N-formylpyrazolines and N-benzoylpyrazolines as novel inhibitors of mammalian cathepsin B and cathepsin H

Article abstract of DOI:10.1016/j.bioorg.2014.07.012

Cathepsins, intracellular proteases, are known to be involved in a number of physiological processes ranging from degradation of extracellular proteins, prohormone processing, progressions of atherosclerosis, etc. High levels of cathepsins have been indicated in various pathological conditions like arthritis, cancer and other tissue degenerative disorders. One of the reasons attributed to these high levels is decrease in inhibitor concentration. Therefore, the work on the identification of small molecular weight compounds as inhibitors of cysteine proteases is of great therapeutic significance. Exploring this work in the same direction, we here present the synthesis of substituted N-formylpyrazolines and N-benzoylpyrazolines and study these as inhibitors to cysteine proteases. After a preliminary screening of the compounds as inhibitors to cysteine proteases in general, studies were carried out to study their inhibitory effects on cathepsin B and cathepsin H. SAR studies show that N-formylpyrazolines were better inhibitors than N-benzoylpyrazolines. The most potent inhibitors among the two series were nitro substituted compounds 1i and 2i with K_ivalues of ～1.1 × 10^-9M and 19.5 × 10^-8M for cathepsin B and K_ivalues of ～5.19 × 10^-8M and 9.8 × 10^-7M for cathepsin H, respectively. Docking experiments showing interaction between N-formylpyrazolines and N-benzoylpyrazolines with enzyme active sites structures also provided useful insights.

Full text of DOI:10.1016/j.bioorg.2014.07.012

Bioorganic Chemistry 57 (2014) 43–50
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
N-formylpyrazolines and N-benzoylpyrazolines as novel inhibitors of
mammalian cathepsin B and cathepsin H
⇑
N. Raghav , S. Garg
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:
Received 10 March 2014
Available online 19 August 2014
Cathepsins, intracellular proteases, are known to be involved in a number of physiological processes
ranging from degradation of extracellular proteins, prohormone processing, progressions of atherosclero-
sis, etc. High levels of cathepsins have been indicated in various pathological conditions like arthritis, can-
cer and other tissue degenerative disorders. One of the reasons attributed to these high levels is decrease
in inhibitor concentration. Therefore, the work on the identification of small molecular weight com-
pounds as inhibitors of cysteine proteases is of great therapeutic significance. Exploring this work in
the same direction, we here present the synthesis of substituted N-formylpyrazolines and N-benzoylpy-
razolines and study these as inhibitors to cysteine proteases. After a preliminary screening of the com-
pounds as inhibitors to cysteine proteases in general, studies were carried out to study their inhibitory
effects on cathepsin B and cathepsin H. SAR studies show that N-formylpyrazolines were better inhibitors
than N-benzoylpyrazolines. The most potent inhibitors among the two series were nitro substituted com-
pounds 1i and 2i with Ki values of ꢀ1.1 ꢁ 10ꢂ9 M and 19.5 ꢁ 10ꢂ8 M for cathepsin B and Ki values of
ꢀ5.19 ꢁ 10ꢂ8 M and 9.8 ꢁ 10ꢂ7 M for cathepsin H, respectively. Docking experiments showing interac-
tion between N-formylpyrazolines and N-benzoylpyrazolines with enzyme active sites structures also
provided useful insights.
Keywords:
N-formylpyrazolines
N-benzoylpyrazolines
Cathepsin B & cathepsin H inhibitors
Endogenous proteolysis
Ó 2014 Published by Elsevier Inc.
1. Introduction
adiazoles [22] (VI) and epoxide [23] (VII) have also been reported
as (peptidyl and non peptidyl) inhibitors of cathepsin B are shown
Cysteine proteases are the center of attention in synthetic pro-
tease inhibitor development for chemotherapy of a number of dis-
eases. It has been revealed through literature studies that
cathepsin B is by far the most abundantly expressed mammalian
cathepsin. Increased expression of cathepsin B levels in patients
with heart failure suggests its role in the genesis and development
of cardiac disease [1]. Furthermore, cathepsin B is negatively corre-
lated with other pathological conditions like pancreatitis [2,3]
osteoarthritis [4], gastric cancer [5], oral cancer metastasis [6],
colorectal cancer [7] and ovarian cancer [8,9]. Cathepsin H, an ami-
nopeptidase, is also associated with various pathological condi-
tions like human fibrous meningioma [10], colorectal cancer [11],
arthritis [12], human prostate tumor [13] and lung cancer [14].
in Fig. 1.
Peptidyl inhibitors are not considered to be viable drug candi-
dates for treating diseases like cancer, apoptosis, etc. because of
gastric instability or the possibility of immunogenic reactions.
Keeping in view the involvement of cathepsins B and H in different
inflammatory and cancerous conditions research on non-peptidyl
drugs has become an important aspect in drug research and devel-
opment [24,25].
Low molecular weight compounds with different functionalities
have also been identified as cysteine protease inhibitors and their
potential use as anti parasitic agents [26–29] has been reported.
Pyrazole nucleus having two aryl substituent increased in vitro
cytotoxic activity against human cancer cell lines [30]. Pyrazolines
have also been reported to possess antibacterial [31], antifungal
[32], antiviral [33], antiparasitic [34], antitubercular [35], insecti-
cidal agents [36], antipyretic [37], diuretic [38], antidiabetic [39],
tranquillizing [40], muscle relaxant [41], psychoanaleptic [42],
anticonvulsant [43], antihypertensive [44], antidepressant [45]
and anticancer [46] activities. They have also been found to be
Nitric oxide synthase (NOS) inhibitors and had shown Cannabinoid
CB1 receptor antagonist activity [47].
Literature survey suggests that
a large work has been
accomplished on peptides or peptidyl analogues as inhibitors to
cysteine proteases [15,16]. Various compounds such as flavones
[17] (I), aldehyde [18] (II), acyloxymethyl ketone [19] (III),
cyclopropenones [20] (IV) and nitriles [21] (V) as peptidyl or thi-
⇑
Corresponding author.
E-mail address: nraghav.chem@gmail.com (N. Raghav).
http://dx.doi.org/10.1016/j.bioorg.2014.07.012
0045-2068/Ó 2014 Published by Elsevier Inc.

44
N. Raghav, S. Garg / Bioorganic Chemistry 57 (2014) 43–50
absolute ethanol (50 mL). The solution was kept for cooling at
room temperature, and the solid formed was filtered off, washed
with water, dried and recrystallized from ethanol.
2.2.2. General method for the synthesis of N-benzoylpyrazolines [59]
Equimolar mixture of chalcone and benzoic hydrazide was
refluxed for 6 h in absolute ethanol in the presence of a few drops
of acetic acid as catalyst. The solution was kept for cooling at room
temperature, and the solid formed was filtered off, washed with
water, dried and recrystallized from ethanol.
The characterization of all the compounds has been done on the
basis of literature melting points, IR and 1H NMR spectral data
which has been provided in the Supplementary data.
Fig. 1. Effect of substituted N-formylpyrazolines and N-benzoylpyrazolines on the
endogenous proteolytic activity for 3 h and 24 h reaction. The data in each bar
represents the % residual activity in presence of individual compound w.r.t. control
taken as 100.
2.3. Proteolytic studies
2.3.1. 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 0.1 M acetate buffer pH 5.5 containing 0.2 M NaCl
in a mixer-cum-blender to obtain 10% (w/v) homogenate and was
stored at 4 °C.
We have previously reported the effect of some semicarba-
zones, thiasemicarbazones, pyrazoles and pyrazolines as inhibitors
of endogenous proteolytic activities [48–53]. Recently we have
reported bischalcones and their derivatives [54]; acylhydrzides
[55]; o-hydroxychalcones and their cyclized derivatives [56] as
novel inhibitors of cathepsins B and H. Evaluation of pyrazolines
as anti-inflammatory and anticancer agents and role of cathepsins
B and H in these diseased conditions emphasize the importance of
study of pyrazoline derivatives as inhibitors [57] of these enzymes.
Toward this endeavor, in the present study we report the synthesis
and screening of N-formyl and N-benzoylpyrazolines as cathepsins
B and H inhibitors not reported earlier. The present study may pro-
vide new therapeutic opportunities in cancer treatment. The
results are further compared with in silico studies which support
the postulation that synthesized compounds may act as enzyme
inhibitors.
2.3.2. Assay for proteolytic activity
The proteolytic activity was estimated at pH 5.0, 37 °C using
0.1 M acetate buffer as the incubation medium. The homogenate
prepared above was incubated with the buffer at 37 °C for 3 h
and 24 h, separately. The reaction was stopped by the addition of
TCA and the resulting solution was centrifuged to precipitate pro-
teins. The acid soluble proteins were quantitated in the superna-
tant using Bradford method [60]. The experiments were
conducted in triplicate and the results are presented in Table 1
(Fig. 1).
2.3.3. Purification of goat liver cathepsin B and cathepsin H
2. Materials and methods
All the purification steps were carried out at 4 °C. Cathepsin B
and H were isolated, separated and purified as reported previously
[54] including the steps of acetone powder preparation, homogeni-
zation, acid-autolysis, 30–80% ammonium sulfate fractionation,
molecular sieve chromatography on Sephadex G-100 column chro-
matography and finally ion-exchange chromatographies on CM-
Sephadex C-50 and DEAE Sephadex A-50 column. The enzymes
were sufficiently pure to carry out enzyme assays in order to see
the inhibitory effects of synthesized compounds with the help of
specific synthetic substrates. The purity of enzymes after the above
mentioned procedure has been checked by electrophoresis to a sin-
gle band homogeneity and has been reported [61,62]. The specific
activities of the cathepsin B and cathepsin H obtained were ꢀ11.15
and ꢀ22.91 nmol/min/mg, respectively.
All the chemicals (analytical grade) and biochemicals Fast Gar-
net GBC (o-aminoazotoluene diazonium salt, substrate a-N-ben-
zoyl-D,L-arginine-2-naphthylamide (BANA) and Leu-ßNA were
purchased either from Sigma Chemical Co., USA or from Bachem
Feinchemikalien AG, Switzerland. The protein sample was concen-
trated using Amicon stirred cells with YM 10 membrane under
nitrogen pressure of 4–5 psi. The source of enzyme, fresh goat liver,
was obtained from local slaughter house.
2.1. General procedure
Melting points were determined in open capillary tubes and are
thus uncorrected. All the chemicals and solvents used were of lab-
oratory grade. IR spectra (KBr, cmꢂ1) 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. Thin layer chromatography on alumi-
num 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) were used to monitor progress of
reaction. ELISA plate reader was used for measuring absorbance
in the visible range. Refrigerated ultracentrifuge Remi C-24BL
was used for centrifugation purpose.
2.3.4. Effect of synthesized compounds on the activity of cathepsin B
and cathepsin H
The activities of cathepsin B were estimated at varying concen-
trations of synthesized compounds (Fig. 2a and b), separately. First
of all, enzyme was equilibrated in 0.1 M phosphate buffer, pH 6.0
at 37 °C. The stock solutions of compounds were prepared in
DMSO. Appropriate amount of individual compounds at different
concentrations were added in the reaction mixture separately.
After incubation time of 30 min. residual enzyme activity was esti-
mated by the usual enzyme assay [54] at pH 6.0 using a-N-ben-
zoyl-D,L-arginine-2-naphthylamide (BANA) as substrate. The
2.2. Synthesis of compounds
experiments were performed in triplicate for each concentration
and averaged before further calculations. The % activity in each
case has been calculated with respect to the control where no com-
pound was added but an equivalent amount of solvent was pres-
ent. The results are presented in Table 1. Similar experiments
2.2.1. General method for the synthesis of N-formylpyrazolines [58]
A
mixture of chalcone (0.01 moles), hydrazine hydrate
(0.02 moles) and formic acid (0.01 moles) was refluxed for 6 h in

N. Raghav, S. Garg / Bioorganic Chemistry 57 (2014) 43–50
45
Table 1
Enzyme inhibition studies in presence of substituted N-formylpyrazolines and N-benzoylpyrazolines.
S. no.
Compound name
(Protease activity)
Enzyme inhibition studies
Cathepsin B
% Residual activity at
10ꢂ4 M concentration of
compounds
Cathepsin H
3 h
24 h
% Residual
Ki (10ꢂ8 M)
% Residual
activity at
Ki (10ꢂ8 M)
incubation incubation activity at
(Z) ꢁ 10ꢂ6
M
(Z) ꢁ 10ꢂ5
M
concentration of
compounds
concentration of
compounds
Control
100
100
100
100
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
N-formyl-3,5-diphenylpyrazoline (1a)
67.2 5.9 69.20 6.2
34.43 3.2 37.75 3.4
32.40 3.2 35.10 3.4 34.610 0.3 (0.1)
52.29 5.8 61.77 6.0
89.11 7.7 89.87 8.9
81.92 6.7 82.00 7.7
76.20 7.5 77.20 7.6
22.17 1.8 22.24 2.1
67.21 6.6 (0.1)
57.12 3.5 (0.1)
2.27 0.21
1.80 0.17
1.46 0.14
2.11 0.20
6.68 0.66
4.55 0.45
4.48 0.44
1.41 0.14
0.11 0.01
1.13 0.11
76.78 6.9 (0.1)
61.38 4.9 (0.1)
49.57 3.0 (0.1)
69.38 6.1 (0.1)
92.97 8.6 (0.1)
86.03 8.0 (0.1)
80.06 7.6 (0.1)
30.20 2.7 (0.1)
14.15 1.0 (0.1)
27.64 1.4 (1.0)
66.19 6.6 (1.0)
55.13 4.4 (1.0)
44.20 3.5 (1.0)
62.47 5.5 (1.0)
79.37 7.6 (1.0)
17.4 1.74
13.5 1.35
13.0 1.30
13.7 1.32
38.7 3.86
27.2 2.70
22.1 2.20
9.63 0.96
5.19 0.51
5.80 0.58
320 3.20
189 0.18
181 1.80
301 3.00
965 9.61
503 50.1
410 41.1
144 1.42
98 9.80
N-formyl-5-(-20-chlorophenyl)-3-phenylpyrazoline (1b)
N-formyl-5-(-30-chlorophenyl)-3-phenylpyrazoline (1c)
N-formyl-5-(-40-chlorophenyl)-3-phenylpyrazoline (1d)
N-formyl-5-(-20-methoxyphenyl)-3-phenylpyrazoline (1e)
N-formyl-5-(-30-methoxyphenyl)-3-phenylpyrazoline (1f)
N-formyl-5-(-40-methoxyphenyl)-3-phenylpyrazoline (1g)
N-formyl-5-(-20-nitrophenyl)-3-phenylpyrazoline (1h)
N-formyl-5-(-30-nitrophenyl)-3-phenylpyrazoline (1i)
N-formyl-5-(-40-nitrophenyl)-3-phenylpyrazoline (1j)
N-benzoyl-3,5-diphenylpyrazoline (2a)
66.25 5.7 (0.1)
91.82 0.9 (0.1)
89.76 8.3 (0.1)
82.87 0.7 (0.1)
28.89 0.2 (0.1)
6.000 0.5 6.280 0.6 10.410 0.9 (0.1)
8.140 0.7 8.20 0.7 23.47 1.1 (1.0)
33.10 3.2 36.20 3.6
51.19 4.1 53.10 5.0
75.51 6.5 76.01 3.3
65.19 5.2 66.20 3.2
90.21 8.9 91.21 7.6
80.20 7.5 81.10 7.4
40.17 3.3 (1.0) 115.2 11.0
N-benzoyl-5-(-20-chloro phenyl)-3-phenylpyrazoline (2b)
N-benzoyl-5-(-30-chloro phenyl)-3-phenylpyrazoline (2c)
N-benzoyl-5-(-40-chloro phenyl)-3-phenylpyrazoline (2d)
N-benzoyl-5-(-20-methoxyphenyl)-3-phenylpyrazoline (2e)
N-benzoyl-5-(-30-methoxy phenyl)-3-phenylpyrazoline (2f)
29.28 2.8 (1.0)
27.51 2.7 (1.0)
32.54 3.0 (1.0)
82.66 5.9 (1.0) 490.1 49.0
58.17 5.2 (1.0) 159.4 15.90 76.06 7.0 (1.0)
51.85 4.0 (1.0) 127.7 12.77 69.69 6.7 (1.0)
26.46 2.1 (1.0)
20.77 1.8 (1.0)
21.46 2.1 (1.0)
77.5 7.75
67.7 6.76
92.5 9.25
N-benzoyl-5-(-40-methoxy phenyl)-3-phenylpyrazoline (2g) 82.59 8.0 88.20 7.5
N-benzoyl-5-(-20-nitrophenyl)-3-phenylpyrazoline (2h)
N-benzoyl-5-(-30-nitrophenyl)-3-phenylpyrazoline (2i)
N-benzoyl-5-(-40-nitrophenyl)-3-phenylpyrazoline (2j)
28.34 2.2 31.08 1.8
7.90 0.8 8.000 0.5
57.2 5.72
19.5 1.95
28.7 2.87
34.77 2.9 (1.0)
27.49 2.7 (1.0)
28.77 2.6 (1.0)
8.20 0.8
8.27 0.7
127 12.65
The TCA soluble peptides were estimated at 630 nm using Bradford method [48] and the results are the mean and S.M.D. of the experiment conducted in triplicate and is
calculated as % residual protease activity in 0.1% liver homogenate w.r.t. control where no compound was added but an equivalent amount of solvent was present. Cathepsin B
and cathepsin H activities were calculated using BANA and Leu-bNA as substrates at pH 6.0 and 7.0, respectively. The enzyme activity was determined at minimum inhibitory
concentration of each compound given in parenthesis. The specific activity of the cathepsin B and cathepsin H were ꢀ11.15 nmol/min/mg and ꢀ22.91 nmol/min/mg,
respectively. In order to determine Ki values, experiments were conducted in triplicates in presence and absence of a fixed concentration of different compound, separately.
The results were then plotted between 1/V and 1/S to obtain Line-weaver Burk plots and all the compounds were established as competitive inhibitors. The Ki values were
calculated using Line-weaver Burk equations for competitive inhibition.
were designed to evaluate the effect of varying concentrations of
synthesized compounds separately on cathepsin H using LeußNA
as substrate [54] at pH 7.0 (Fig. 2c and d).
drug screening setting with population size 200, generation 70 and
no of solutions 3. The results presented in Table 2 pertain to the
interaction data. Fitness is the total energy in kcal/mol of a pre-
dicted pose in the binding site. The empirical scoring function of
iGemdock is the sum total of Van der Waal, H-bonding and electro-
static energy. The best docked pose of the substrates in the active
site of cathepsins B and H along with the most inhibitory or the ref-
erence compounds are shown in Fig. 4.
2.3.5. Kinetic studies of synthesized compounds on cathepsin B and
cathepsin H
After establishing the inhibitory action of synthesized com-
pounds on cathepsin B, experiments were designed to evaluate
the type of inhibition and to determine the Ki value of these com-
pounds on respective enzymes. For that, enzyme activities were
evaluated at different substrate concentrations in presence and
absence of a fixed concentration of inhibitor. The enzyme concentra-
tion was kept constant in all the experiments. Line-weaver Burk plot
were drawn between 1/S and 1/V in presence and absence of differ-
ent series of compounds on cathepsin B (Fig. 3a and b) and cathepsin
H (Fig. 3c and d). And the Ki values were calculated using the Line-
3. Result and discussion
All the synthesized substituted N-formylpyrazolines and substi-
tuted N-benzoylpyrazolines (Scheme 1) show a characteristic IR
absorption peak. The structure elucidation of compounds was based
on the spectral data (IR & 1H NMR). The IR spectra showed mainly
stretching bands at 1610–1580, 1600–1450 and 2950–2800 cmꢂ1
assigned to (C@N), aromatic (C@C) and (CAH) functionalities,
respectively. In the 1H NMR spectrum, an ABX pattern was observa-
ble; Ha, Hb and Hx appear as double doublets at d 3.10–3.30, 3.75–
3.80 and 5.40–5.50 ppm with (Jab = 16.2–18.0 Hz, Jax = 4.5–7.2 Hz,
Jbx = 10.8–12.3 Hz). The protons of the aromatic ring were observed
at 7.03–7.76 ppm in case of N-benzoylpyrazolines and the formyl
0
weaver Burk equation for competitive inhibition Km = Km(1 + I/Ki).
2.4. Drug modeling 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 [63] and cathep-
sin H [64] were retrieved from Protein Data Bank as (cav2IPP
B_PYS.pdb), and (cav8PCH H_NAG.pdb), respectively. The active
site box was selected as such provided in the pdb file of 8 Å radius
excluding the reference ligands. 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
proton appeared as
benzoylpyrazolines.
a singlet at 8.90 ppm in case of N-
3.1. Effect of synthesized compounds on in vitro endogenous
proteolysis in liver homogenate
Table 1 presents the results of proteolytic studies conducted at
pH 5.0 on endogenous protein substrate in presence of different

46
N. Raghav, S. Garg / Bioorganic Chemistry 57 (2014) 43–50
Fig. 2. Effect of N-formylpyrazolines, 1a–1j, (a) and N-benzoylpyrazolines, 2a–2j, (b), on cathepsin
B activity. Effect of N-formylpyrazolines, 1a–1j, (c) and N-
benzoylpyrazolines, 2a–2j, (d) on cathepsin H activity. Results are mean of experiments conducted in triplicate. % Residual activities are presented w.r.t. control which contain
equivalent amount of solvent.
Fig. 3. Line-weaver Burk plot for cathepsin B at varying concentrations of BANA in presence of 1 ꢁ 10ꢂ7 M concentration of N-formylpyrazolines (a), 1 ꢁ 10ꢂ6
M
concentration of N-benzoylpyrazolines (b), respectively at pH 6.0. The Km value for control have been found to be 3.64 ꢁ 10ꢂ4 M. Line-weaver burk plot for cathepsin H on
varying concentrations of leu-ßNA in presence of 1 ꢁ 10ꢂ6 M concentration of N-formylpyrazolines (c), 1 ꢁ 10ꢂ5 M concentration of N-benzoylpyrazolines (d), respectively at
pH 7.0. The Km value for control have been found to be 5.34 ꢁ 10ꢂ4 M. The Ki values as calculated from this graph are presented in Table 1.
compounds separately at 10ꢂ4 M. It can be observed that proteolytic
activity is inhibited appreciably in presence of these compounds.
Further, it was found that the inhibition was more at 3.0 h and less
at 24.0 h suggesting that inhibition caused by the compounds is of
reversible type because initial inhibition caused by the compounds
is reversed when incubated for a longer time. The compounds bear-
ing nitro group led to a dramatic decrease in proteolytic activity and
showed higher inhibition than those of the corresponding

N. Raghav, S. Garg / Bioorganic Chemistry 57 (2014) 43–50
47
Fig. 4. Docking results showing the alignment of most inhibitory compounds along with the substrate in the active site of cathepsin B (cav2IPP B_PYS.pdb) and LeußNA in the
active site of cathepsin H (cav8PCH H_NAG.pdb). Here (a and b) show alignment of 1i and 2i, along with BANA in the active site of cathepsin B (cav2IPP B_PYS.pdb),
respectively; and (c and d) show the alignment of 1i and 2i along with Leu ßNA in the active site of cathepsin H (cav8PCH H_NAG.pdb), respectively.
R'
C6H5CONHNH2
O
H2NNH2.2H2O
HCOOH
C2H5OH
CH3COOH
Ha
Hb
N
Hx
O
R'
Ha
N
Hb
N
Hx
N
C
R'
CHO
N-formyl-3-subsitutedphenyl-5-phenylpyrazoline
N-benzoyl-3-subsitutedphenyl-5-phenylpyrazoline
Scheme 1.
compounds with methyl or methoxy group at the same position
indicating that activity decreases in presence of electron withdraw-
ing groups whereas it is less affected by the presence of electron
donating groups. A similar trend has previously been reported for
semicarbazones [49], phenylhydrazones [50] and arylhydrazones
[51]. Concrete conclusions about the structure activity relationship
and the potency of compounds cannot be drawn at this stage
because at this pH a large number of proteases are present in the
homogenate which are responsible for hydrolysis of proteins. At
pH 5.0, where most of the proteolytic activity is attributed to cys-
teine proteases it was thought proper to study the effect of synthe-
sized compounds on purified cathepsin B and cathepsin H.
individual enzymes. The results also imply that in addition to
cathepsins B and H other cysteine proteases are also active at pH
5.0 and are responsible for the proteolytic activities which are
insensitive toward the designed compounds signifying that the
compounds act as specific inhibitors of cathepsins B and H. Among
the various compounds tested, N-formyl-5-(-30-nitrophenyl)-3-
phenylpyrazoline (1i) was found to be most inhibitory to cathep-
sin B and cathepsin H activity in series of N-formylpyrazolines.
Similar trend was observed in case of N-benzoylpyrazolines where
2i was evaluated as most inhibitory. It has been found that cathep-
sin B and cathepsin H activity is maximally inhibited by the nitro
substituent in both the series.
3.2. Effect of synthesized compounds on the activities of cathepsin B
and cathepsin H
3.3. Enzyme kinetic studies
Line-weaver Burk plots drawn in presence and absence of inhib-
itors for cathepsin B (Fig. 3a and b) and for cathepsin H (Fig. 3c and
d) separately show that the plots of 1/V vs 1/S were straight lines
intersecting at the Y-axis with constant Vmax values in all the com-
pounds whereas the value of Km changed in presence of each com-
pound. These studies suggested a competitive type of inhibition
exhibited by these compounds. Using the Line-weaver Burk equa-
tion for competitive inhibition the Ki values were calculated, which
The activities of cathepsin B and cathepsin H were estimated at
varying concentrations of title compounds (Fig. 2a–d), respectively.
The figures show the relationship between the enzyme activity and
concentration of different compounds. It can be observed that at
10ꢂ4 M concentration of each compound, where proteolytic activ-
ity was not completely blocked, cathepsins B and H were com-
pletely inhibited emphasizing the importance of study on

48
N. Raghav, S. Garg / Bioorganic Chemistry 57 (2014) 43–50
Table 2
Docking studies showing decrease in different energies of cathepsin B & H in presence of substituted N-formylpyrazolines and N-benzoylpyrazolines.
Compound Enzyme
Cathepsin B
Cathepsin H
Total energy, kcal/
mol
VDW, kcal/
mol
HBond, kcal/
mol
Electronic, kcal/
mol
Total energy, kcal/
mol
VDW, kcal/
mol
HBond, kcal/
mol
Electronic, kcal/
mol
BANA
Leupeptin
Leu-bNA
LeuCH2Cl
1a
1b
1c
1d
1e
ꢂ128.61
ꢂ113.44
–
ꢂ87.34
ꢂ88.45
–
ꢂ38.19
ꢂ25.00
–
ꢂ3.08
–
–
–
–
–
–
–
–
0
0
0
0
0
0
0
–
–
0
0
0
0
0
0
0
0
0.49
0.29
0
0
0
0
ꢂ77.01
ꢂ59.12
ꢂ77.06
ꢂ77.60
ꢂ80.82
ꢂ79.40
ꢂ79.63
ꢂ83.50
ꢂ84.89
ꢂ94.42
ꢂ93.28
ꢂ90.38
ꢂ82.09
ꢂ84.34
ꢂ82.04
ꢂ84.54
ꢂ88.21
ꢂ87.09
ꢂ88.37
ꢂ93.00
ꢂ99.53
ꢂ95.23
ꢂ61.81
ꢂ46.12
ꢂ70.16
ꢂ68.09
ꢂ73.84
ꢂ69.18
ꢂ69.13
ꢂ76.50
ꢂ72.98
ꢂ68.37
ꢂ70.78
ꢂ71.08
ꢂ71.16
ꢂ73.93
ꢂ71.028
ꢂ74.94
ꢂ76.21
ꢂ76.59
ꢂ74.59
ꢂ76.90
ꢂ68.57
ꢂ76.90
ꢂ15.21
–
–
–
ꢂ13
ꢂ80.16
ꢂ83.90
ꢂ82.27
ꢂ81.27
ꢂ84.11
ꢂ82.33
ꢂ83.66
ꢂ93.63
ꢂ89.40
ꢂ84.79
ꢂ91.38
ꢂ99.23
ꢂ96.42
ꢂ96.36
ꢂ97.53
ꢂ95.52
ꢂ96.89
ꢂ102.48
ꢂ110.71
ꢂ106.98
ꢂ63.99
ꢂ68.50
ꢂ65.47
ꢂ64.33
ꢂ66.30
ꢂ71.85
ꢂ66.27
ꢂ65.00
ꢂ59.53
ꢂ67.80
ꢂ77.80
ꢂ84.80
ꢂ82.94
ꢂ82.24
ꢂ83.87
ꢂ78.43
ꢂ82.44
ꢂ73.86
ꢂ81.43
ꢂ78.58
ꢂ16.18
ꢂ15.40
ꢂ16.80
ꢂ16.94
ꢂ17.82
ꢂ10.49
ꢂ17.39
ꢂ28.63
ꢂ30.36
ꢂ17.28
ꢂ13.57
ꢂ14.43
ꢂ13.48
ꢂ14.12
ꢂ13.66
ꢂ17.09
ꢂ14.44
ꢂ29.07
ꢂ29.28
ꢂ28.10
ꢂ6.89
ꢂ9.5
ꢂ6.97
ꢂ10.22
ꢂ10.50
ꢂ7.00
ꢂ11.92
ꢂ25.66
ꢂ22.50
ꢂ18.66
ꢂ10.92
ꢂ10.41
ꢂ11.02
ꢂ9.60
ꢂ12.00
ꢂ10.50
ꢂ13.78
ꢂ15.70
ꢂ30.43
ꢂ18.34
0
0
0
1f
1g
1h
1i
1j
2a
2b
2c
2d
2e
ꢂ0.38
0
ꢂ0.64
0
0
0
0
0
0
0
0
0
0
0.46
0
2f
2g
2h
2i
ꢂ0.39
ꢂ0.52
0
2j
ꢂ0.30
The results are one of the docking experiments run using iGemdock under drug screening settings. The ligands were loaded as MDL mol file. The active site was extracted
from the structure of cathepsin B and cathepsin H were retrieved from Protein Data Bank (http://www.rcsb.org/) as cav2IPP B_PYS.pdb [48], and (cav8PCH H_NAG.pdb) [49],
respectively.
It can be observed that leupeptin showed ꢀ98.8% inhibition at
have been compiled in Table 1. The Ki value of most inhibiting
10ꢂ6 M concentration for cathepsin B whereas it showed ꢀ51.8%
compound for cathepsin B in the corresponding series i.e. N-form-
inhibition at 10ꢂ5 M concentration for cathepsin H which is in
ylpyrazolines and N-benzoylpyrazolines has been evaluated as
ꢀ1.1 ꢁ 10ꢂ9 M and 19.5 ꢁ 10ꢂ8 M for compounds 1i and 2i, respec-
accordance with the previously reported results. Similarly, Leu-
CH2ACl showed ꢀ9.3% inhibition at 10ꢂ5 M concentration for
tively; similarly for cathepsin H, these compound showed maxi-
cathepsin B whereas it showed ꢀ93.5% inhibition at 10ꢂ5 M con-
centration for cathepsin H. The results obtained are comparable
with earlier results reported for brain cathepsin B [68] and cathep-
sin H [62]. In the present context, we observed that the inhibition
of cathepsin B caused by the compound, 1i, is quite comparable to
leupeptin where the Ki value for the title compound has been
found to be 1.1 ꢁ 10ꢂ9 M. Inhibition caused by 1i is quite signifi-
cant where there is no complementary binding site in the molecule
as is present in leupeptin. N-formylated pyrazolines show more
inhibition due to better electrophilic center as compared to N-ben-
zoylated pyrazolines and therefore are better inhibitors for these
enzymes. In addition, the significance of benzoylated pyrazolines
can be well correlated with indomethacin which is p-chloro ben-
zoyl derivatives of indole acetic acid, the compound act as a non
steroid anti-inflammatory drug and is known to inhibit cyclooxy-
genase enzyme responsible for inflammation. It may be worth
mentioning here that lysosomes have long been reported as agents
of inflammation in polyarthritis, bacterial endotoxicity and rheu-
matoid arthritis [69–71]. The anti-inflammatory activity of indo-
methacin can be assigned due to cyclooxygenase inhibition as
well as inhibition of cathepsins [72]. The synthesized pyrazoline
derivatives have been found to inhibit cathepsin B and H up to
sub nanomolar range indicating the possible use of title com-
pounds as anti-inflammatory compounds.
mum inhibition with Ki values ꢀ5.19 ꢁ 10ꢂ8 M and 9.8 ꢁ 10ꢂ7
M
respectively. We have demonstrated here that in general, these
compounds showed more inhibition on cathepsin B activity in
comparison to cathepsin H. Analysis of the effect of these com-
pounds on cathepsin B and cathepsin H by the combination of ser-
endipitous biological selectivity with target-directed chemical
specificity suggests an optimistic future for their use as cysteine
protease inhibitors as therapy in a number of disease processes.
In order to ascertain inhibition ability of the studied compounds,
results were compared with potential inhibitors of cathepsin B
e.g. Leupeptin and cathepsin H e.g. Leu-CH2ACl, respectively. As
reported in literature, Leupeptin being a potential peptide inhibitor
of cathepsin B [65], inhibited the goat brain cathepsin B [61] com-
petitively with Ki value of 12.5 ꢁ 10ꢂ9 M whereas Ki value for
human liver cathepsin B [66] was reported to be 7.0 ꢁ 10ꢂ9 M. In
contrast, Ki value for human liver cathepsin [67] H was reported
to be 9.2 ꢁ 10ꢂ6 M. Inhibition by leupeptin has been attributed to
the exact complementarily of peptidyl side chain and the active
site directed formyl group that is susceptible to nucleophilic attack
by the thiol group present at the active site of both these enzymes.
3.4. Molecular docking experiment
Docking methods can provide valuable insight into the binding
mode between the ligand and the enzyme active site thereby have
an important role in the understanding of ligand–enzyme interac-
tions. On the basis of the interaction data of docking experiments

N. Raghav, S. Garg / Bioorganic Chemistry 57 (2014) 43–50
49
that include total energy and individual energy terms, an indicative
of the fitness of a predicted pose in the binding site, it is suggested
that the level of interaction is highest for N-benzoylpyrazolines
(2a–2j) followed by N-formylpyrazolines (1a–1j) with the active
site of cathepsin B and the same order is found in case of cathepsin
H (Table 2). In cathepsin B, all the compounds showed a lesser
interaction than the peptidyl inhibitor, leupeptin. Decrease in total
energy (kcal/mol) for leupeptin–cathepsin B has come out be
ꢂ113.44 which is quite close to substrate BANA-128.61 in compar-
ison to the compounds under consideration. This is due to peptide
protein interaction. Leupeptin is peptidyl in nature and flexibility
in the peptide molecule provide better interactions and therefore
binds effectively with the enzyme active site resulting in higher
binding energy. The synthesized compounds are smaller in struc-
ture and provide lesser interaction area. This is further explained
while discussing the docking results of cathepsin H. From molecu-
lar docking experiments, it can be interpreted that the compounds
should inhibit cathepsin B to a lesser extent in comparison to leu-
peptin. We have found that the most inhibitory compound, 1i,
inhibited the enzyme with the Ki value of 1 ꢁ 10ꢂ9 M which is
equivalent to leupeptin (detailed before). Thus, the present study
justifies its importance that mere computational data may not be
helpful in designing the enzyme inhibitors that eventually can
evolve as therapeutic agent. An experimental in vitro study is
equally or more informative.
Fig. 4a and b shows the docked view of most inhibitory com-
pounds, 1i and 2i along with the substrate BANA in the active site
of cathepsin B. The docked view of standard inhibitor leupeptin
along with BANA has been displayed in Fig. 4c for comparison.
The amino acid of the active site involved in the H-bonding with
the compound is detailed as green, the structure of the compound
is shown as thin lines. It can be observed that active site amino
acids Cys-29, His-199 interact with the most inhibitory compound
as well as with the leupeptin. Therefore, we can observe that inter-
action of designed compounds with the active site of the enzyme is
similar to that of leupeptin. Literature reports that leupeptin is a
competitive inhibitor of the enzyme cathepsin B therefore we
expect that the compounds should also inhibit the enzyme in a
competitive manner. Docking experiments supports the in vitro
studies. It can further be distinguished from these figures that
the compounds and leupeptin are in alignment with substrate
BANA and bind at the same site of enzyme. On comparing the
docking energies of the most inhibitory formyl and benzoyl pyraz-
olines, 1i and 2i, with the most inhibitory previously reported [57]
most inhibitory 1,3,5-triphenylpyrazolines and 3,5-diphenylpyraz-
olines we found that for cathepsin B compounds, 2d and 4d, which
are also nitro substituted show a decrease in total energy of inter-
action approx 90–86 kcal/mol. It can further be observed that func-
tionalization of pyrazolines resulted in significant inhibition of
cathepsin B thus validating the significance of present study. The
most inhibitory 1,3,5-triphenylpyrazolines and 3,5-diphenylpyraz-
olines, 2d and 4d, previously reported from our work exhibited Ki
values for cathepsin B in 42 nM and 53 nM, respectively however
in the present study we have found that formylated pyrazoline
1i, exhibited a Ki value in 1 nM, equivalent to leupeptin, is also a
formylated peptidyl inhibitor.
in Fig. 4d and e. For comparison Fig. 4f, detailing the interaction of
leu-CH2Cl along with leu-bNA is also provided here. All these com-
pounds interact with amino acyl acceptor site of the enzyme.
Amino acids Ser-69, Gln-78 and Asn-112 interact with the 1i, 2i
and leu-CH2Cl as well as leu-bNA, therefore indicating a competi-
tive inhibition caused by these compounds, which is confirmed
by enzyme kinetic studies. The results reported here are slightly
different from previously reported 1,3,5-triphenylpyrazolines and
3,5-diphenylpyrazolines, where 2b and 4b which are chloro-
substituted were the most inhibitory compounds with the Ki val-
ues of 1.17 and 1.56
nitro substituted compounds were also found to exhibit significant
Ki values of 2.3 and 3.3 M. Overall we can conclude that electron
lM, respectively. However the corresponding
l
withdrawing groups affected the inhibitory potential of the
compounds.
In the present work we can see that although the in vitro inhibi-
tion and in silico interaction data do not correlate well but type of
inhibition exhibited by these compounds can be visualized from
the docked poses and interacting amino acids. In both the enzymes,
the enzyme–ligand interaction data is more for benzoylated pyraz-
olines than formylated pyrazolines. A plausible explanation can be
proposed on the basis of more interaction surface area provided by
phenyl group in computational studies and a prone electrophilic
center of formyl group responsible for more inhibitory potential
of formyl group. In these two series of compounds, benzoyl group
can provide more interaction sites due to the larger surface area
as compared to H-present in formylated compounds, therefore
the decrease in energy as found in computational study is more
as compared to formyl group. However in formylpyrazolines, the
electrophilic center i.e. HAC@O is more susceptible to ASH nucleo-
philic attack when compared with C6H5AC@O site present in ben-
zoylpyrazolines. The in vitro studies show the actual inhibitory
potency of the compounds therefore evaluation of formylated pyr-
azolines as more potent inhibitors of these thiol enzymes, cathepsin
B and cathepsin H is properly justified.
In order to explore nonpeptidyl novel inhibitors to cathepsin B
& H, we here for the first time report the comparative study of
these compounds on clinically significant enzymes cathepsin B &
H. Such studies are important where the structurally related com-
pounds showing potential biological activities are evaluated for
their inhibitory effect on physiologically significant enzyme
cathepsin B and cathepsin H.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgments
One of the authors, Shweta Garg is thankful to UGC New Delhi,
India for award of JRF and also to Kurukshetra University, Kuruksh-
etra for providing necessary research laboratory facilities.
Appendix A. Supplementary material
The data regarding the characterization of compounds can be
found in the supplementary file. Supplementary data associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bioorg.2014.07.012.
In cathepsin H, the decrease in total energy for the reference
inhibitor Leu-CH2Cl was less as compared to all the designed com-
pounds (Table 2). Here, it can be seen that though Leu-CH2Cl is spe-
cific inhibitor for cathepsin H [71], but possess only one amino acid
residue as compared to leupeptin–cathepsin B complex. Therefore,
the leu-CH2Cl-cathepsin H interaction causes a lesser decrease in
energy of ꢂ59.12 kcal/mol only. The interaction energy data sug-
gest the compounds are better inhibitors than the standard leu-
CH2Cl. The docked view of most inhibitory compounds, 1i and 2i
along with leu-bNA in the active site of cathepsin H are presented
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