The literature reports that inhibitors of these
cysteine proteases have great implications as anti-parasitic
agents. N-Acylhydrazones6 have been found to be potent
inhibitors of cruzain, a cysteine protease, and the compounds
showed a comparable magnitude of IC50 when tested against
the standard drug nifurtomox. Brak et al.7 reported another
non-peptidic cruzain inhibitor with a triazole moiety.
Cathepsins B, H and L, lysosomal cysteine proteases, have
also emerged as important targets for the development of a
variety of therapeutic agents because of their involvement in
various disease conditions. Cathepsin B and H have long been
reported to be associated with cancer progression because of
their ability to degrade extracellular matrices facilitating inva-
sion, angiogenesis and metastasis, which is evident from
cysteine proteases have also been reported in various inam-
matory conditions14 such as periodontitis, rheumatoid arthritis,
etc. An imbalance between the ratio of cellular inhibitors and
cathepsin has been considered as one of the reasons for the
elevated levels of these enzymes.15,16 Therefore, the identica-
tion of compounds which act as potent inhibitors to cathepsins
is a major thrust area in drug development and chemotherapy.
Inhibitors targeting cysteine cathepsins have been used in vitro
to show that these enzymes play an important role in tumor
invasion.17
Cathepsin B, a lysosomal cysteine protease possessing car-
boxydipeptidase and endopeptidase activities18 responsible for
intracellular protein turnover, antigen processing, proenzyme
activation and hormone maturation, has received much atten-
tion for its potential role in conditions such as cancer,19
muscular dystrophy20 and rheumatoid arthritis.21 Cathepsin B
has been found to be overexpressed in different malignancies
like prostrate,22,23 brain,24 lung25 and colorectal cancers.26
Inhibitors of cathepsin B have been found effective in reducing
the invasive potential of tumor cells.27
Cathepsin H, another equally important and unique lyso-
somal cysteine protease with endoaminopeptidase activity, has
also been found to be associated with various diseases. There is
also a growing body of evidence that the level of cathepsin H or
cathepsin H-like enzymes28 is increased in disease states,
including breast carcinoma,29 melanoma and tumor
Department of Chemistry, Kurukshetra University, Kurukshetra-136119, Haryana,
India. E-mail: nraghav.chem@gmail.com; Tel: +91 9896918277
† Electronic supplementary information (ESI) available: Tables and
characterization of synthesized compounds can be found in the ESI. See DOI:
10.1039/c5ra00357a
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metastasis.30 A cathepsin H-like enzyme has been found in cells
The biological activities, such as antitumor and anti-
from a human melanoma cell line. This enzyme can degrade inammatory activity, possessed by chalcones and pyrazolines
brinogen and bronectin and thus may, together with other and the role of cathepsins B, H and L in these disease condi-
proteases, be involved in the destruction of extracellular matrix tions motivated us to evaluate the effect of chalcone based
components, leading to cancer proliferation, migration and pyrazolines on the activity of cathepsin B and H.50 Previously, we
metastasis.28 Deletion of cathespin H impaired angiogenic have reported various compounds as inhibitors of hydrolysis
switching of the pre-malignant hyperplastic islets signicantly, activity on endogenous protein substrates at pH 5.0, where
and a reduction in the subsequent number of tumors was cysteine proteases are active. Some of these compounds which
observed.31
are identied as inhibitors of these proteases have been found
Cathepsin L is another of the powerful lysosomal cysteine to inhibit cathepsin B and H reversibly.37 Systematic changes in
proteases which degrades many proteins and has been impli- the structures of inhibitors can lead to a potential therapeutic
cated as participating in bone collagen degradation by osteo- molecule. It is a known fact that in order to develop an appro-
clasts.32 Overexpression of cathepsin L and/or abnormal activity priate therapeutic agent, the number of molecules synthesized
has also been implicated in a number of disease states.33 For and screened is great.
example, cathepsin L is responsible for bone resorption
We have previously observed that systematic alterations in
through the degradation of collagen type I; this deregulation is the structure of chalcones led to the identication of 40-phe-
believed to lead to osteo- and rheumatoid arthritis.34 In addi- nylchalcones as activators of one enzyme i.e. acid phosphatase
tion, several infectious organisms, such as the SARS and Ebola and inhibitors of another enzyme i.e. alkaline phosphatase,
viruses, utilize cathepsin L-like proteins for replication in known to perform similar physiological functions.51 Their
human cells.35 The large number of disease states associated simpler structural analogs either had no effect52,53 or lesser
with cathepsin L calls for an understanding of its biological effect on these enzymes.54
function.33 Inhibitors of cathepsin L-like cysteine proteases also
The introduction of a phenyl group at the 40-position of the
have potential utility in the treatment of protozoan infections chalcones led to such a change in the structure that an appre-
such as trypanosomiasis and malaria.3 The involvement of ciably differential effect of the compounds was observed on acid
cathepsins B, H and L in the above mentioned disease states and alkaline phosphatases. With this background we observed
emphasizes the need for the development of potential inhibi- the effect of 40-phenylchalcones and their pyrazoline derivatives
tors of these enzymes. As such, the identication of inhibitors on the activities on cathepsins B, H and L, and two signicant
of these cathepsins would provide valuable tools to probe the enzymes emerged as potential drug targets for melanoma
role of these enzymes in biological systems, as well as to provide therapy.
potential starting points for drug discovery efforts.
A large amount of work has been previously reported on
peptide based inhibitor molecules,36 but due to the immuno-
logical problems and gastric instability encountered with the
2. Results and discussion
use of peptidyl molecules, in the past decade this research area Chalcones and their cyclized derivatives, pyrazolines, has wide
has been focused on the designing and development of non- potential roles as antitumor agents and anti-inammatory
peptidyl inhibitors of cysteine proteases. Toward this agents (Scheme 2). Different pyrazolines (1a, 1b and 1c) have
endeavour we have recently reported some non-peptidyl inhib- been reported to possess signicantly high cytotoxic activity
itors of cathepsins B and H, for example acyl hydrazide and against MCF-7 and HCT 116 cell lines.55 Manna et al. has also
triazoles,37 o-hydroxy chalcones and their cyclised derivatives reported anticancer activity of some pyrazolines.56 3,5-Diaryl
avones38 and bischalcone based quinozolines, as novel inhib- pyrazolines (3a) and their N1-acetylated derivatives (3b) showed
itors of cathepsin B and H.39
potent and selective activity in the NCI 60 human cancer lines
The chalcones chemically known as 1,3-diarylprop-2-en-1- panel.57 Chalcones and simple pyrazolines having potential
ones (Scheme 1) are known for their diverse pharmacological anticancer activities ignited a thought to evaluate these
activities, including antitumor and anti-inammatory activi- designed molecules as inhibitors to cysteine proteases,
ties.40 The literature has revealed that chalcones have been cathepsins B and H, because of the overexpression and dimin-
found to be potential inhibitors to calpains and cathepsin B and ished level of inhibitors of these cathepsins in various
H.41 Pyrazolines (Scheme 1) also possess a variety of biological cancerous conditions.58–62
activities such as antimicrobial, antifungal, antidepressant,
Preliminary studies of some chalcone hydrazone based pyr-
anticonvulsant,42 immunosuppressive,43 antitumor, anti- azolines on the inhibition of endogenous protein substrates
amoebic,44 antiarthritic, cerebroprotective effect and antibac- have also been reported.63,64 Some of these pyrazolines inhibited
terial45 properties. Some of these compounds have also anti- cathepsins B and H appreciably.50 Based on these observations,
inammatory, anti-diabetic, anaesthetic, analgesic, tranquil- we in the present work have explored 40-phenyl substituted
lizing, muscle relaxant, psychoanaleptic, and antihypertensive chalcones and their pyrazoline derivatives as inhibitors of
activities46–48 and potent selective activity such as nitric oxide proteolytic activity on endogenous protein substrates, followed
synthase (NOS) inhibitor and cannabinoid CB1 receptor by detailed enzyme inhibition studies on cathepsins B and H. In
antagonist activity.49
addition the effect of the synthesized compounds was also
screened on cathepsin L. To accomplish the project, rst of all
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Scheme 1 Representative chalcones and pyrazolines as inhibitors of cathepsins B, H and L.
the pyrazolines were synthesized and characterised from the chiral center, whose hydrogen has a chemical shi of 5.2 ppm
respective chalcones (Scheme 3) as discussed below.
(H5x, J ¼ 12.2, 7.2 Hz). These spectral data unequivocally proved
In the rst step, the 40-phenylchalcones (1a–1i) were the 2-pyrazoline structure. The 1H NMR spectra of the
obtained by a solvent free method from 40-phenylacetophenone compounds in CDCl3 show signals at 8.8–9.0 and 8.0–8.85 ppm
and aromatic aldehydes under basic conditions. In the second assignable to CHO protons in 2a–2i and NH protons in 4a–4i
step, reaction of the synthesized chalcones with hydrazine respectively. The multiplet signals observed in the 6.6–8.5 ppm
hydrate/formic acid, phenyl hydrazine hydrate and hydrazine region are assigned to the protons of the phenyl rings. The
hydrate in alcohol resulted in the formation of N-for- sharp singlets observed at 3.3–3.8 and 3.33–3.90 ppm are
mylpyrazolines (2a–2i), N-phenylpyrazolines (3a–3i) and pyr- assigned to the diaminomethyl and methoxy protons.
azolines (4a–4i) respectively.
The structures of the synthesized compounds were eluci-
dated on the basis of mp, 1H NMR and IR spectra. The ring
2.1. Biological activity
closure is demonstrated by the IR spectra of compounds 2a–2i,
In vitro endogenous proteolysis studies at pH 5.0 in goat liver
homogenate were carried out for 3 h and 24 h reaction time for
the preliminary exploration of inhibition by these compounds.
It was found that the compounds 1b, 1d, 1e, 2h, 3a, 3f, 3g and 4g
3a–3i and 4a–4i, which showed the characteristic band for NH at
3100–3300 cmꢀ1 and a band at 1590–1605 cmꢀ1 corresponding
1
to C]N stretching. The H NMR spectra showed an ABX spin
system caused by the coupling of three hydrogen atoms
attached to the C-4 and C-5 of the heterocyclic ring. The
inhibited 100% of the proteolytic activity at a nal concentra-
tion of 1 ꢁ 10ꢀ4 M. A number of cysteine proteases are
methylene protons of the heterocyclic ring appeared as two
responsible for this proteolytic activity; therefore it is neither
double doublets, one at d ¼ 3.75–3.82 ppm (H4a, J ¼ 17, 12.3 Hz)
feasible nor proper to explore any structural activity relation-
ship between the substituent and enzyme activity at this stage.
and the other at 3.05–3.12 ppm (H4b, J ¼ 17, 7.2 Hz). The exis-
tence of these double doublets clearly indicates the magnetic
The only conclusion drawn from these results is that the inhi-
nonequivalence of these two protons due to being adjacent to a
bition caused by the designed compounds is of reversible type
Scheme 2 Some pyrazolines as antitumor and anti-inflammatory agents.
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Scheme 3 Synthesis of chalcones and their cyclized derivatives.
because the inhibition is greater at 3h incubation time and less in the concentration range of 10ꢀ4 to 10ꢀ9 M. 40-Phenyl-
at 24 h incubation time. It can be interpreted that either the chalcones (1a–1i), N-formyl substituted pyrazoline derivatives
inhibition is reversed with time, or some proteases which are (2a–2i), N-phenyl substituted pyrazoline derivatives (3a–3i) and
insensitive toward the designed compounds have become active N-hydro-substituted pyrazoline derivatives (4a–4i) were tested;
in the due course of time. It was evident from experimental data all groups showed satisfactory inhibitory effect, but the
that the compounds inhibited proteolysis of endogenous compounds 2a–2i showed the maximum inhibitory effect in the
substrates at this pH. In the literature it was found53 that most case of cathepsins B and L, however 4a–4i showed the
of proteolytic activity at this pH is due to cysteine proteases, maximum inhibitory effect on cathepsin H.
therefore it was thought proper to evaluate their inhibitory
2.2.1. Effect of 40-phenylchalcones (1a–1i) on the activity of
effect on cathepsins B, H and L, important cysteine proteases. cathepsins B, H and L. Fig. 1–3 show the relationship between
Table S1† presents the results of the endogenous proteolytic the enzyme activity and concentration of the substituted chal-
activity in the presence of 40-phenylchalcones and their pyr- cones (1a–1i) for cathepsins B, H and L respectively. It has been
azoline derivatives.
found that enzyme activity is inhibited and is affected by the
substituents present in chalcones. Among the various
compounds tested, at 1 ꢁ 10ꢀ5 M concentration 1b and 1d
exhibited the maximum inhibitory effect in case of cathepsin B
and cathepsin H respectively, and in the case of cathepsin L 1b
shows maximum inhibition at 10ꢀ9 M.
2.2. Enzyme inhibition studies
The inhibitory effects of the synthesized compounds were
studied on cathepsins B, H and L. Each derivative was examined
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Fig. 1 The results are depicted as the % residual activities of cathepsin B in the presence of different concentrations (0.1, 0.2, 0.3, 0.50, 0.75 and
1.0 ꢁ 10ꢀ5 M) of various chalcones (1a–1i), 0.1, 0.2, 0.3, 0.50, 0.75 and 1.0 ꢁ 10ꢀ6 M of N-formylpyrazolines (2a–2i), 0.1, 0.2, 0.3, 0.50, 0.75 and
1.0 ꢁ 10ꢀ4 M of N-phenylpyrazolines (3a–3i) and 0.1, 0.2, 0.3, 0.50, 0.75 and 1.0 ꢁ 10ꢀ5 M of diarylpyrazolines (4a–4i) after an interaction time of
30 min. The experiments were conducted in triplicate and were calculated w.r.t. the control having no compound, but an equivalent amount of
solvent was added.
2.2.2. Effect of N-formyl-2-pyrazolines on the activity of
2.2.5 Enzyme kinetic studies. All the synthesized
cathepsins B, H and L. On evaluating the inhibitory effect of N- compounds, aer establishing the inhibitory effect, were
formyl-2-pyrazolines it was found that electron withdrawing screened to evaluate the inhibition type and to determine the
substituents exhibited a greater inhibitory effect as compared to inhibition constant Ki value. It was found that all the
electron releasing substituents. The activity of cathepsin B was compounds inhibited cathepsin B and cathepsin L in a
reduced to 54% at 1 ꢁ 10ꢀ7 M, and the activity of cathepsin L competitive manner, whereas cathepsin H was inhibited in a
was reduced to 35% for 2b, revealing this compound as the noncompetitive manner except for in the case of N-phenyl pyr-
most potent inhibitor among all tested compounds (Table S2†). azolines, in which competitive inhibition was found (Fig. 4–6).
In the case of cathepsin H, 2d shows the maximum inhibition The Ki values have been summarised in Table S3.†
i.e. 32% at 1 ꢁ 10ꢀ5 M.
2.2.3. Effect of N-phenyl-2-pyrazolines on the activity of
2.3. SAR studies
cathepsins B, H and L. The experiments were designed to study
the effect of N-phenyl-2-pyrazolines on the cysteine proteases,
cathepsins B, H and L. It was found that 70% inhibition for
cathepsin B activity was achieved at 1.0 ꢁ 10ꢀ5 M concentration
in the case of 3b and 58% in case of 3g. Cathepsin H was
maximally inhibited by 3d and 3e i.e. 35% at 1 ꢁ 10ꢀ5 M.
Cathepsin L was inhibited by 3b up to 50% at 1 ꢁ 10ꢀ6 M.
2.2.4. Effect of 3-(4-biphenyl)-5-substituted phenyl-2-
pyrazolines on cathepsins B, H and L. In the case of the 3-(4-
biphenyl)-5-substituted phenyl-2-pyrazolines a denite pattern
of inhibition could not be established. Almost all substituents
showed 80–85% inhibition at 1 ꢁ 10ꢀ6 M. But in the case of
cathepsin L, 4b showed the maximum inhibition, i.e. 55% at 1
ꢁ 10ꢀ8 M.
The literature reports that cysteine proteases, including
cathepsins B, H and L, play important roles in cancer, inam-
mation and tissue degenerative processes, where proteolysis in
one aspect or another is an important phase for the spread of
these diseases. Targeting these enzymes is therefore one of the
strategies in the development of new chemotherapeutic agents.
In vitro inhibition studies clearly suggest that for cathepsin B
the order of inhibition caused by the different series of
compounds was 2a–2i > 4a–4i > 1a–1i > 3a–3i, for cathepsin L it
was 2a–2i > 1a–1i > 4a–4i > 3a–3i, whereas for cathepsin H it can
be observed that the order of inhibition was 1a–1i > 4a–4i > 2a–
2i > 3a–3i as observed from the Ki values. In these enzymes, the
synthesized compounds show different tendencies. In
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Fig. 2 The results are depicted as the % residual activities of cathepsin H in the presence of different concentrations (0.1, 0.2, 0.3, 0.50, 0.75 and
1.0 ꢁ 10ꢀ5 M) of various chalcones (1a–1i), 0.1, 0.2, 0.3, 0.50, 0.75 and 1.0 ꢁ 10ꢀ4 M of N-formylpyrazolines (2a–2i), 0.1, 0.2, 0.3, 0.50, 0.75 and
1.0 ꢁ 10ꢀ4 M of N-phenylpyrazolines (3a–3i) and 0.1, 0.2, 0.3, 0.50, 0.75 and 1.0 ꢁ 10ꢀ6 M of diarylpyrazolines (4a–4i) after an interaction time of
30 min. The experiments were conducted in triplicate and were calculated w.r.t. the control having no compound, but an equivalent amount of
solvent was added.
cathepsins B and L the formyl group probably plays an impor- results have previously been reported from our lab for o-hydroxy
tant role and interacts with the nucleophilic thiol group present chalcones and their cyclized derivatives,38 where the Ki value for
at the active site. Cathepsin L is sensitive toward a–b unsatu- the most inhibitory compound has been found to be 6.18 ꢁ
rated carbonyl compounds like cathepsin H. The cysteine of 10ꢀ8 M. In case of cathepsin H, we evaluated that halogen
cathepsin H does not seem to interact with this group so substituted pyrazolines were most inhibitory. The results are
effectively. In cathepsin H the unsaturated carbonyl group similar to those reported previously50 where halogen
seems to play an important role in the binding and inhibition. substituted pyrazolines inhibited cathepsin H appreciably.
N-Phenylpyrazolines seem to cause less inhibition to both the However, in series 1, nearly the same inhibition was observed
enzymes as the Ki value is sub-micromolar. Similar results have invariably, regardless of the substituents.
been previously obtained in the case of 1,3,5-triphenylpyrazo-
lines and related structural analogs48 where the phenyl
substituent pyrazolines were least inhibitory.
2.4. Molecular docking
The docking approach used in this study was aimed at the
identication of compounds that selectively bind to the active
site of cathepsin B and modulate the enzymatic activity. The
individual binding poses of each compound were assessed, and
their interactions in the active site of the enzyme were analysed.
Docking alterations are based on the interaction force eld
scoring, which includes van der Waals and electrostatic inter-
actions between the active site and ligand. Table S3† represents
the data of the docking studies of the 40-phenylchalcones (1a–1i)
and the pyrazoline derivatives (2a–2i), (3a–3i) and (4a–4i) with
the cathepsin B active site (available through RCSB Protein Data
Bank, PDB entry cav2IPP B_PYS.pdb). The binding energies
To further explore the structure–activity relationship, the
substitution pattern was carefully altered in the designed
compounds, keeping in mind their different electronic effects.
It can be further observed that for cathepsin B and cathepsin L,
within the series (1a–1i), the compound 1b greatly inuenced
enzyme activity with Ki values of 7.0 ꢁ 10ꢀ6 and 0.54 ꢁ 10ꢀ9 M,
respectively, and among pyrazolines (2a–2i) the most inhibitory
compound was 2b with Ki values of 7.2 ꢁ 10ꢀ8 and 0.26 ꢁ 10ꢀ9
M respectively.
In all the series the electron withdrawing –NO2 group was
found to be most inhibitory for cathepsin B and L. Similar
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Fig. 3 The results are depicted as the % residual activities of cathepsin L in the presence of different concentrations (0.1, 0.2, 0.3, 0.50, 0.75 and
1.0 ꢁ 10ꢀ5 M) of various chalcones (1a–1i), 0.1, 0.2, 0.3, 0.50, 0.75 and 1.0 ꢁ 10ꢀ4 M of N-formylpyrazolines (2a–2i), 0.1, 0.2, 0.3, 0.50, 0.75 and
1.0 ꢁ 10ꢀ4 M of N-phenylpyrazolines (3a–3i) and 0.1, 0.2, 0.3, 0.50, 0.75 and 1.0 ꢁ 10ꢀ6 M of diarylpyrazolines (4a–4i) after an interaction time of
30 min. The experiments were conducted in triplicate and were calculated w.r.t. the control having no compound, but an equivalent amount of
solvent was added.
(kcal molꢀ1) of the 40-phenylchalcones (1a–1i) and pyrazoline Like leupeptin, which has been reported as a competitive
derivatives (2a–2i), (3a–3i) and (4a–4i) vary in the ranges ꢀ80.44 inhibitor to cathepsin B, the designed compounds show
to ꢀ91.82, ꢀ86.39 to ꢀ95.35, ꢀ96.78 to ꢀ101.15 and ꢀ94.45 to competitive inhibition.
ꢀ98.95, respectively, and the binding energies of the most
Fig. 7(a–d) shows the results of the best docking pose of 1b,
inhibitory compounds in each series 1b, 2b, 3b, and 4b show 2b, 3b and 4b, respectively, in the active site of cathepsin B. It
decreases in total energy of ꢀ83.30, ꢀ89.11, ꢀ99.42 and ꢀ97.74, can be observed that Cys-29, Trp-30 and Gly-198 have been
respectively. On the basis of the interaction data of the docking found to interact with the substrate as well as the compound
experiments, it was observed that all the compounds showed under consideration. This supports the results obtained during
less interaction than the reference leupeptin, a peptidyl inhib- the in vitro studies, because the compounds are evaluated to be
itor. The maximum interaction is shown by BANA with a score competitive inhibitors and to compete with the binding site of
of ꢀ124.91. The decrease in total energy for leupeptin– the substrate. The proposed mechanism is shown in Scheme 4.
cathepsin B came out to be ꢀ119.17, of which the contributions The results clearly indicate the signicance of the in vitro
of the van der Waals interactions was greatest with a score of inhibition studies.
ꢀ91.77 as compared to the H-bond with a score of ꢀ28.00. The
However for cathepsin H the decrease in total energy for the
leupeptin–cathepsin B binding energy is due to peptide–protein reference inhibitor leu-CH2Cl was less as compared to all the
interaction. Leupeptin is peptidyl in nature, and therefore, designed compounds. Here, it can be seen that although leu-
being a exible molecule, it binds effectively with the enzyme CH2Cl is a specic inhibitor for cathepsin H65,66 it possesses
active site, resulting in a higher binding energy. iGemDOCK only one amino acid residue in contrast to leupeptin–cathepsin
provides algorithms for exible docking approach for both B. Therefore the leu-CH2Cl–cathepsin H interaction causes a
ligands and proteins,66 therefore exible ligands like leupeptin decrease in energy of only ꢀ59.99, of which ꢀ43.49 is the van
will show a larger decrease in total energy as compared to the der Waals interaction and ꢀ16.50 is due to H-bonds.
molecules under study. The compounds under consideration
As listed in Table S4† all the designed compounds have been
are smaller in structure and possess lower exibility compared found to show a greater decrease in ligand–cathepsin H inter-
to leupeptin. Therefore, the binding energy of the title action energy than leu-CH2Cl–cathepsin H. From docking
compounds is less than leupeptin, but the in vitro analysis studies, the compound 1a was evaluated to be the most inhib-
reveals that the compounds are good inhibitors to cathepsin B. itory to cathepsin H among the 40-phenylchalcones, showing a
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Fig. 4 Effect of the compounds 1b, 2b, 3b and 4b on cathepsin B activity. The experiment was done in three different concentrations in triplicate.
All compounds showed competitive inhibition.
total decrease in energy of ꢀ81.56, of which ꢀ72.77 is assigned ꢀ106.21, ꢀ99.83 to ꢀ115.61 and ꢀ94.09 to ꢀ106.17, respec-
to van der Waals interactions whereas ꢀ8.79 is from H-bonding. tively, and the most inhibitory compounds in each series 1b, 2b,
The compound 2d was supported as being the best inhibitor to 3b, and 4b show a decrease in total energy of ꢀ88.05, ꢀ105.4,
cathepsin H among the N-formylpyrazolines, because it showed ꢀ105.55 and ꢀ103.14 kcal molꢀ1. When these docking energies
a total decrease in energy of ꢀ81.94, of which ꢀ68.45 is were compared with those of the substrate Z-Phe-Arg-4mbNA
assigned to van der Waals interactions whereas ꢀ13.48 is from and the peptidyl inhibitor leupeptin, it was found that they are
H-bonding. As evidenced by the docking results, among the very low. For Z-Phe-Arg-4mbNA it was ꢀ130.74 kcal molꢀ1, and
phenylpyrazolines, 3d was found to be most inhibitory to for leupeptin it came out to be ꢀ116.8 kcal molꢀ1. Leupeptin,
cathepsin H, showing a total decrease in energy of ꢀ85.26, of possessing a peptidyl chain, exhibits a larger decrease in energy;
which ꢀ81.65 is assigned to van der Waals interactions whereas however, the extent of the inhibition caused by 1b and 2b is of
ꢀ3.61 is from H-bonding. However among the N-pyrazolines, 4d the order of 10ꢀ9 M, which is greater than that of leupeptin,
was evaluated to be the best inhibitor to cathepsin H, showing a where the Ki value is reported to be 1.5 ꢁ 10ꢀ9 M.71
total decrease in energy of ꢀ83.12, of which ꢀ72.92 is assigned
Fig. 9(a–d) shows the result of the best docking pose of 1b, 2b,
to van der Waals interactions whereas ꢀ10.19 from H-bonding. 3b and 4b respectively in the active site of cathepsin L. The amino
Fig. 8(a–d) shows the docked poses of the most inhibitory acids Gln-19, Leu-69, Asp-162 and His-163 have been found to
compounds 1d, 2d, 3d and 4d in the aminoacyl binding site of interact with the compounds, as well as the with substrate Z-Phe-
cathepsin H, respectively. As can be seen, the designed Arg-4mbNA, thus indicating a competitive type of inhibition.
compound and the substrate leu-bNA bind at two different sites.
Fig. 10(a–c) represents the correlation between the total
Gln-73 and Ser-69 have been found to interact with leu-bNA energy vs. log Ki of the designed compounds for cathepsin B, H
Ha–H bonds. However Gln-78, Asn-116 and Gln-23 interact with and L respectively. It can be observed from Fig. 10(a), that the
the most inhibitory compound in each series. The docked poses most effective binding of compound 2b followed by 2g causing
and interactions clearly suggest that a non-competitive type of maximum inhibitory potential is well correlated in this graph.
inhibition is exerted by the compounds on cathepsin H. In vitro The N-formylpyrazoline series (2a–2i) exhibits maximum
inhibition studies also suggest that all the N-phenylpyrazoline inhibitory power, followed by the N-pyrazolines (4a–4i), the 40-
series exerted a non-competitive inhibition on cathepsin H.
phenylchalcones (1a–1i) and N-phenylpyrazolines (3a–3i) in that
In the case of cathepsin L the ranges of the binding energies order. The correlation plot for cathepsin H, 10b, depicts the
of the 40-phenylchalcones (1a–1i) and pyrazoline derivatives (2a– chalcones as the most inhibitory series followed by N-pyrazo-
2i), (3a–3i) and (4a–4i) vary from ꢀ88.05 to ꢀ101.48, ꢀ91.34 to lines, N-formylpyrazolines and N-phenylpyrazolines. The plot of
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Fig. 5 Effect of the compounds 1d, 2d, 3d and 4d on cathepsin H activity. The experiment was done in three different concentrations in triplicate.
3d showed competitive inhibition, and the other compounds showed non-competitive inhibition.
binding energy vs. inhibitory constant for cathepsin
L
purchased from Bachem Feinchemikalein AG (Switzerland).
(Fig. 10(c)) shows that N-formylpyrazolines are the most inhib- Fast Garnet GBC was purchased from Sigma USA. The chro-
itory, followed by 40-phenylchalcones, N-pyrazolines and N- matographic media Sephadex G-100, CM-Sephadex C-50 and
phenylpyrazolines.
DEAE Sephadex, A-50 were supplied by Pharmacia Fine Chem-
In the present work, we have used the decrease in total icals, Uppsala (Sweden). All the solutions used were prepared
energy of the enzyme–ligand complex as a measure of the fresh in glass-distilled conductivity water. The source of the
binding affinity of the ligand within the active site of the enzymes was fresh goat liver obtained from a local slaughter-
enzyme. However, Zhang et al. used a free energy based house. 40-Phenyl acetophenone and aldehydes were purchased
approach in understanding such interactions.68–70
from Himedia, Bombay. An Elisa plate reader was used for
It may be worth mentioning here that the in silico studies measuring absorbance in the visible range. IR spectra were
have been used only as supporting tool for enzyme inhibition recorded on a Horizon 300 MHz spectrometer. 1H NMR spectra
studies, therefore the advanced details of the docking studies were recorded on a Bruker 300 MHz instrument. The chemical
are not included here. The in silico predicted behaviour of the shis are expressed in ppm units from an internal TMS stan-
enzyme–ligand interaction can give an idea about the interac- dard. The refrigerated ultracentrifuge Remi C-24BL was used for
tion between these two, but cannot substitute for the in- centrifugation purposes under cold conditions.
solution studies.
Melting points were taken in open capillaries and are
In the present study we found that different effects have been uncorrected. The progress of the reactions was monitored on
exerted by the related classes of compounds on cathepsin B and silica gel G plates using iodine vapor as the visualizing agent.
H activities. The electron withdrawing –NO2 substitution proved
to be a good inhibitor of cathepsins B and L, and the chloro- and
uoro-substituted compounds were more inhibitory to H.
3.1. Synthesis
3.1.1. Procedure for syntheses of 3-substitutedphenyl-1-(4-
biphenyl) propen-1-ones (1a–1i). A series of 1-biphenyl-3-
(substituted phenyl)-2-propen-1-ones was synthesized by the
3. Experimental section
The
substrates
a-N-benzoyl-d,L-arginine-2-naphthylamide grinding of substituted aldehydes (0.01 mol) with 4-phenyl-
(BANA)/Z-Arg-Arg-4mbNA, LeubNA and Z-Phe-Arg-4mbNA were acetophenone (0.01 mol) in the presence of potassium
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Fig. 6 Effect of the compounds 1b, 2b, 3b and 4b on cathepsin L activity. The experiment was done in three different concentrations in triplicate.
All compounds showed competitive inhibition.
hydroxide (0.01 mol) with a mortar and pestle. The progress of
the reaction and the purity of the products were conrmed
through TLC. The mixture was poured in ice water, ltered,
washed with ethanol, dried and recrystallised with ethanol;
yield: 85–90% depending on substitution.
3.1.4. General procedure for syntheses of 3-(4-biphenyl)-5-
substituted phenyl-2-pyrazolines (4a–4i). Compounds 1a–1i
(0.001 mol) and hydrazine hydrate (0.001 mol) in ethanol (25
ml) were reuxed at 60–70 ꢂC for 6–8 h. The reaction progress
was monitored with the help of TLC. Once the reaction was
complete the reaction mixture was allowed to cool at
room temperature. The ne crystals that thus separated
out were ltered, washed with cold ethanol, dried and
recrystallised with ethanol; yield: 83–90% depending on
substitution.
3.1.2. General procedure for syntheses of N-formyl-3-(4-
biphenyl)-5-substituted
phenyl-2-pyrazolines
(2a–2i).
Compounds 1a–1i (0.001 mol), hydrazine hydrate (0.001 mol)
and 15–20 ml ofꢂformic acid in ethanol (25 ml) were then
reuxed at 60–70 C for 12–14 h. The progress of reaction was
monitored with the help of TLC. Aer the completion of the
reaction, the mixture was allowed to cool at room temperature
and was kept in a refrigerator for 4–5 h. The ne crystals that
thus separated out were ltered, washed with cold ethanol,
dried and recrystallised with ethanol; yield: 75–85% depending
on substitution.
3.2. Proteolytic studies
3.2.1. Preparation of liver homogenate. Goat liver was
purchased freshly from a local slaughterhouse. The fresh goat
liver was rst washed with cold isotonic saline solution. The
tissue was then homogenized a in 0.1 M sodium acetate buffer
at pH 5.5 containing 0.2 M NaCl in a mixer-cum-blender to
3.1.3. General procedure for syntheses of N-phenyl-3-(4-
biphenyl)-5-substituted
phenyl-2-pyrazolines
(3a–3i).
ꢂ
Compounds 1a–1i (0.001 mol), phenyl hydrazine hydrate (0.001
obtain 10% (w/v) homogenate. It was then stored at 4 C.
mol) and added acetic acid in a catalytic amount in ethanol (25
3.2.2. Assay for proteolytic activity. The proteolytic studies
ꢂ
ml) were reuxed at 60–70 C for 6–8 h. The reaction progress
on the endogenous protein substrate were carried out at pH 5.0
ꢂ
was monitored using TLC. The reaction mixture was allowed to
cool at room temperature. The ne crystals that thus separated
out were ltered, washed with cold ethanol, dried and recrys-
tallised with ethanol; yield: 87–95% depending on
substitution.
at 37 C using 0.1 M acetate buffer as the incubation medium.
The homogenate was mixed with the buffer and was incubated
ꢂ
at 37 C for 3 h. The reaction was stopped by the addition of
TCA, and the resulting solution was centrifuged to precipitate
proteins. The acid soluble proteins were quantitated in the
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Fig. 7 Docking results showing the alignment of the most inhibitory Fig. 8 Docking results showing the alignment of the most inhibitory
compounds, 1b, 2b, 3b and 4b, in the active site of cathepsin B compounds, 1d, 2d, 3d and 4d, in the active site of cathepsin H
(cav2IPP B_PYS.pdb), along with the substrate BANA in a, b, c and d, (cav8PCH H_NAG.pdb) along with the substrate leubNA in a, b, c and d,
respectively. The binding site radius is 8 A˚. The results are for docking
respectively. The binding site radius is 8 A˚. The results are for docking
at drug screening settings. The amino acids Cys-29, Gly-198 and Trp- at drug screening settings. The inhibitor and substrate bind at two
30 can be observed interacting with the inhibitor and the amide bond different sites. The amino acids Ser-69 and Glu-73 can be observed
of the substrate BANA through H-bonds as shown in green. The interacting with the substrate Leu-b-NA, and Gln-70, Gln-78, Gln-86
and Asn-112 interact with the inhibitor through H-bonds as shown in
green. The hydrophobic interactions are shown in grey.
hydrophobic interactions are shown in grey.
Scheme 4 Proposed mechanism of inhibition for chalcones.
supernatant using the Bradford method.72 The experiment was
conducted in triplicate and the results are presented in
Table S1.†
3.3. Purication of goat liver cathepsin B, cathepsin H and
cathepsin L
All the purication steps were carried out at 4 ꢂC. Cathepsin B,
H50 and L69 were isolated, separated and puried from goat liver
using the following procedure. Goat liver acetone powder was
homogenized in cold 0.1 M sodium acetate buffer pH 5.5 con-
taining 0.2 M NaCl and 1 mM EDTA followed by acid-autolysis at
pH 4.0 and 30–80% ammonium sulphate fractionation,
Sephadex G-100 column chromatography and nally cation and
anion exchange chromatography on a CMSephadex C-50 and
DEAE-Sephadex A-50. The specic activities of the cathepsin B,
cathepsin H and cathepsin L were 13.46 nmol per min mg, 25.56
Fig. 9 Docking results showing the alignment of the most inhibitory
compounds, 1b, 2b, 3b and 4b, in the active site of cathepsin L
(cav3BC3 L_CSW.pdb) along with the substrate Z-Phe-Arg-4mbNA in
a, b, c and d, respectively. The binding site radius is 8 A˚. The results are
for docking at drug screening settings. Only hydrophobic interactions
are visible, except for in compound 4b, where Gln-19 interacts
through H-bonding with the substrate as well as with the compound.
Hydrophobic interactions are exerted by Gln-19, Gln-23, Trp-26, Gly-
68, Leu-69, Asp-162, His-163 and Gly-164.
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Fig. 10 Correlation plot between binding energies and log Ki values for cathepsin B (a), cathepsin H (b), and cathepsin L (c). ,, D, ꢁ and >
represent 1a–1i, 2a–2i, 3a–3i and 4a–4i, respectively.
nmol per min per mg and 15.42 mmol per min per mg 4mbNA as the substrate.74 The results are shown in Table S1†
respectively.
and Fig. 1–3.
3.5. Determination of Ki values
3.4. Enzyme inhibition studies
Aer establishing the inhibitory action of the synthesized
compounds on cathepsin B, cathepsin H and cathepsin L,
experiments were designed to evaluate the type of inhibition
and to determine the Ki value of these compounds. For that, the
enzyme activity was evaluated at different substrate concentra-
tions in the presence and absence of a xed concentration of the
inhibitor. The enzyme concentration was kept constant in all
the experiments. A Lineweaver–Burk plot was drawn of 1/S and
1/V in the presence and absence of the inhibitor (Fig. 4–6).
The activities of cathepsin B were estimated at varying
concentrations of synthesized 40-phenylchalcones (1), N-for-
mylpyrazolines (2), N-phenylpyrazolines (3) and pyrazolines (4).
First of all, the enzyme was equilibrated in 0.1 M phosphate
buffer of pH 6.0 at 37 ꢂC. The stock solutions of the compounds
were prepared in DMSO. Then an appropriate amount of the
individual compounds was added to the reaction mixture
separately to effect the nal concentration of each compound.
Aer an incubation time of 30 min, the residual enzyme activity
was estimated by the usual enzyme assay73 at pH 6.0 using a-N-
benzoyl-d,L-arginine-2-naphthylamide (BANA) as a substrate.
3.6. Drug modeling studies
Similarly, for cathepsin H, the activities were estimated at All docking studies were performed using iGemdock. For these
varying concentrations of the synthesized compounds using studies the small molecular weight ligands were prepared and
leubNA as the substrate.67
the enzyme structure active site was retrieved from the Protein
The activities for cathepsin L were estimated at varying Data Bank. The structures were prepared using marvin sketch
concentrations of the synthesized compounds using Z-Phe-Arg- and were saved as MDL Mol Files. The structure of cathepsins B,
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H and L were retrieved from the Protein Data Bank (http://
www.rcsb.org/) as cav2IPP B_PYS.pdb,75 cav8 PCH-NABpdb76
and cav3BC3L_CSW77 respectively. Aer loading the prepared
ligands and the binding site docking experiments were run with
drug screening settings, and the results are presented in Table
S4–S6† and Fig. 7–9.
E. J. Barreiro and L. M. Lima, Bioorg. Med. Chem., 2009, 17,
641–652.
7 K. Brak, I. D. Kerr, K. T. Barrett, K. N. Fuchi, M. Debnath,
M. K. Ang, J. C. Engel, J. H. McKerrow, P. S. Doyle,
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4. Conclusions
In the present work we synthesized 40-phenylchalcones and
their pyrazoline derivatives and screened their effect on
cathepsins B, H and L. The synthesized compounds were shown 11 M. M. Mohamed and B. F. Sloane, Nat. Rev. Cancer, 2006, 6,
to be potent inhibitors of cathepsins B, H and L. The kinetic 764–775.
studies revealed that all compounds showed reversible 12 J. Kos and T. T. Lah, Oncol. Rep., 1998, 5, 1349–1361.
competitive inhibition on cathepsin B and L with affinities in 13 M. J. Duffy, Clin. Cancer Res., 1996, 2, 613–618.
the subnanomolar range. Except for the N-phenylpyrazolines, 14 I. Assfalg-Machleidt, M. Jochum, D. Nast-Kolb, M. Siebeck,
all compounds showed reversible non-competitive inhibition
on cathepsin H with affinities in the micromolar range.
Cathepsin B is more susceptible as compared to cathepsin H.
A. Billing, T. Joka, G. Rothe, G. Valet, R. Zauner,
H.-P. Scheuber and W. Machleidt, Biol. Chem. Hoppe-Seyler,
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The maximum inhibition was shown by 1-formyl-3-(4-biphenyl- 15 H. Fritz, M. Jochum, R. Geiger, K. H. Duswald, H. Dittmer,
5-(40-nitrophenyl)-2-pyrazoline, 2b in case of cathepsins B and L,
and for cathepsin H, 2d was found most inhibitory. The results
H. Kortmann, S. Neumann and H. Lang, Folia Histochem.
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presented in this study can add to the existing knowledge of 16 M. Jochum, K. H. Duswald, S. Neumann, J. Witte, H. Fritz
¨
cathepsins B, H and L inhibitors not reported earlier.
and U. Seemuller, Proteinases and their inhibitors in
inammation: basic concepts and clinical implications, in
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Conflicts of interest
The authors have declared no conict of interest.
Acknowledgements
18 H. Kirschke, A. J. Barrett and N. D. Rawlings, Protein Prole,
1995, 2, 1587–1643.
Among the authors, Indu Ravish is thankful to UGC New Delhi,
India for the award of SRF and also to Kurukshetra University, 19 B. F. Sloane, K. Moin, E. Krepela and J. Rozhin, Cancer
Kurukshetra for providing necessary research laboratory
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