2,5-Diaryloxadiazoles and their precursors as novel inhibitors of cathepsins B, H and L

Article abstract of DOI:10.1016/j.bioorg.2016.05.003

High levels of cathepsins indicated in various pathological conditions like arthritis, cancer progressions, and atherosclerosis explains the need to explore potential inhibitors of these proteases which can be of great therapeutic significance. We, in the present work, report the synthesis of some 2,5-diaryloxadiazoles from N-subsitutedbenzylidenebenzohydrazides. The synthesized compounds were screened for their inhibitory potential on cathepsins B, H and L. Structure Activity Relationship studies show that 2,5-diaryloxadiazoles were less inhibitory than their precursors. 1i and 2k have been found to be most inhibitory to cathepsins B and L. Their K_i values have been calculated as 11.38 × 10^-8 M and 66.4 × 10^-8 M for cathepsin B and 4.2 × 10^-9 M and 47.31 × 10^-9 M for cathepsin L, respectively. However, cathepsin H activity was maximally inhibited by compounds, 1e and 2c with K_i values of 4.4 × 10^-7 M and 5.6 × 10^-7 M, respectively. Enzyme kinetic studies suggest that these compounds are competitive inhibitors to the enzymes. The results have been compared with docking results obtained using iGemDock.

Full text of DOI:10.1016/j.bioorg.2016.05.003

Accepted Manuscript
2, 5-Diaryloxadiazoles and their precursors as novel inhibitors of cathepsins B,
H and L
Shweta Garg, Neera Raghav
PII:
S0045-2068(16)30045-1
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.05.003
YBIOO 1908
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 January 2016
1 May 2016
13 May 2016
Please cite this article as: S. Garg, N. Raghav, 2, 5-Diaryloxadiazoles and their precursors as novel inhibitors of
cathepsins B, H and L, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.05.003
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2, 5-DIARYLOXADIAZOLES AND THEIR PRECURSORS AS NOVEL
INHIBITORS OF CATHEPSINS B, H AND L
Shweta Garg and Neera Raghav*
Department of Chemistry, Kurukshetra University, Kurukshetra-136119 (INDIA)
Tel.: +919896918277
E-mail: nraghav.chem@gmail.com
Abstract
High levels of cathepsins indicated in various pathological conditions like arthritis, cancer
progressions, atherosclerosis etc. explains the need to explore potential inhibitors of these
proteases which can be of great therapeutic significance. We, in the present work, report the
synthesis of some 2,5-diaryloxadiazoles from N-subsitutedbenzylidenebenzohydrazides. The
synthesized compounds were screened for their inhibitory potential on cathepsins B, H and L.
Structure Activity Relationship studies show that 2,5-diaryloxadiazoles were less inhibitory than
their precursors. 1i and 2k have been found to be most inhibitory to cathepsins B and L. Their Ki
values have been calculated as 11.38x10-8 M and 66.4 x10-8 M for cathepsin B and 4.2 x10-9 M
and 47.31 x10-9 M for cathepsin L, respectively. However, cathepsin H activity was maximally
inhibited by compounds, 1e and 2c with Ki values of 4.4x10-7 M and 5.6x10-7 M, respectively.
Enzyme kinetic studies suggest that these compounds are competitive inhibitors to the enzymes.
The results have been compared with docking results obtained using iGemDock.
Keywords: 2,5-diaryloxadiazoles, N-subsitutedbenzylidenebenzohydrazides, Cathepsin B,
cathepsin H and Cathepsin L inhibitors.
1. Introduction
Enzyme inhibition studies have been a great tool in the development of newer medicines. Almost
60% of present drugs act by inhibiting enzymes responsible for pathological conditions.
Discovering novel therapeutic targets and identification of their inhibitors can provide different
outlook for treatment of diseases. Cathepsins B, H and L, three important lysosomal cysteine
proteases have become important therapeutic targets because of their underlying proteolytic role
in cell degenerative processes and cleavage of extracellular matrix1. Their potential role is not at
all deniable in diseases like inflammation2 particularly arthritis, periodontitis; cell proliferation
and tumor growth3,4, Alzeimers disease5,6 accentuate the need for development of specific
inhibitors of these enzymes which can be of great therapeutic potential. The quest for their
1

identification is also a result due to a decrease in the concentration of natural inhibitors of these
cathepsins which is one of the consequences in the mentioned diseased conditions. In past
various peptidyl inhibitors have been synthesized and evaluated as inhibitors to cathepsins7 but
the need for identification of newer therapeutic agents never ends in order to obtain better
efficacy than and to avoid untoward effects of available drugs. With oral instability and
immunogenic problems faced using peptidyl inhibitors, in recent past research has found its way
to discover non-peptidyl inhibitors that can act as therapeutic agents in diseased conditions
associated with the cysteine proteases. In this direction, we have reported8-11 some non-peptidyl
inhibitors of cathepsins B and H such as acyl hydrazides and triazoles, o-hydroxy chalcones and
their cyclised derivatives and bischalcone based quinozolines and 4’-phenylchalcones and their
derivatives as novel inhibitors of cathepsins B and H. Work has also been carried out on
semicarbazones and thiosemicarbazones of carbonyl compounds12 and chalcones13. The present
work has been proposed on the basis of different biological activities possessed by 2,5-
diaryloxadiazoles and N-subsitutedbenzylidenebenzohydrazides and to explore their possible
role in the treatment of conditions led due to imbalanced activities of cathepsin B, cathepsin H
and cathepsin L such as in cancer, arthritis etc.
The compounds with similar scaffold have been reported as cysteine protease inhibitors; for
example, hydrazone, 1 as parasitic cysteine protease inhibitor14, hydrazone, 2 as cathepsin S
Inhibitor15 and 1,2,4- oxadiazole-N-acylhydrazone16, 3 as cysteine protease cruzain inhibitor17.
O
H
Y
H
H
O
N
N
N
N
N
N
N
O
N
N
O
H
N
O
O
N
H
N
N
H
(2)
(1)
(3)
R
Variety of therapeutically active compounds having Oxadiazole nucleus currently being used
in clinical medicine are: HIV – integrase inhibitor Raltegravir®, 4 an antiretroviral drug18;
Zibotentan®, 5 an anticancer agent19; a nitrofuran antibacterial, furamizole; antihypertensive
agents tiodazosin and nesapidil, 6.
2

O
O
N
N
O
N
H
O
S
N
O
N
N
OH
N
OH
O
O
N
N
N
H
H
N
N
N
N
N
O
N
(5)
O
(6)
O
O
(4)
N-substituted hydrazides are also of wide interest because of their diverse biological activities
and clinical applications. Novel Hydrazide-Hydrazone derivatives, 7 with Antitumor activity20
Arylidene hydrazides, 3,4-methylenedioxy/4-methyl/4-nitro benzylidene hydrazide21, 8 showing
antidepressant activity, para-subsituted hydrazone, 9 and N-arylidene-2-(2-phenoxyphenyl)
acetohydrazides, 10 exhibited good and persistent anti-inflammatory activity22,23 are reported.
R
O
N
H
N
N
O
N
H
CN
R
N
N
H
H2NO2S
(7)
C6H5
R=3,4- methylenedioxy
a) R= H
b) R=Cl
c)
(8)
R= OCH3
H
N
Br
O
O
N
O
N
R
N
N
H
R
(9)
(10)
The present work deals with the synthesis, characterization of some 2,5-diaryloxadiazoles from
their precursors N-subsitutedbenzylidenebenzohydrazides. It has been observed by our research
group that in most of cases open chain analogues have been evaluated as better inhibitors of
these enzymes therefore to evaluate potential role of synthesized compounds as cathepsins B, H
and L inhibitors we here report the comparative studies of cyclized derivatives as well as their
open chain precursors. The results were extended to elaborate the type of inhibition and were
also correlated with in-silico studies.
3

2. Results and Discussion
2.1. Chemistry
N-subsitutedbenzylidenebenzohydrazides were synthesized by the reaction of benzoic hydrazide
with substituted benzaldehydes in presence of a few drops of acetic acid as catalyst and 2,5-
diaryl-1,3,4-oxadiazole
were
synthesized
by
the
cyclization
with the
of
N-
of
subsitutedbenzylidenebenzohydrazides
in dichloromethane
help
iodobenzenediacetate in presence of a few drops of acetic acid (scheme 1). The structure
elucidation of compounds was based on the spectral data (IR and 1H NMR). All the synthesized
N- substitutedbenzylidenebenzohydrazides show a characteristic IR absorption peak. The IR
spectra of N-subsitutedbenzylidenebenzohydrazides showed mainly stretching bands at 1531-
1585and 1645-1681 cm-1 assigned to imine of Schiff bases and (C=O) functionalities,
1
respectively. In the H NMR spectrum, singlets at δ 8.40-8.94 ppm for imine protons were
observed. In case of 2,5-diaryl-1,3,4-oxadiazole, above mentioned IR and NMR spectra was
missing that confirm the cyclization of N- substitutedbenzylidenebenzohydrazides and formation
of 2,5-diaryl-1,3,4-oxadiazole.
2.2. Pharmacological evaluation
2.2.1. Proteolysis of endogenous protein substrates
The effect of synthesized compounds i.e. N- substitutedbenzylidenebenzohydrazides and 2,5-
diaryl-1,3,4-oxadiazole, on proteolysis of endogenous protein substrates was observed at pH 5.0,
where most of the proteolytic activity is attributed to cysteine proteases24. The studies were
conducted in homogenate containing endogenous protein substrates for 3h and 24h (Table 1).
Some compounds completely inhibited proteolysis at 1×10-4M concentration. Inhibition after 3.0
hr was more than at 24.0 hr (Figure 1) suggesting a reversible type of inhibition. The electron
withdrawing nitro- substituted compounds exhibited greater inhibition than electron releasing
similarly positioned methyl or methoxy group indicating that inhibition is guided by electronic
forces in the synthesized ligands. The inhibition pattern of compounds in series 1 is more or less
similar to that of series 2 (Table 1)
The proteolysis studies suggested that open chain precursors are more effective inhibitors as
compared to cyclized derivatives; this may be due to the better interaction of imine group with
the active site which is lost during cyclization. The inhibition of endogenous proteolytic activity
by these compounds at pH 5.0 which is attributable to cysteine proteases opens up further scope
4

to study these compounds as inhibitors to cathepsins B, H and L.
2.2.2. Effect of synthesized compounds on the activity of Cathepsin B
The activities of cathepsin B were estimated at different concentrations each of synthesized
compounds. Effect of N-subsitutedbenzylidenebenzohydrazides 1(a-k) and 2,5-diaryl-1,3,4-
oxadiazole 2(a-k) on cathepsin B activity is presented in Figure 2(i-ii). Among N-
subsitutedbenzylidenebenzohydrazides and 2,5-diaryl-1,3,4-oxadiazole, 1i and 2k with nitro
substitution were evaluated as most inhibitory to cathepsin B.
2.2.3. Effect of synthesized compounds on the activity of Cathepsin H
Figure 3 (i-ii) present the effect of N-subsitutedbenzylidenebenzohydrazides 1(a-k) and 2,5-
diaryl-1,3,4-oxadiazole 2(a-k) at varying concentrations on cathepsin H activity. In each series
N’-(4-chlorophenylmethylene)benzohydrazide, 1e and 2 -phenyl -5-(2-chlorophenyl)-1,3,4-
oxadiazole, 2c were evaluated as most inhibitory compounds. It has been found that cathepsin H
activity is maximally inhibited by the chloro substituted compounds in both the series with
opposing inductive and resonating effect.
2.2.4. Effect of synthesized compounds on the activity of Cathepsin L
The activities of cathepsin
L
estimated at varying concentrations of N-
subsitutedbenzylidenebenzohydrazides 1(a-k) and 2,5-diaryl-1,3,4-oxadiazole 2(a-k) are
presented in figure 4 (i-ii), respectively. Among the various compounds in each series most
inhibitory compound to catnepsin B N’-(2-nitrophenylmethylene)benzohydrazide (1i) and 2,5-
diaryl-1,3,4-oxadiazoles, 2-phenyl -5-(4-nitrophenyl)- 1,3,4-oxadiazole (2k) were found best
inhibitors to cathepsin L also.
2.2.5. Enzyme kinetic studies
Enzyme kinetic studies are important in order to establish the type of inhibition exhibited by
compounds and Lineweaver-Burk plots have been a great tool in differentiating between various
reversible inhibitors in an efficient manner, therefore, after ascertaining the inhibition of
synthesized compounds on cathepsins B, H and L, experiments were designed to evaluate the
type of inhibition and to find out their Ki values. Line-weaver Burk plots drawn in 1/S and 1/V in
presence and absence of a fixed concentration of inhibitor at different substrate concentrations
for cathepsin B, cathepsin H and cathepsin L are presented in figure 2 (iii, iv), figure 3 (iii, iv)
and figure 4(iii, iv), respectively. These results indicated that all compounds exhibited a
competitive type of inhibition as evidenced by the plots between 1/V and 1/S, where straight
5

lines intersecting at the Y-axis with Vmax remaining constant and changing Km have been
obtained. The Ki values calculated using Line-weaver Burk equation for competitive inhibition
has been presented in Table 2.
Among the series of compounds (1-2), it was found that for cathepsin B and cathepsin L, para-
methyl substituted compounds such as N’-(4-methylphenylmethylene)benzohydrazide (1b) and 2
-phenyl -5-(4-methylphenyl)- 1,3,4-oxadiazole (2b) showed negligible inhibitory effect on
enzyme activity. Nitro substituted compounds in both the series were most inhibitory to
cathepsin B as well as cathepsin L. N’-(2-Nitrophenylmethylene)benzohydrazide (1i) and 2-
phenyl-5-(4-nitro phenyl)-1,3,4-oxadiazole (2k) have been evaluated as best inhibitors with Ki
values of 1.138x10-7 M and 6.64 x10-7 M, respectively for cathepsin B. And, for cathepsin L the
Ki values were evaluated as 4.2 x10-9 M and 47.31 x10-9M, respectively (Table 2). The inhibition
of cathepsins B and L by 1i and 2k can be explained on the basis of the binding sites. The
binding sites in proteases designated as S1, S2….Sn corresponding to enzyme site designated as
P1, P2….Pn account for the reaction specificities of proteases25. The substrate specificity of
catehpsins B and L suggest that S1 binding sites of both these enzymes very well accommodate
arginine amino acid however S2 is responsible for differentiating the specificities in these two
enzymes. For cathepsin L specificity S2 site is described by phenyl alanine whereas in cathepsin
B it is again specified by argininine26. The similar behavior of cathepsins B and L toward
potential inhibitors 1i and 2k, can be due to similar binding site S.
S1
NH2
NH
S1
HN
HN
NH2
NH
C6H5
O
CO O
O
H
H
HN
N
N
N
C6H5
N
H
H
O
O
C6H5
BANA
Z-Phe-Arg-4mβNA
Chloro substituted compounds i.e., N’-(4-chlorophenylmethylene)benzohydrazide (1e) and 2 -
phenyl -5-(2-chlorophenyl)- 1,3,4-oxadiazole (2c) showed maximum inhibitory effect on
cathepsin H activity with Ki values of 4.4x10-7 M and 5.6x10-7, respectively (Table 2).
It can be observed (Table 2) that open chain analogues were potent and selective inhibitor as
6

compared to their cyclized derivatives for cathepsins B, H and L. The order of inhibition for both
of series has been evaluated for cathepsin L > cathepsin B > cathepsin H.
The results when compared with other recently reported non-peptidyl inhibitors of these
cathepsins precisely displayed some trends. An important observation was that among various
open chain and their cyclized derivatives former have been evaluated as more potent inhibitors.
In case of flavanones and flavones when compared with respective chalcones, we observed that
open chain precursors were better inhibitors than cyclized derivatives with Ki value of most
inhibitory compound in each series was ~6.18x10-8M, 4.8x10-7M and 7.85 x10-7M for cathepsin
B and ~2.8 x10-7M, 31.8 x10-6M and 33.7 x10-6M for cathepsin H for chalcone, flavanone and
flavones, respectively9. In case of acyl hydrazides, the open chain precursors for respective
triazoles8 also have been found to be better inhibitors than cyclized derivatives with Ki value for
the most inhibitory compound as 0.64µM, 1.43µM, 2.4 µM, 0.95 µM for cathepsin B and
8.2µM, 8.6µM, 3.3 µM, 2.0 µM for cathepsin H, respectively. Similar results have been
observed for chalcone semicarbazones and their pyrazoline derivatives13 where most inhibitory
chalcone semicarbazone and its pyrazoline have been shown a Ki value of ~0.207x10-6M and
9.40 × 10-6 M, for cathepsin B, ~2.10x10-5M and 2.5 × 10-5 M, for cathepsin H and 0.40 x10-10M
and 0.117 × 10-8 M, for cathepsin L, respectively.
The cyclized derivatives have been found to be more inhibitory only when a highly susceptible
nucleophilic group was introduced at any position in cyclic compound11,27
.
Another important similarity was that nitro compounds have been found to be most inhibitory to
cathepsins B and L whereas chloro-substituted compounds have been found best inhibitors to
cathepsin H11,28. It has been found that in all previously reported non-peptidyl inhibitors of
cathepsins under consideration is that cathepsin L has been found to be more prone to inhibition
by various inhibitors than cathepsin B and cathepsin H in that order13,28. In the present series
also, we could find that cathepsin L is inhibited more than cathepsin B and cathepsin H. The
results are quite encouraging and support the need to carry out such studies in future so that
selective inhibitors of each cathepsin may be searched.
After establishing the in-vitro inhibitory potential of the synthesized compounds in-silico studies
were also carried out to correlate the results as discussed below.
2.2.6. Molecular docking experiment
Molecular docking experiments were conducted using iGemDock, an automated drug design
system for docking, screening and post analysis and has been successfully used to correlate the in
7

vitro and in silico studies of enzymes8-13. Using iGemDock, the predicted poses generated can
be directly visualized in the active site of the target enzyme and simultaneous analysis can be
carried out to predict the affinity of compound with active site by decrease in total energy. The
software provides a tool based on docked poses (protein-ligand interactions) and the compound
properties29. In the present work we have tried to find out a correlation between enzyme
inhibition potency and the docking scores.
The interaction data of docking experiments (Table suppl) include total energy and individual
energy terms indicating the fitness of a predicted pose in the binding site suggest that the level of
interaction is highest for N-subsitutedbenzylidenebenzohydrazides followed by 2,5-
diaryloxadiazoles within the active site of cathepsin B and the same order is found in case of
cathepsin H and cathepsin L. The results can be correlated with the observed Ki values. The Ki
values are lower for open chain analogues suggesting higher interaction of compounds with
active site. In cathepsin B, all the compounds showed a lesser interaction than leupeptin, a
peptidyl inhibitor of the enzyme. Decrease in total energy for leupeptin-cathepsin B has come
out be -127.7 which is almost equal to that of substrate BANA, -125.55. This is due to
complementary substrate of peptidyl nature in enzyme active site. Leupeptin binds more
effectively with the enzyme active site than any of the synthesized compounds (Table 3)
resulting in higher binding energy. Docking methods provide valuable information regarding the
binding mode between the ligand and the enzyme active site and can play an important role in
understanding the ligand-enzyme interactions. From molecular docking experiments, we
observed that all of compounds inhibit the enzyme in a competitive manner and because these
compete at the binding site of enzyme with substrate. Figures 5i and 5ii show the docked view of
most inhibitory compounds, 1i and 2k in the active site of cathepsin B, respectively. It is visible
that active site group Cys-29 and Gly-198 interact with these compounds as well as with the
substrate through H-bonds.
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 that though Leu-CH2Cl is specific
inhibitor for cathepsin H30, but possess only one amino acid residue as compared to leupeptin–
cathepsin B. Therefore, the leu-CH2Cl-cathepsin H interaction causes a decrease in energy of -
59.84. Table 3 presents the docking energies of the most potent inhibitory compound in each
series which show higher decrease in ligand-cathepsin H interaction energy than leu-CH2Cl-
8

cathepsin H. The docked view of most inhibitory compounds, 1e and 2c in the active site of
cathepsin H are presented in figures 5iii and 5iv. All these compounds interact with the amino
acyl acceptor site of the enzyme. Amino acids Gln-70 and Gln-78 interact with 1e and 2c. These
amino acids interact with the compounds through H-bonds as visualized by green coloured
ribbon of enzyme. However grey colored Asn-112 and pro-77 interact through vander waals
forces of interaction. The compounds can be visualized superimposed with the substrate
indicating competitive inhibition of cathepsin H by these compounds, which is in accordance
with the enzyme kinetic studies.
In cathepsin L, all the compounds showed a lesser interaction than the peptidyl inhibitor,
leupeptin. Decrease in total energy for leupeptin-cathepsin L has come out be -102.07. As
compared to this the binding energy of most inhibitor compounds of each series are less (Table
3). The docked view of most inhibitory compounds, 1i and 2k in the active site of cathepsin L
are presented in figures 5v and 5vi. From molecular docking experiments, we observed that all of
compounds inhibit the enzyme in a competitive manner and because these compete at the
binding site of enzyme with substrate. It is clearly observed that 1i and 2k interact with Gln-19,
Gly-68 and Gly-164 through hydrogen bonds. A portion of their binding region overlaps with
the interaction zone of Z-Phe-Arg-4mβNA suggesting a competitive inhibition that is also
observed by enzyme inhibition studies. Fig. 6 presents the correlation plot between Ki value and
the docking scores of the most potent inhibitor in each series.
The present study can be helpful in explaining the role of larger contact area between enzyme
and ligand in acquiring greater enzyme inhibition. The results are in consensus with the docking
data where similarly substituted N-subsitutedbenzylidenebenzohydrazide show a larger decrease
in total energy as compared to respective 2,5-diaryloxadiazole. In addition, greater inhibition by
N-subsitutedbenzylidenebenzohydrazides can also involve the direct interaction of active site
sulfhydryl group of enzymes with either of the nucleophilic carbonyl- or imine- centers present
therein which are lacking in cyclized 2,5-diaryloxadiazole derivatives. The study can add to
designing of existing non-peptidyl inhibitors that may contribute further to increase the potency
and specificity of cysteine protease inhibitors.
In the present work decrease in total energy of enzyme–ligand complex is presented as binding
affinity of ligand within the active site of enzyme. However, Zhang et al. have used free energy
based approach in understanding such interactions31.
9

3. Materials and methods
3.1. Materials
All the chemicals were of analytical grade. Fast Garnet GBC (o-aminoazotoluene diazonium salt,
α-N-benzoyl-D, L-arginine-2-naphthylamide (BANA), Z-Phe-Arg-4mβNA and Leu-βNA were
purchased from Bachem 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.
3.2. Methods
Melting points determined in open capillary tubes are thus uncorrected. All the chemicals and
solvents used were of laboratory grade. IR spectra (KBr, cm-1) were recorded on a Perkin-Elmer
1
spectrometer. H NMR spectra was recorded on Bruker 300 MHz NMR spectrometer (chemical
shifts in δ 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.
3.2.1. General Procedure for the synthesis of N-subsitutedbenzylidenebenzohydrazides
To the ethanolic solution of benzoic hydrazide (0.01mol) substituted benzaldehyde (0.011mol)
was added and the resulting solution was refluxed in the presence of catalytic amount of acetic
acid for 1-2 hours. The solvent was evaporated in air at room temperature. The solid thus
obtained was filtered and washed with cold ethanol to get desired product. The structure
elucidation of N-subsitutedbenzylidenebenzohydrazides was based on melting point and the
spectral data (IR, 1H-NMR and 13C-NMR).
3.2.2. General method for the synthesis of 2,5-diaryl-1,3,4-oxadiazole
To the stirred solution of N-substitutedbenzylidenebenzohydrazides (0.003mol) in
dichloromethane, iodobenzenediacetate (0.003mol) was added. The contents were stirred for 2 h
and the progress of the reaction was monitored by TLC. The solvent was removed and the
residue was taken in petroleum ether and stirred for 30 min. The solid thus obtained was filtered,
washed with petroleum ether and dried to afford desired product (scheme 1). The structure
elucidation of 2,5 -diphenyl- 1,3,4-oxadiazoles were based on melting point and the spectral data
(IR, 1H-NMR and 13C-NMR) which has been provided in the supplementary data.
10

3.3. Enzymatic studies
3.3.1. 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 0.1M acetate buffer pH 5.5 containing 0.2M NaCl
in a mixer-cum-blender to obtain 10% (w/v) homogenate and was stored at 4oC.
3.3.2. Assay for proteolytic activity
The proteolytic activity was estimated at pH 5.0, 37οC using 0.1 M acetate buffer as the
incubation medium. The homogenate prepared above was incubated with the buffer at 37oC for 3
h and 24 h. (Figure 1), separately. 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 supernatant using Bradford method35. The experiments were conducted in
triplicate and the results are presented in Table 1.
3.3.3. Purification of cathepsin Β, cathepsin H and cathepsin L:
All the purification steps were carried out at 4°C. Cathepsin B, H and L were isolated, separated
and purified from goat liver using the following procedure36. Goat liver acetone powder
homogenisation in cold 0.1 M sodium acetate buffer pH 5.5 containing 0.2 M NaCl and 1mM
EDTA, Acid-autolysis by lowering pH from 5·5 to 4·0 by gradual addition of cold 1 Ν HCl, 30-
80% ammonium sulphate fractionation. Fractionation of proteases based on molecular weight on
Sephadex G-100 column chromatography and finally cation-exchange chromatography on CM-
Sephadex C-50 and DEAE Sephadex A-50 column. The specific activities of the cathepsin B,
cathepsin H and cathepsin L were ~11.15 nmoles/min/mg, ~22.91 nmoles/min/mg and ~16.78
nmoles/min/mg, respectively.
Enzyme assays
The purified cathepsin B, H and L were first activated in presence of thiol activators at pH 6.0,
pH 7.0 and pH 6.0, respectively. Activated enzyme solution (100µl) was equilibrated with 0.1M
phosphate buffer at respective pH containing 1mM EDTA separately for 10 min at 37oC.
Cathepsins B, H and L activities were then analyzed using BANA, leu-βNA and Z-Phe-Arg-
4mβNA as substrates, respectively. The released β-naphthylamine was measured colorimetrically
at 620nm by the use of fast garnet GBC dye using established procedures8,36. The experiments
were performed in triplicates.
11

3.4. Pharmacology
3.4.1. Enzyme inhibition studies
Enzyme inhibition studies were carried out in the similar manner as detailed above. To the pre-
activated enzyme 20µl of stock solution (5mM prepared in DMSO) of different compounds
under study was added separately to the activated enzyme assay mixtures to effect final
concentrations as 10-4 M in 1 ml assay. After 30 min, 25µl of 100mM substrate stock solution
was added to start the reaction. In control experiments, equivalent amount of DMSO was added
and percent residual activities were calculated with reference to control (Table 1).
The compounds exhibiting inhibition at 1×10-4M concentration (Table 1) were further studied for
their inhibitory effect at their varying concentrations by adding appropriate amount of individual
compounds in the reaction mixture separately to effect the final concentration of N-
subsitutedbenzylidenebenzohydrazides as 0.1×10-5M, 0.2×10-5M, 0.4×10-5M, 0.6×10-5M,
0.8×10-5M, 1.0×10-5M for cathepsin B; 0.1×10-6M, 0.2×10-6M, 0.4×10-6M, 0.6×10-6M, 0.8×10-
6M, 1.0×10-6M for cathepsin L and 0.1×10-4M, 0.2×10-4M, 0.4×10-4M, 0.6×10-4M, 0.8×10-4M,
1.0×10-4M for cathepsin H and 2,5-diaryloxadiazoles as 0.1×10-4M, 0.2×10-4M, 0.4×10-4M,
0.6×10-4M, 0.8×10-4M, 1.0×10-4M for cathepsins B & H and 0.1×10-5M, 0.2×10-5M, 0.4×10-5M,
0.6×10-5M, 0.8×10-5M, 1.0×10-5M for cathepsin L (Figures 2-4(i-ii)).
3.4.2. Enzyme kinetic studies
After establishing the inhibitory action of synthesized compounds on cathepsins B, H and L,
experiments were designed to evaluate the type of inhibition and to determine their K values. For
i
that, enzyme activity was evaluated at six different substrate concentrations in presence 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 2-4(iii-iv). The values
represent Mean±S.M.D of at least three individual experiments. The Ki values of compounds
were calculated using the Line weaver-Burk equation Km’=Km(1+[I]/Ki) for competitive
inhibition (Table 2).
3.4.3. Molecular Docking Studies
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, cathepsin H
and cathepsin L were retrieved from Protein Data Bank as cav2IPP B_PYS.pdb32, and cav8PCH
12

H_NAG.pdb33 and cav3BC3L_CSW34, respectively. 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 3 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, H and L along with their respective
substrates and most inhibitory compounds of each series are shown in figure 5.
3.5. Conclusion
We have evaluated structurally related two series of 22 compounds for their inhibitory activity
against cathepsin B, H and L and have identified some synthetic non-peptidyl inhibitors for
cathepsin B, H and L. The compounds inhibited the enzyme cathepsin L to maximum extent
showing Ki value of the order of nM followed by cathepsin B and were least inhibitory to
cathepsin H. In general, the inhibitory potency of compounds was related to the electro-
negativity property of substituent. Through enzyme kinetics, it was revealed that all compounds
inhibit cathepsin B, H and L in a competitive manner. The in-vitro results presented in this study
have been compared with in-silico studies.
The authors have declared no conflicts of interest.
Acknowledgements
One of the authors, Shweta Garg is thankful to UGC New Delhi, India for award of SRF and also
to Kurukshetra University, Kurukshetra for providing necessary research laboratory facilities.
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16

Figure 1: Effect of Substituted N-subsitutedbenzylidenesbenzohydrazides and 2,5-
diaryloxadiazoles on the endogenous proteolytic activity for 3 hr and 24 hr reaction. The
data in each bar represents the % Residual Activity in presence of individual compound
w.r.t. control taken as 100.
Figure 2: Effect of varying concentration of most inhibitory compounds from each series,
on cathepsin B activity, figure 2(i-ii), respectively. Results are mean of experiments
conducted in triplicate. % Residual activities are presented w.r.t control which contain
equivalent amount of solvent.
Line - weaver Burk plot for cathepsin B at varying concentrations of BANA in presence of
1x10-6M and 1x10-5M concentration of N-subsitutedbenzylidenebenzohydrazides and 2,5-
diaryl-1,3,4-oxadiazoles at pH 6.0 figure 2(iii-iv), respectively. The Km value for control
have been found to be 3.64x10-4 M. The Ki values as calculated from this graph are
presented in table 2
Figure 3: Effect of varying concentration of compounds from each series, on cathepsin H
activity, figure 3(i-ii), respectively. Results are mean of experiments conducted in triplicate.
% Residual activities are presented w.r.t control which contain equivalent amount of
solvent.
Line - weaver Burk plot for cathepsin H at varying concentrations of leu-ßNA in presence
of 1x10-5M concentration of N-subsitutedbenzylidenebenzohydrazides and 2,5-diaryl-1,3,4-
oxadiazoles at pH 7.0 figure 3(iii-iv),, respectively. The Km value for control has been found
to be 5.34x10-4M. The Ki values as calculated from this graph are presented in table 2.
Figure 4: Effect of varying concentration of compounds from each series, on cathepsin L
activity, figure 4(i-ii), respectively. Results are mean of experiments conducted in triplicate.
% Residual activities are presented w.r.t control which contain equivalent amount of
solvent.
Line - weaver Burk plot for cathepsin L at varying concentrations of z-Phe-Arg-4mβNA in
presence
of
1x10-6M
and
1x10-5M
concentration
of
N-
subsitutedbenzylidenebenzohydrazides and 2,5-diaryl-1,3,4-oxadiazoles at pH 6.0,figure
4(iii-iv), respectively. The Km value for control has been found to be 6.024x10-5 M. The Ki
values as calculated from this graph are presented in table 2.
Figure 5: Docking results showing the alignment of most inhibitory compounds along with
the substrates. The circles drawn show overlapping of substrate as well as the compound.
Here fig. 5(i-ii) show alignment of 1i and 2k, along with BANA in the active site of
cathepsin B (cav2IPP B_PYS.pdb32 ), respectively. Fig. 5(iii-iv) show alignment of 1e and
2c, along with LeußNA in the active site of cathepsin H (cav8PCH H_NAG.pdb33),
17

respectively. Fig. 5(v-vi) show alignment of 1i and 2k, along with Z-Phe-Arg-4mβNA in the
active site of cathepsin L (cav3BC3L_CSW.pdb34), respectively.
Figure 6: Correlation plot between binding energies and log Ki values of most inhibitory
compound in each series for cathepsin B, cathepsin H and cathepsin L.
18

3 hours reaction time
24 hours reaction time
120
100
80
60
40
20
0
Figure 1
19

Figure 2
20

Figure 3
21

Figure 4
22

(i)
(ii)
(iv)
(iii)
(v)
(vi)
Figure 5
23

log Ki
0
-20
-9
-7
-5
-3
-1
1i,2k(cathepsin B)
1e,2c(cathepsin H)
1i,2k(cathepsin L)
-40
-60
-80
-100
-120
Figure 6
N-subsitutedbenzylidenebenzohydrazides and their cyclized derivatives
1,2
a
R
H
O
O
NH2
OH
N
H
b
c
d
e
f
p- CH3
o-Cl
NH2NH2.2H2O
O
R
m-Cl
p-Cl
N
N
O
o-OCH3
m-OCH3
p- OCH3
O-NO2
m-NO2
p-NO2
R
N
O
IBD
N
g
h
i
R
H
dichloromethane
(2)
(1)
j
Scheme 1
k
24

Table-1: Enzyme inhibition studies in presence of Substituted N-subsitutedbenzylidenebenzohydrazides and their derivatives
(Protease Activity)
Enzyme inhibition studies
Cathepsin L
% residual activity at 10-4M
Concentration of
Cathepsin B
Cathepsin H
Compound
code
S.No.
% residual activity at
(Z)x10-6M
% residual activity
at (Z)x10-5M
Concentration of
compounds
% residual activity
at (Z)x10-5M
Concentration of
compounds
compounds
Concentration of
compounds
3 hours
incubation
100
24 hours
incubation
100
Control
1a
100
100
100
1.
2.
50.33±5.04 69.32±4.63
47.25±4.69(0.1)
69.90±5.30
44.05±5.23(0.1)
1b
89.60±8.06 99.81±8.38
46.57±4.33 64.57±4.59
47.52±4.68 72.99±5.46
44.44±3.83 60.37±4.09
54.70±5.22 77.05±5.95
68.35±6.88 79.11±6.24
50.85±4.75 72.34±4.54
13.52±1.21 36.95±1.85
70.86±6.73(0.1)
46.12±4.53(0.1)
47.10±4.67(0.1)
42.55±4.20(0.1)
49.70±5.42(0.1)
56.74±5.64(0.1)
48.59±4.88(0.1)
23.89±1.10(0.1)
33.28±3.13(0.1)
28.39±2.74(0.1)
39.93±3.59
25.66±2.53
39.45±3.84
24.75±2.43
41.11±4.04
49.59±4.44
40.2±3.99
61.40±7.59(0.1)
39.11±3.77(0.1)
41.77±4.02(0.1)
36.67±3.62(0.1)
41.24±3.93(0.1)
56.06±5.57(0.1)
46.94±4.68(0.1)
22.07±2.54(0.1)
32.74±2.86(0.1)
27.60±2.79(0.1)
3.
4.
1c
1d
1e
1f
5.
6.
7.
1g
1h
1i
8.
9.
90.07±8.95
73.01±7.25
75.07±7.30
10.
11.
1j
24.11±2.32
48.15±2.93
1k
23.38±1.91 41.74±2.32
25

12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
2a
51.94±4.18
62.53±4.56
48.68±4.85
50.19±5.02
47.13±4.67
2b
68.35±6.15 99.02±8.11
42.25±4.21 56.39±3.92
41.09±4.07 51.37±3.42
46.99±4.66 58.03±4.11
67.57±6.71 77.47±5.99
57.16±5.69 69.63±5.27
70.90±7.07 82.70±6.48
24.14±2.41 39.4±2.24
31.13±3.12 46.70±2.99
78.04±7.59
43.75±3.87
40.60±3.24
45.25±4.51
57.32±5.71
54.03±5.40
66.77±6.67
37.71±2.07
39.68±2.71
49.89±4.97
25.12±2.51
36.19±3.61
30.98±3.09
45.35±4.49
48.08±4.79
43.49±4.34
92.07±9.20
59.47±5.94
76.46±7.61
41.40±4.21
40.20±3.04
42.08±4.21
55.92±5.48
52.09±5.17
65.02±6.48
34.30±0.22
37.10±2.73
2c
2d
2e
2f
2g
2h
2i
2j
2k
14.44±1.42 32.95±1.62
33.30±1.29
1.5±0.10(0.1)
90±8.90 (1)
69.33±6.92
50.11±5.01(1)
8.11±5.01(1)
28.30±1.29
90.98±0.88 (0.001)
6.01±0.60 (10)
leupeptin
Leu-CH2-Cl
--
--
--
--
The TCA soluble peptides were estimated at 630 nm using Bradford method[31]. The results are presented as % Residual Activity
w.r.t. control taken as 100% where no compound was added but an eqvivalent amount of solvent was present. Where no
compound was added but an equivalent amount of solvent was present. Cathepsin B, Cathepsin H and Cathepsin L activities were
calculated using BANA, Leu-βNA and Z-Phe-Arg-4mβNA as substrates at pH 6.0, 7.0 and 6.0, 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 nanomoles/min/mg, ~22.91 nanomoles/min/mg and 16.78 nanomoles/min/mg,
respectively.
26

Table-2: Ki values exerted by N-subsitutedbenzylidenebenzohydrazides and their
derivatives on Cathepsin B, Cathepsin H and Cathepsin L
S.
No.
1.
Code of
compound
Cathepsin B
Ki (10-7M)
2.90
Cathepsin H
Ki (10-7M)
21.40
12.60
6.80
Cathepsin L
Ki (10-9M)
13.00
95.90
7.30
1a
1b
1c
1d
1e
1f
2.
87.20
2.10
3.
4.
2.60
11.30
4.40
8.63
5.
1.90
6.34
6.
8.30
14.00
16.80
13.30
48.40
36.40
41.60
27.20
25.50
5.60
8.14
7.
1g
1h
1i
1.80
28.60
19.76
4.20
8.
4.40
9.
1.13
10.
11.
12.
13.
14.
15.
16
17
18
19
20
21
22
1j
1.40
5.86
1k
2a
2b
2c
2d
2e
2f
1.20
5.53
26.90
113.00
16.17
12.81
19.21
68.50
44.40
92.20
7.90
124.00
1357.0
92.10
70.20
99.30
216.00
144.00
337.00
57.00
63.40
47.31
11.30
10.00
14.70
16.10
13.30
66.90
38.50
49.60
2g
2h
2i
2j
10.00
6.64
2k
The data present the Ki values calculated using Lineweaver – Burk equation for competitive
inhibition evidenced through Lineweaver – Burk plots drawn between 1/V and 1/S. The
experiments were conducted using cathepsin B, H and L preparation having specific activities
11.15, 22.91 and 16.78 nmoles/min/mg, respectively.
27

Table-3: Docking energies of Cathepsins B, H and L in presence of most inhibitory compounds
and their analogues
Compound
Total Energy (kcalmol-1)
CATHEPSIN B
VDW
H Bond
Electronic
BANA
Leupeptin
1i
-125.56
-127.72
-100.84
-85.12
-89.68
-96.85
-69.33
-67.02
-32.21
-30.87
-32.76
-18.35
-3.67
0
1.25
0.25
2k
CATHEPSIN H
leuCH2Cl
LeuβNA
1e
-59.84
-76.89
-78.67
-78.07
-43.88
-67.30
-65.27
-64.07
-15.96
-9.60
-13.40
-14.00
0
0
0
0
2c
CATHEPSIN L
Z-Phe-Arg-4mβNA
-127.24
-102.07
-92.53
-87.75
-94.83
-34.83
-79.54
-70.44
-28.90
-65.06
-13.00
-17.71
-3.50
-2.17
0
Leupeptin
1i
2k
0
The results are one of the docking experiments run using iGemdock under drug screening
settings. The active sites were retrieved from Protein Data Bank; cathepsin B as cav2IPP
B_PYS.pdb[29], cathepsin H as cav8PCH H_NAG.pdb[30] and cathepsin L as
cav3BC3L_CSW[31] and the ligands were loaded as MDL mol files.
28

Graphical Abstract
N-subsitutedbenzylidenebenzohydrazides and their cyclized derivatives i.e, 2,5-diaryl-1,3,4-
oxadiazole are reported as novel inhibitors of cathepsin B, cathepsin H and cathepsin L with
potency in nanomolar range.
Cathepsins(Cath)
N
Goat liver
N
O
IBD
R
N
O
N
H
R
N-subsitutedbenzylidenebenzohydrzides
(1)
2,5-diaryl-1,3,4-oxadiazole
(2)
cyclization
InhibitionStudies
CathB
CathH
CathL
-9
1i(4.2x10 M)
Potent
inhibitor
Compound
-7
-8
1e(4.4x10 M )
1i(11.38x10 M)
2k(66.4x10 M)
-9
2k(47.31x10 M)
-7
-8
2c(5.6x10 M)
(with K
i
values)
29