2,3-Dihydroquinazolin-4(1H)-one derivatives as potential non-peptidyl inhibitors of cathepsins B and H

Article abstract of DOI:10.1016/j.bioorg.2015.01.005

A direct correlation between cathepsin expression-cancer progression and elevated levels of cathepsins due to an imbalance in cellular inhibitors-cathepsins ratio in inflammatory diseases necessitates the work on the identification of potential inhibitors to cathepsins. In the present work we report the synthesis of some 2,3-dihydroquinazolin-4(1H)-ones followed by their evaluation as cysteine protease inhibitors in general and cathepsin B and cathepsin H inhibitors in particular. 2,3-Dihydroquinazolin-4(1H)-ones, synthesized by the condensation of anthranilamide and carbonyl compound in presence of PPA-SiO₂ catalyst, were characterized by spectral analysis. The designed compounds were screened as inhibitors to proteolysis on endogenous protein substrates. Further, a distinct differential pattern of inhibition was obtained for cathepsins B and H. The inhibition was more to cathepsin B with K_i values in nanomolar range. However, cathepsin H was inhibited at micromolar concentration. Maximum inhibition was shown by compounds, 1e and 1f for cathepsin B and compounds 1c and 1f for cathepsin H. The synthesized compounds were established as reversible inhibitors of cathepsins B and H. The results were also compared with the energy of interaction between enzyme active site and compounds using iGemdock software.

Full text of DOI:10.1016/j.bioorg.2015.01.005

Bioorganic Chemistry 59 (2015) 12–22
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
2,3-Dihydroquinazolin-4(1H)-one derivatives as potential non-peptidyl
inhibitors of cathepsins B and H
⇑
Mamta Singh, Neera Raghav
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 27 September 2014
Available online 28 January 2015
A direct correlation between cathepsin expression–cancer progression and elevated levels of cathepsins
due to an imbalance in cellular inhibitors-cathepsins ratio in inflammatory diseases necessitates the
work on the identification of potential inhibitors to cathepsins. In the present work we report the synthe-
sis of some 2,3-dihydroquinazolin-4(1H)-ones followed by their evaluation as cysteine protease
inhibitors in general and cathepsin B and cathepsin H inhibitors in particular. 2,3-Dihydroquinazolin-
Keywords:
2
,3-Dihydroquinazolin-4(1H)-one derivatives
4
(1H)-ones, synthesized by the condensation of anthranilamide and carbonyl compound in presence of
PPA-SiO catalyst, were characterized by spectral analysis. The designed compounds were screened as
inhibitors to proteolysis on endogenous protein substrates. Further, a distinct differential pattern of inhi-
bition was obtained for cathepsins B and H. The inhibition was more to cathepsin B with K values in
Cathepsin B
Cathepsin H
Non peptidyl inhibitors
Endogenous proteolysis
2
i
nanomolar range. However, cathepsin H was inhibited at micromolar concentration. Maximum inhibition
was shown by compounds, 1e and 1f for cathepsin B and compounds 1c and 1f for cathepsin H. The syn-
thesized compounds were established as reversible inhibitors of cathepsins B and H. The results were also
compared with the energy of interaction between enzyme active site and compounds using iGemdock
software.
Ó 2015 Elsevier Inc. All rights reserved.
1
. Introduction
Cathepsins are challenging therapeutic targets of drug design.
islets significantly and a reduction in the subsequent numbers of
tumor has been observed [11]. Inhibitors that target cysteine
cathepsins have been used in vitro to show that these enzymes play
an important role in tumor invasion [12]. In the past decade, vari-
ous studies have been carried out to identify small molecular
weight compounds as inhibitors of cysteine proteinases such as
aldehydes (1), pyrimidone (2), quinazolone (3) and 4-quinolinones
(4) [13–16]. In a recent study, some acyl hydrazides (5) [17] and
bischalcones and their quinazoline-2(1H)-one derivatives (6–7)
[18] as inhibitors to cathepsin B and H have been reported.
Cathepsins B and H have long been associated with cancer progres-
sion because of their ability to degrade extracellular matrices facil-
itating invasion, angiogenesis and metastasis as is evident from
numerous clinical reports and experimental models [1–4].
Increased levels of these enzymes in tumor state have been predic-
tive factors for cancer patients [5,6]. In addition, elevated levels of
these cysteine proteases have also been reported in various inflam-
matory conditions [7] such as rheumatoid arthritis and periodonti-
tis. One of the reasons for these elevated levels of the enzymes has
been attributed to an imbalance between cellular inhibitors and
cathepsins ratio [8,9]. Therefore identification of compounds
which act as potent inhibitors to cathepsins is a major thrust area
in the drug development and chemotherapy.
Inhibitors of cathepsin B have been found effective in reducing
the invasive potential of tumor cells [10]. Deletion of cathepsin H
impaired angiogenic switching of the pre-malignant hyperplastic
⇑
Corresponding author.
E-mail address: nraghav.chem@gmail.com (N. Raghav).
http://dx.doi.org/10.1016/j.bioorg.2015.01.005
045-2068/Ó 2015 Elsevier Inc. All rights reserved.
0

M. Singh, N. Raghav / Bioorganic Chemistry 59 (2015) 12–22
13
Literature reports that quinazolinone moieties have been
2.2. Methods
explored as a key functional group in a variety of anticancer agents.
Many quinazolinones have contributed to the quest for an ultimate
antitumor chemotherapeutic agent. 4(3H)-Quinazolinone deriva-
tives (8–13) [19–24] and 2,3-dihydro quinazolin-4(1H)-ones (14)
2.2.1. Proteolytic studies
2.2.1.1. Preparation of liver homogenate. The fresh goat liver was
first washed with cold isotonic saline solution. The tissue was then
homogenized in 0.1 M sodium acetate buffer pH 5.5 containing
[
25] showed promising antitumor potency.
0
.2 M NaCl, 1 mM EDTA and 0.1% Triton X-100 in a mixer-
cum-blender to obtain 10% (w/v) homogenate [33–38]. It was then
stored at 4 °C.
2
.2.1.2. Assay for proteolysis study. The proteolysis was carried out
at pH 5.0 at 37 °C using 0.1 M acetate buffer as the incubation med-
ium. Then, 100 l of homogenate was mixed with 880 l buffer
and 20 l of compounds at this pH and was incubated at 37 °C
l
l
l
for 3 h and 24 h, separately. The % residual activity is calculated
w.r.t. control where no compound was added but an equivalent
amount of solvent was present. The reaction was stopped by the
addition of 400
to precipitate proteins. The acid soluble proteins were quantitated
in the 200 l supernatant using 50 l Bradford dye according to
ll TCA and the resulting solution was centrifuged
l
l
Bradford method [39]. The experiments were conducted in tripli-
cate and the results are presented in Table 1. Endogenous Protein
ꢀ4
Hydrolysis is calculated in 0.1% liver homogenate at 10 M con-
centration of compounds and is calculated as proteolytic activity
in mg/h/ml in enzyme homogenate.
It may be worth mentioning here that lysosomes have been
reported as agents of inflammation in polyarthritis, bacterial endo-
toxicity and rheumatoid arthritis [26–28]. Involvement of cathep-
sin B and H in various inflammatory diseases also emphasizes the
significance of designing and development of these inhibitors as
anti-inflammatory agents [29]. It is also an established fact that
non steroidal anti-inflammatory drugs (NSAIDs) inhibit the prolif-
eration rate and induces apoptosis in colon cancer cell line in a pros-
taglandin independent pathway [30]. The role of piroxicam, an
NSAID is known to exert its pharmacological action by inhibiting
lysosomal enzymes [31]. The drug, used as a therapeutic agent in
osteoarthritis and rheumatoid arthritis, has been found in the che-
moprevention of colon carcinogenesis [32]. These literature reports
confirm a direct correlation between lysosomal enzyme inhibition
and development of anti-tumor and anti-inflammatory agents.
Keeping in view the presence of quinazolinone moiety in
compounds exhibiting anticancer activity it was thought proper
to synthesize some compounds with these structural background
in order to develop some potential inhibitors to cathepsins B and H.
In continuation of our previous work on low molecular weight
compounds as inhibitors to endogenous proteolytic activities
of cysteine proteases [33–38], in the present study, we report
2.2.2. Purification of goat liver cathepsin B and cathepsin H
All the purification steps were carried out at 4 °C. Cathepsins B
and H were isolated, separated and purified from goat liver by the
already established procedure as reported previously [17,18]. Fol-
lowing the steps of liver acetone powder preparation, homogeniza-
tion in cold 0.1 M sodium acetate buffer pH 4.76 containing 0.2 M
NaCl and 1 mM EDTA, acid-autolysis at pH 4.0 and 30–80% ammo-
nium sulfate fractionation. Further fractionation of proteases was
based on molecular weight on Sephadex G-100 column chroma-
tography, cation-exchange chromatography on CM-Sephadex
C-50 and DEAE-Sephadex A-50.
The specific activities of the cathepsin B and cathepsin H
were ꢁ10.38 nanomoles/min/mg and ꢁ22.56 nanomoles/min/mg,
respectively.
2.2.3. Enzyme assays
Stock solutions of the compounds (5 mM) were prepared in
DMSO. The purified cathepsins B and H were first activated in pres-
ence of thiol activators at pH 6.0 and pH 7.0, respectively. Then,
1
5
l
l of the enzyme solution was mixed with 940
sodium phosphate buffer containing 1 mM EDTA separately, for
0 min at 37 °C. Then, 20 l of stock solution of different com-
ll of 0.1 M
2
,3-dihydroquinazolin-4(1H)-ones as cathepsin B and H inhibitors.
SAR, inhibitory potency and type of inhibition exerted by these
compounds are reported which may provide new therapeutic
opportunities in cancer treatment. The results are compared with
in silico studies to rationalize our findings.
1
l
pounds under study were added separately to the activated
enzyme assay mixtures to effect final drug concentrations as
ꢀ
4
1
1
ꢂ 10 M in 1 ml assay (4.5% DMSO). After 30 min, 25
ll of
00 mM substrate stock solution was added to start the reaction.
2
. Experimental protocols
The released b-naphthylamine was quantitated colorimetrically
at 520 nm by the usual assay procedure as adopted previously
[17,18]. In control experiments, the equivalent amount of respec-
tive solvents was added and percent residual activities were calcu-
lated with reference to control.
2.1. Materials
All the chemicals were of analytical grade. Fast Garnet GBC
ꢀ4
(o-aminoazotoluene diazonium salt,
a
-N-benzoyl-
D,L
-arginine-2-
The compounds which showed 100% inhibition at 1 ꢂ 10
M
naphthylamide (BANA) and Leu-bNA 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.
concentration were further studied for their inhibitory effect at
their lower concentrations (Table 1) by adding appropriate amount
of individual compounds in the reaction mixture separately to
effect the final concentration of each compound as
ꢀ4
ꢀ4
ꢀ4
ꢀ4
0.001 ꢂ 10 M, 0.005 ꢂ 10 M, 0.01 ꢂ 10 M, 0.05 ꢂ 10 M,
ꢀ4
ꢀ4
ꢀ4
ꢀ4
0.1 ꢂ 10 M, 0.25 ꢂ 10 M, 0.5 ꢂ 10 M, 0.75 ꢂ 10
M and
ꢀ4
1.0 ꢂ 10 M (Figs. 1 and 2).

1
4
M. Singh, N. Raghav / Bioorganic Chemistry 59 (2015) 12–22
Table 1
Effect of substituted 2,3-dihydro-2-phenylquinazolin-4(1H)-one on hydrolysis of endogenous protein substrates, cathepsin B and cathepsin H activities.
O
O
NH
NH
N
R
H
N
H
(1a-1h)
(1i)
Code No. Endogenous Protein Hydrolysis
Cathepsin B activity
Mean ± SMD
Cathepsin H activity
% Residual activity Mean ± SMD
3
h
24 h
% Residual activity
Mean ± SMD % Residual Activity Mean ± SMD % Residual Activity
Control
4.78 ± 0.04
0 ± 0.00
100
0
4.95 ± 0.02
0 ± 0.00
0 ± 0.00
0 ± 0.00
4.84 ± 0.05
0 ± 0.00
0 ± 0.00
0 ± 0.00
0.53 ± 0.05
0 ± 0.00
100
0
0
0
97.78
0
0
0
5.65 ± 0.06
5.58 ± 0.10(0.1)
2.77 ± 0.008(0.05) 49.02
5.43 ± 0.10(0.1)
3.34 ± 0.08(0.05)
3.11 ± 0.01(0.001) 55.04
0.89 ± 0.02(0.001) 15.75
5.22 ± 0.01(0.1)
3.92 ± 0.02(0.05)
4.95 ± 0.07(0.1)
100
98.76
3.70 ± 0.25
2.95 ± 0.03(0.1)
100
79.73
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
4.73 ± 0.03
4.39 ± 0.02
4.55 ± 0.02
4.20 ± 0.05
4.67 ± 0.08
0 ± 0.00
98.95
91.84
95.19
87.86
97.69
0
3.03 ± 0.10(0.05) 81.89
1.45 ± 0.04(0.1) 39.18
3.55 ± 0.08(0.05) 95.94
3.63 ± 0.03(0.01) 98.11
3.48 ± 0.02(0.01) 94.05
3.39 ± 0.06(0.1)
3.27 ± 0.05(0.05) 88.37
3.51 ± 0.02(0.1) 94.86
96.11
59.11
92.38
69.38
87.61
91.62
2.98 ± 0.07
3.82 ± 0.04
62.34
79.92
10.70
0
The results are presented as Mean ± S.M.D. of the experiment conducted in triplicate. The % residual activity is calculated w.r.t. control where no compound was added but an
equivalent amount of solvent was present. Endogenous Protein Hydrolysis is calculated as % residual activity in 0.1% liver homogenate at 10–4 M concentration and is
calculated as proteolytic activity in mg/h/ml in enzyme homogenate. The TCA soluble peptides were estimated at 630 nm using Bradford method. Cathepsins B and H
ꢀ
4
activities were calculated as % residual activity and measured at 10 M concentration using BANA and Leu-bNA as substrates in enzyme preparations having specific activity
-Cl taken as positive
as ꢁ10.38 nmol/min/mg and ꢁ22.56 nmol/min/mg, respectively. Value of Mean ± S.M.D. and % residual activity in presence of leupeptin and Leu-CH
2
control for cathepsin B and cathepsin H has been calculated as 0.067 ± 0.0012 and 1.20 (0.001 mM) for cathepsin B and 0.241 ± 0.015 and 6.50 (0.01 mM) for cathepsin H,
respectively.
2.2.4. Kinetic measurements
2.2.5. General procedure
After establishing the inhibitory action of synthesized com-
Melting points were determined in open capillary tubes and are
uncorrected. All the chemicals and solvents used were of labora-
pounds on cathepsins B and H, experiments were designed to eval-
uate the type of inhibition and to determine their K values. For
ꢀ1
i
tory grade. IR spectra (KBr, cm ) were recorded on a Perkin–Elmer
1
that, enzyme activity was evaluated at six different substrate con-
spectrometer. H NMR spectra was recorded on Brucker 300 MHz
ꢀ
ꢀ4
4
ꢀ4
ꢀ4
centrations
(2.5 ꢂ 10 M,
2.0 ꢂ 10 M,
1.5 ꢂ 10 M,
NMR spectrometer (chemical shifts in d ppm) using TMS as an
internal standard. The purity of the compounds was ascertained
by thin layer chromatography on aluminum plates percoated with
silica gel G (Merck) in various solvent systems using iodine vapors
as detecting agent or by irradiation with ultraviolet lights
(254 nm). ELISA plate reader was used for measuring absorbance
in the visible range.
ꢀ
4
ꢀ4
ꢀ4
1
0
.0 ꢂ 10 M, 0.50 ꢂ 10 M, 0.30 ꢂ 10 M, 0.25 ꢂ 10 M and
ꢀ
4
.20 ꢂ 10 M) in presence and absence of a fixed concentration
of inhibitor indicated in Table 2. The enzyme concentration was
kept constant in all the experiments as detailed previously (Figs. 3
and 4). The values represent Mean ± S.M.D. of at least three indi-
vidual experiments.
Fig. 1. Effect of varying concentrations of 2,3-dihydroquinazoline-4(1H)-one at pH 6.0 on cathepsin B activity in presence of 2.5 mM concentration of BANA. Results are the
mean of the experiments conducted in triplicates at respective concentrations of compounds. Activities are expressed as percent of control which contains equivalent amount
of solvent.

M. Singh, N. Raghav / Bioorganic Chemistry 59 (2015) 12–22
15
Fig. 2. Effect of varying concentrations of 2,3-dihydroquinazoline-4(1H)-one at pH 7.0 on cathepsin H activity in presence of 2.5 mM concentration of Leu-bNA. Results are
the mean of the experiments conducted in triplicates at respective concentrations of compounds. Activities are expressed as percent of control which contains equivalent
amount of solvent.
(ANH str), 1643 (AC@O str), 512, 1426 (AC@CA str); 1H NMR
CDCl , 300 MHz, d ppm): 5.57 (1H, br s, ANH), 5.90 (1H, br s,
ANH), 6.65 (1H, s, ACH), 6.75 (1H, d, J = 7.8 Hz, ArAH), 7.13 (1H,
m, ArAH), 7.24 (1H, m, ArAH), 7.68 (1H, d, J = 7.8 Hz, ArAH), 7.77
(2H, d, J = 9.0 Hz, ArAH), 8.15 (2H, d, J = 9.0 Hz, ArAH); 13C NMR
Table 2
values and type of Inhibition exerted by 2,3-dihydroquinazoline-4(1H)-ones on
cathepsins B and H.
K
i
(
3
S.no.
Compound name
Type of inhibition
i
K values (lM)
Cathepsin B
Cathepsin H
(
1
75 MHz, CDCl
28.44, 127.93, 120.09, 117.28, 115.25, 113.68, 68.65.
2-(4-Methylphenyl)-2,3-dihydroquinazolin-4(1H)-one (1c):
Yield: 78%, m.p.°C: 232–234 [41]; IR (KBr, cm ): 3310, 3156
3
, d ppm): 169.30, 152.72, 147.21, 146.76, 133.76,
1
2
3
4
5
6
7
8
9
1a
1b
1c
1d
1e
1f
1g
1h
1i
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
111.100
1.570
52.630
2.810
14.410
10.420
2.500
56.820
23.250
8.180
52.630
18.580
90.090
ꢀ1
%
0.041
ANH str), 1651 (AC@O str), 1605, 1465 (AC@CA str); 1H NMR
(
(
0.0064
32.520
10.640
14.980
3 3
CDCl , 300 MHz, d ppm): 2.40 (s, 3H, ACH ), 5.03 (1H, br s,
ANH), 5.78 (1H, s, ANH), 6.09 (1H, s, ACH), 6.59 (2H, d,
J = 6.0 Hz, ArAH), 6.77 (1H, m, ArAH), 7.08 (1H, d, J = 9.0 Hz, ArAH),
7
.26 (1H, m, ArAH), 7.39 (2H, d, J = 6.0 Hz, ArAH), 7.81 (1H, d,
The experiments were conducted in triplicate in presence and absence of a fixed
concentration of different compound, separately (experimental value is given in
Table 1 in parenthesis). The results were then plotted between 1/V and 1/S to obtain
1
3
J = 9.0 Hz, ArAH);
147.28, 141.94, 136.22, 133.86, 128.27, 128.76, 126.09, 117.62,
3
C NMR (75 MHz, CDCl , d ppm): 165.44,
i
Lineweaver–Burk plots and then the K values were calculated using Lineweaver–
1
15.24, 114.88, 68.72, 23.08.
-(4-Methoxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (1d):
Burk equations for competitive and non-competitive inhibition depending upon the
results. Enzyme assays were conducted using BANA and Leu-bNA as substrates for
cathepsin B and cathepsin H activities having specific activity as ꢁ10.38 nmol/min/
mg and ꢁ22.56 nmol/min/mg, respectively.
2
ꢀ1
%
Yield: 92%, m.p.°C: 188–189 [41]; IR (KBr, cm ): 3299, 3109
1
(ANH str), 1659 (AC@O str), 1610, 1474 (AC@CA str); H NMR
(
CDCl , 300 MHz, d ppm): 3.75 (s, 3H, AOCH ), 5.05 (1H, br s,
3
3
ANH), 5.76 (1H, br s, ANH), 6.11 (1H, s, ACH), 6.62 (1H, d,
J = 9.0 Hz, ArAH), 6.76 (1H, m, ArAH), 6.84 (2H, d, J = 6.0 Hz, ArAH),
7.22 (1H, m, ArAH), 7.42 (2H, d, J = 6.0 Hz, ArAH), 7.82 (1H, d,
2
.2.6. Synthesis of 2,3-dihydroquinazolin-4-(1H)-ones
Anthranilamide (0.50 g, 0.0037 mol), benzaldehyde (0.10 g,
1
3
0
.0073 mol) and PPA-SiO
2
(0.0012 mol) were added to alcohol
J = 9.0 Hz, ArAH);
3
C NMR (75 MHz, CDCl , d ppm): 168.25,
(
2 ml) [40]. The mixture was refluxed for the 5–6 h. The progress
150.44, 143.27, 135.52, 133.58, 130.92, 129.59, 121.28, 117.32,
116.71, 116.46, 114.22, 70.76.
of the reaction was monitored by TLC. After completion, solvent
was evaporated at reduced pressure, and solid was recrystallized
from alcohol. The structure elucidations of compounds were based
on the spectral data (IR, H NMR and C NMR).
-Phenyl-2,3-dihydroquinazolin-4(1H)-one (1a): %Yield: 84%,
2-(4-Chlorophenyl)-2,3-dihydroquinazolin-4(1H)-one (1e):
ꢀ1
%Yield: 91%, m.p.°C: 200–202 [41]; IR (KBr, cm ): 3310, 3186
1
13
1
(ANH str), 1651 (AC'O str), 1605, 1465 (AC@CA str); H NMR
2
(CDCl , 300 MHz, d ppm): 5.32 (1H, br s, ANH), 5.78 (1H, br s,
3
ꢀ1
m.p.°C: 216–218 [41]; IR (KBr, cm ): 3325, 3178 (ANH str), 1651
ANH), 6.48 (1H, s, ACH), 6.62 (1H, d, J = 6.0 Hz, ArAH), 6.74 (1H,
1
(
AC@O str), 1454–1602 (AC@CA str); H NMR (CDCl
ppm): 5.40 (1H, br s, ANH), 5.76 (1H, br s, ANH), 6.35 (1H, s, ACH),
.60 (1H, d, J = 9.0 Hz, ArAH), 6.71 (1H, m, ArAH), 7.17 (1H, m,
ArAH), 7.28–7.31 (5H, m, ArAH), 7.73 (1H, d, J = 9.0 Hz, ArAH);
3
, 300 MHz, d
t, J = 9.0 Hz, ArAH), 7.19 (1H, m, ArAH), 7.28 (2H, d, J = 7.5 Hz,
ArAH), 7.44 (2H, d, J = 7.5 Hz, ArAH), 7.77 (1H, m, ArAH); 13
C
6
NMR (75 MHz, CDCl , d ppm): 169.80, 147.15, 142.38, 133.45,
3
132.67, 128.78, 128.64, 128.16, 117.35, 115.23, 113.40, 71.48.
1
3
C NMR (75 MHz, CDCl
28.65, 128.04, 127.68, 126.82, 117.58, 115.80, 113.95, 68.60.
-(4-Nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (1b):
Yield: 88%, m.p.°C: 205–206 [40]; IR (KBr, cm ): 3279, 3178
3
, d ppm): 169.84, 147.95, 144.64, 133.92,
2-(4-Fluorophenyl)-2,3-dihydroquinazolin-4(1H)-one (1f):
ꢀ1
1
%
%Yield: 84%, m.p.°C: 203–204 [41]; IR (KBr, cm ): 3302, 3178
(ANH str), 1651 (AC@O str), 1605, 1454 (AC@CA str); 1H NMR
2
ꢀ1
(CDCl , 300 MHz, d ppm): 5.57 (1H, br s, ANH), 6.65 (1H, br s,
3

1
6
M. Singh, N. Raghav / Bioorganic Chemistry 59 (2015) 12–22
Fig. 3. Lineweaver–Burk plots for cathepsin B activity on varying concentrations of BANA in presence of minimum concentration of 2,3-dihydroquinazoline-4(1H)-one as
and 1nVmax value for Control have been found to be 4.0 ꢂ 10ꢀ M and 0.120.
4
reported in Table 1 at pH 6.0. The K
m
Fig. 4. Lineweaver–Burk plots for cathepsin H activity on varying concentrations of Leu-bNA in presence of minimum concentration of 2,3-dihydroquinazoline-4(1H)-one at
and 1nVmax value for control have been found to be 5.0 ꢂ 10ꢀ M and 0.170.
4
pH 7.0 as reported in Table 1. The K
m
ANH), 6.75 (1H, s, ACH), 7.09 (1H, s, ArAH), 7.19–7.27 (3H, m,
ArAH), 7.51–7.62 (3H, m, ArAH), 8.28 (1H, s, ArAH); 1 C NMR
str); 1H NMR (CDCl
, 300 MHz, d ppm): 3.01 (6H, s, A(NCH ) ),
3 3 2
3
4.34 (1H, br s,ANH), 5.70 (1H, br s, ANH), 5.82 (1H, s, ACH), 6.68
(1H, d, J = 6.0 Hz, ArAH), 6.90 (1H, m, ArAH), 6.95 (2H, d,
J = 9.0 Hz, ArAH), 7.28 (1H, m, ArAH), 7.44 (2H, d, J = 9.0 Hz, ArAH),
(
1
75 MHz, CDCl
3
, d ppm): 170.23, 160.18, 147.44, 140.70, 133.54,
28.65, 128.25, 117.65, 115.32, 115.22, 113.65, 71.84.
-(4-Bromophenyl)-2,3-dihydroquinazolin-4(1H)-one (1g):
1
3
2
3
7.95 (1H, d, J = 6.0 Hz, ArAH); C NMR (75 MHz, CDCl , d ppm):
ꢀ1
%
(
(
Yield: 82%, m.p.°C: 202–203 [42]; IR (KBr, cm ): 3310, 3186
168.24, 147.98, 147.72, 133.98, 133.64, 128.45, 127.78, 117.25,
115.21, 114.65, 113.62, 68.76, 41.24, 41.30.
ANH str), 1651 (AC@O str), 1605, 1474 (AC@CA str); 1H NMR
CDCl , 300 MHz, d ppm): 5.58 (1H, br s, ANH), 5.69 (1H, br s,
3
1H-Spiro(cyclohexane-1,2-quinazolin)-4(3H)-one (1i): %Yield:
ꢀ1
ANH), 6.57 (1H, s, ACH), 6.60 (1H, d, J = 6.0 Hz, ArAH), 6.92 (2H,
d, J = 7.5 Hz, ArAH), 7.14 (1H, m, ArAH), 7.28 (1H, m, ArAH), 7.31
82%, m.p.°C: 219–220 [40]; IR (KBr, cm ): 3362, 3179 (ANH str),
1
1647 (AC@O str), 1618, 1458 (AC@CA str); H NMR (CDCl
300 MHz, d ppm): 1.35 (2H, m, >CH ), 1.37–1.53 (4H, m, >CH
1.72–1.76 (4H, m, >CH ), 4.86 (1H, br s, ANH), 6.55 (1H, br s,
ANH), 6.59 (1H, d, J = 9.0 Hz, ArAH), 6.83 (1H, t, J = 7.5 Hz, ArAH),
7.15 (1H, t, J = 7.5 Hz, ArAH), 7.72 (1H, d, J = 9.0 Hz, ArAH); 13
NMR (75 MHz, CDCl , d ppm): 167.33, 148.28, 133.25, 128.42,
117.04, 115.25, 113.82, 68.51, 35.77, 28.89, 20.05.
3
,
(
(
1
2H, d, J = 7.5 Hz, ArAH), 7.68 (1H, d, J = 6.0 Hz, ArAH); 1 C NMR
3
2
2
),
75 MHz, CDCl
29.12, 128.44, 121.46, 117.46, 115.28, 113.78, 70.94.
-(4-(Dimethylamino) phenyl)-2,3-dihydroquinazolin-4(1H)-
3
, d ppm): 170.24, 147.66, 143.58, 133.08, 131.85,
2
2
C
ꢀ1
one (1h): %Yield: 92%, m.p.°C: 224–228 [40]; IR (KBr, cm ):
294, 3186 (ANH str), 1651 (AC@O str), 1625, 1454 (AC@CA
3
3

M. Singh, N. Raghav / Bioorganic Chemistry 59 (2015) 12–22
17
Table 3
org/) as cav2IPP B_PYS.pdb [43] and cav8PCH H_NAG.pdb [44],
respectively. The structures were used for docking purpose assum-
ing that there is not much alteration in amino-acid sequence of dif-
ferent organisms. These enzymes are known to retain the main
ordered structures of papain superfamily [45]. It is also reported
that overall folding pattern of polypeptide chain is grossly same
in these proteases and utilize same catalytic mechanism [46]. After
loading the prepared ligands and the binding site, docking was
started at Standard Docking Accuracy Settings. Fitness is the total
energy of a predicted pose in the binding site which is the sum
total of electronic, H-bonding and Van der Waal interactions. The
results for cathepsin B and H are presented in Tables 3 and 4,
respectively. The docked poses of the ligands in the active site of
cathepsin B along with the substrate BANA and the peptide inhib-
itor, leupeptin are shown in Fig. 5 and the docked poses of the
ligands in the amino acyl binding site of cathepsin H along with
Docking studies showing decrease in different energies of cathepsin B in presence of
different 2,3-dihydroquinazoline-4(1H)-ones.
Ligand
Total energy
VDW
H bond
Elec
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
ꢀ79.7252
ꢀ82.7275
ꢀ77.7906
ꢀ78.518
ꢀ65.7884
ꢀ63.0387
ꢀ65.6278
ꢀ63.3019
ꢀ65.3722
ꢀ65.7997
ꢀ65.2799
ꢀ68.3925
ꢀ58.2401
ꢀ73.9838
ꢀ85.236
ꢀ13.9368
ꢀ19.6888
ꢀ12.1628
ꢀ15.2161
ꢀ12.1806
ꢀ12.1223
ꢀ12.1677
ꢀ12.5513
ꢀ13.4453
ꢀ48.2915
ꢀ19.7728
0
0
0
0
0
0
0
0
ꢀ77.5528
ꢀ77.922
ꢀ77.4476
ꢀ80.9438
ꢀ71.6854
ꢀ124.953
ꢀ105.009
0
BANA
Leupeptin
ꢀ2.67805
0
The results are one of the docking experiments run using iGemdock under standard
docking settings. The ligands were prepared in Marvin sketch and saved as MDL
mol file. The active site was extracted from the structure of cathepsin B retrieved
from protein data bank (http://www.rcsb.org/) as cav2IPP B_PYS.pdb [43].
2
the substrate Leu-bNA and the peptide inhibitor, Leu-CH Cl are
shown in Fig. 6.
Table 4
3
. Results and discussion
Docking studies showing decrease in different energies of cathepsin H in presence of
different 2,3-dihydroquinazoline-4(1H)-ones.
3.1. Chemistry
Ligand
Total energy
VDW
H bond
Elec
Leu-bNA
ꢀ85.5961
ꢀ59.3601
ꢀ73.3826
ꢀ83.2776
ꢀ69.2345
ꢀ71.2896
ꢀ69.2709
ꢀ72.3316
ꢀ71.9699
ꢀ76.2175
ꢀ73.121
ꢀ70.7725
ꢀ42.9381
ꢀ60.7197
ꢀ58.847
ꢀ14.8236
ꢀ16.4221
ꢀ12.6629
ꢀ24.145
0
0
0
2
,3-Dihydroquinazoline-4(1H)-one derivatives were synthe-
sized in excellent yields using silica-supported polyphosphoric
acid (PPA-SiO as heterogeneous and reusable catalyst
(Scheme 1). PPA-SiO is safe, easy to handle, environmentally gen-
Leu-CH
2
Cl
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
2
)
a
ꢀ0.28557
ꢀ61.1921
ꢀ58.6849
ꢀ61.4746
ꢀ59.6535
ꢀ59.3417
ꢀ63.1457
ꢀ62.9103
ꢀ8.0424
0
0
0
0
0
0
0
2
ꢀ12.6047
ꢀ7.79627
ꢀ12.678
tle with less disposal problems.
The synthesized compounds were characterized using spectral
data (IR and H NMR). The IR spectra showed mainly stretching
bands at 3300–3100, 3100–3000, 1655–1643 and 1600–
1450 cm assigned to (NAH), (CAH), (C@O) and aromatic (C@C)
functionalities, respectively.
1
ꢀ12.6282
ꢀ13.0718
ꢀ10.2107
ꢀ1
The results are one of the docking experiments run using iGemdock under standard
docking settings. The ligands were prepared in Marvin sketch and saved as MDL
mol file. The active site was extracted from the structure of cathepsin H retrieved
from Protein Data Bank (http://www.rcsb.org/) as (cav8PCH H_NAG.pdb) [44].
1
The H NMR spectrum of compounds in DMSO-d
6
shows signals
at d 4.30–6.65 assignable to ANH protons. The characteristic ACH
protons of tetrahydro-4-quinazolinones were observed as a singlet
at d 5.82–7.13 which is absent in case of spiroquinazolinone Mul-
tiplets observed in the d 6.34–8.45 ppm region are assigned to pro-
tons of phenyl rings. The sharp singlet observed at d 2.38–2.43,
3.01–3.12 and 3.63–3.80 ppm is assigned to methyl, N-methyl
and methoxy protons. The above spectral data suggested the suc-
cessful synthesis of title compounds.
2
.2.7. Drug modeling studies
All docking studies were performed using iGemDOCK. The
structures of ligands were prepared in Marvin sketch minimized
and were saved as MDL Mol File. The structure of cathepsins B
and H were retrieved from Protein Data Bank (http://www.rcsb.
Fig. 5. Docking results (I) and (II) showing the alignment of most inhibitory compounds 1e and 1f, respectively with BANA in the active site of cathepsin B (cav2IPP
B_PYS.pdb).

1
8
M. Singh, N. Raghav / Bioorganic Chemistry 59 (2015) 12–22
Fig. 6. Docking results (I) and (II) showing the alignment of most inhibitory compounds 1c and 1f, respectively with Leu-bNA in the active site of cathepsin H (cav8PCH
H_NAG.pdb).
potential inhibitors to cathepsins B and H. Comparative inhibitory
behavior of chalcones and their cyclic derivatives has also been
reported on these enzymes [56]. Keeping in view, the potential
shown by different quinazolinones as anticarcinogenic agents pre-
viously briefed, and role of cathepsins in cancer 2,3-dihydroqui-
nazolin-4(1H)-one derivatives were screened in vitro for
evaluating their inhibitory potency of cysteine proteases on endog-
enous protein substrates as well as against activity of cathepsins B
and H. The potential of cathepsin inhibitors as anti-inflammatory
agents can also be explored. We have reported these compounds
act as potent inhibitors of cathepsins B and H with effective K
i
values.
3.2.1. Effect of 2,3-dihydroquinazoline-4(1H)-one on in vitro
endogenous proteolysis in liver homogenate
Table 1 presents the inhibition of endogenous proteolytic activ-
Scheme 1. Scheme for synthesis of 2,3-dihydroquinazoline-4(1H)-ones.
ity in presence of different 2,3-dihydroquinazoline-4(1H)-one (1a–
1
h) at pH 5.0, where most of the proteolytic activity is attributed to
cysteine proteases [33] at 3 h and 24 h reaction time. It can be
observed that proteolytic activity is inhibited appreciably in pres-
ence of these compounds. In some cases ꢁ100% inhibition is
3
.2. Pharmacological evaluation
ꢀ4
Elevated levels of cathepsins and low levels of their inhibitors
achieved at 1 ꢂ 10 M concentration. Compounds bearing meth-
oxy 1d and bromo group 1g at the 4-position of benzene ring led
to a dramatic decrease in proteolytic activity. It can be concluded
that nitro 1b, methyl 1c, chloro 1e and fluoro 1f group at the
4-position of benzene ring and spiro compound 1i played a crucial
role in inhibiting the proteolytic activities at 24 h reaction. How-
ever, the unsubstituted compound showed negligible effect on
enzyme activity, i.e. enzymatic activities were influenced by the
substituents on the compounds under consideration. When we
compared the results of 3 h and 24 h reaction, it was found that
the inhibition to endogenous proteolysis increases with time in
presence of these compounds i.e., inhibition of proteolytic activity
was more at 24 h reaction in comparison to 3 h reaction. After
establishing the inhibitory potential of designed compounds on
cysteine proteases in general, the effect of synthesized compounds
on purified cathepsins B and H was studied.
have been observed in tumor cells and suggest the role of cathep-
sins as biological markers of malignant tumors [47]. The identifica-
tion and development of low molecular weight compounds as
inhibitors of specific cysteine proteases has been an active area
of research for cancer therapy. Literature survey suggests that a
large work has been accomplished on peptidyl or peptidyl analogs
as inhibitors to cysteine proteases [48]. However, these inhibitors
are not considered to be viable drug candidates for treating
diseases like cancer, apoptosis etc. because of the possibility of
immunogenic reactions or gastric instability. Therefore, research
on non-peptidyl drugs has become an important aspect in drug
research and development. More recently, a variety of benzophe-
none and thiosemicarbazone analogs [49,50], thiocarbazate [51]
and pyrazoles [52] as small molecular weight inhibitors of cathep-
sin L have been synthesized. Thiosemicarbazones and semicarba-
zones have also been reported to inhibit cathepsin B [53]. We
have also reported some acyl hydrazides [17] and bischalcones,
their quinazoline-2(1H)-one derivative and quinazoline-2(1H)-thi-
one derivatives [18] as inhibitors to cathepsins B and H. In addi-
tion, few pyrazoline derivatives [54,55] have also proved to be
3.2.2. Effect of 2,3-dihydroquinazoline-4(1H)-one on the activity of
cathepsin B and H
Table 1 also present the % residual activities of cathepsins B and
H at a MIC indicated in parenthesis. It can be observed that 1e and

M. Singh, N. Raghav / Bioorganic Chemistry 59 (2015) 12–22
19
1
f are most inhibitory to cathepsin B inhibiting 50% and 85% at
increasing inhibitor concentrations, showing
inhibition.
a
competitive
0
.001 M concentration. The results are quite encouraging in com-
parison to inhibitory potential of leupeptin run alongwith as a
positive control. At the same concentration ꢁ98% inhibition is
observed. The results are further confirmed later while conducting
enzyme kinetic studies. The activities of cathepsin B were esti-
mated at varying concentrations of designed compounds as
detailed in Sections 2.1 and 2.2. Fig. 1 shows the relationship
between the enzyme activity and concentration of substituted
This behavior is consistent with a mutually exclusive binding
mode between inhibitor and substrate; therefore, these inhibitors
compete with substrate for the free enzyme active site.
The K
aver–Burk equation K
which are presented in Table 2.
i
values of compounds were calculated using the Linewe-
0
= K (1+[I]/K ) for competitive inhibition
m
m
i
Preliminary inhibition studies of designed compounds on
endogenous protein substrates suggested that inhibition increases
with increase in time. The inhibition was more at 24 h incubation
time when compared with 3 h incubation. It may be worth men-
tioning here that homogenate consist of mixture of cysteine prote-
ases and all of these may not be susceptible toward the
synthesized compounds and therefore initial studies carried out
on homogenate need study on purified enzymes. During kinetic
studies on purified enzymes, we analyzed that the enzymes are
inhibited in a reversible manner indicating the importance of pres-
ent work. With this background we proposed the mechanism of
inhibition which has been shown in Scheme 2. The mechanism is
based on the molecular docking experiments (explained later in
the text) as well as on in vitro endogenous proteolysis studies.
The thiolate of cysteine-29 is acylated as shown with the assis-
tance of His-199. A similar mechanism has been proposed for inhi-
bition of inhibition of cathepsin L by benzophenone functionalized
thiosemicarbazones [49] where the active site is regenerated.
Here, we have proposed that the active site is acylated as in case
of leupeptin. Similar to these compounds, leupeptin is also
reported a reversible inhibitor of cathepsin B.
2
,3-dihydroquinazoline-4(1H)-ones.
Among the various compounds tested, 2-(4-fluorophenyl)-2,3-
dihydro quinazolin-4(1H)-one 1f was found to be most inhibitory
which showed 100% inhibition at 25
l
M concentration and ꢁ50%
inhibition was achieved at 100 nM concentration.
Similarly, the activities of cathepsin H was estimated at varying
concentrations of synthesized compounds at pH 7.0 using Leu-bNA
as
phenyl)quinazolin-4(1H)-one 1c was most inhibitory to cathepsin
H exhibiting 100% inhibition at 10 M concentration and 50% inhi-
bition at 750 M concentration followed by 2-(4-fluorophenyl)-
,3-dihydro quinazolin-4(1H)-one 1f. The results of inhibition of
cathepsins B and H are presented in Table 2. The inhibitory studies
a substrate. Fig. 2 shows that 2,3-dihydro-2-(4-methyl-
l
l
2
were further extended to determine the type of inhibition and K
i
values of respective compounds.
In order to discover novel non-peptidyl inhibitors of cathepsins
B and H, the present work focused on the synthesis of some 2,3-
dihydroquinazoline-4(1H)-one derivatives has been accomplished
which can lead to the development of new chemotherapeutic
agents in cases where these cathepsins are responsible for invasion
and metastasis of cancer cells. In addition, the compounds can be
useful in the treatment of rheumatoid arthritis and other tissue
degenerative disorders.
3.2.4. Structure–activity relationship (SAR) studies
It is a well known fact that cysteine protease inhibitors play an
important role in several diseases including cancer, metastasis,
inflammation and other tissue degenerative processes. Targeting
this enzyme family is therefore one of the strategies in the develop-
mentof new drugmolecules for chemotherapy in these diseases. The
substitution pattern was carefully altered in the designed com-
pounds to achieve a proper SAR study between the electronic envi-
ronment of the inhibitor and enzyme active site. In the present
study, we found that cathepsin B is inhibited more than cathepsin
3
.2.3. Mechanism of inhibition
The Lineweaver–Burk double-reciprocal plots (Figs. 3 and 4)
show intercepts of all lines converging at the y-axis (1/Vmax),
whereas the slope (K /Vmax) and x-axis intercepts (1/K ) vary
with inhibitor concentration. Consequently, the max values
remain constant, whereas the apparent values of K increase with
m
m
V
m
Scheme 2. Proposed mechanism for inhibition of cathepsin B by 2,3-dihydroquinazoline-4(1H)-ones.

2
0
M. Singh, N. Raghav / Bioorganic Chemistry 59 (2015) 12–22
H by 2,3-dihydroquinazoline-4(1H)-one derivatives and at the same
time when we studied the kinetics of inhibition, it was found that
both cathepsins B and H were inhibited in a competitive manner
Leu-CH
caused by test compounds is comparable. Compound, 1c and 1f
have been found to inhibit cathepsin H effectively with K values
of 2.5 M and 8.2 M, respectively. Cathepsins B and H, both are
inhibited by fluoro substituted compounds.
2
-Cl on cathepsin H activity, it was found that the inhibition
i
(Table 2).
l
l
During SAR studies, we could analyse that the potential of inhi-
bition shown by the synthesized compounds can be related to (i)
the electronic effects of the substitutents and (ii) steric factors. It
seems that these factors greatly contribute to the binding of inhib-
itor with the enzyme as there are not much stereochemical possi-
bilities in the ligand itself. The designed molecules possess only
one chiral centre and the possible orientation of substituent do
not have much possibilities. (Stereochemical view of energy mini-
mzed structures used for molecular docking purpose has been sup-
plied as supplementary data.)
It can be observed from the results that in general for cathepsin
B, electron withdrawing substituent inhibited the enzymes more
In the present work, where we have reported the synthetic non-
peptidyl novel inhibitors for cathepsin B and H, the enzyme inhibi-
tions achieved are comparable with peptidyl inhibitors and there-
fore, the work has a great significance. Investigation of the
inhibition potential of these compounds on activity of cathepsin
B and cathepsin H suggests a positive future for their use as cys-
teine protease inhibitors as therapeutic agents in a number of dis-
ease processes. The compounds under investigation add to the
existing knowledge of non peptidyl inhibitors of cathepsins B and
H and can be potential candidates for anti-cancer and anti-inflam-
matory drug development and chemotherapy.
as compared to electron releasing substituent For example AOCH
ACH , AN(CH exhibited lesser inhibitory effect. However, ANO
3
,
,
3
3 2
)
2
ACl and AF substituted compounds proved to be potential inhibi-
tors to the enzymes. It can further be observed that within a group,
the inhibitory potential is affected by the size as well as electro-
negativity of the substituent. Bromo affected the enzyme inhibition
to a lesser extent as compared to chloro followed by fluoro substi-
tuted compounds. The high inhibitory potential of chloro and flu-
oro compounds, 1e and 1f also possessing high log p value (in
the range of 3.19–3.65), and an indicative of lipophilicity makes
them suitable drug candidate for the treatment of diseases in cases
where cathepsin B is responsible.
3.3. Molecular docking experiment
The results indicated that substituent greatly affected the inhib-
itory potential of the compounds for cathepsin B and cathepsin H.
In order to establish inhibition ability of the studied com-
pounds, results were also compared with potential inhibitors of
The empirical scoring function of iGemDOCK is the estimated
sum total of Van der Waal, H-bonding and electrostatic energy.
From molecular docking experiments, we observed that all of com-
pounds inhibited the enzyme in a competitive manner as these
compete at the binding site of enzyme with substrate.
2
cathepsin B e.g. leupeptin and cathepsin H e.g. Leu-CH -Cl, respec-
tively. As reported in literature, leupeptin is a potential peptidyl
inhibitor of cathepsin B [57] and preferentially inhibit cathepsin
On the basis of the interaction data of docking experiments
(Table 3), it was observed that all the compounds showed a lesser
interaction than the reference leupeptin, a peptidyl inhibitor. The
maximum interaction is observed for BANA with a score of
B in comparison to cathepsin H, K
i
value for human liver cathepsin
H was reported to be 9.2 M [58].
l
ꢀ
124.953. Decrease in total energy for leupeptin–cathepsin B has
come out be ꢀ105.009 of which the contribution of Van der Waal
interactions are more with a score of ꢀ85.236 as compared to H-
bonds with a score of ꢀ19.7728. Leupeptin–cathepsin B binding
energy is due to peptide protein interaction. Leupeptin is peptidyl
in nature and therefore being a flexible molecule binds effectively
with the enzyme active site resulting in higher binding energy.
iGemDOCK provide algorithms for flexible docking approach for
both ligands and proteins [62] therefore flexible ligands like leu-
peptin will show a larger decrease in total energy as compared to
the molecules under study as these are smaller in structure and
possess lesser flexibility compared to leupeptin. Therefore, the
binding energy of title compounds is less than leupeptin, but the
in vitro analysis reveals that the compounds show comparable
inhibition to this reference inhibitor. The proposed mechanism is
previously shown in Scheme 2. The results clearly indicate the sig-
nificance of the in vitro inhibition studies.
2
-(4-Fluorophenyl)-2,3-dihydroquinazolin-4(1H)-one 1f, 2-(4-
nitrophenyl)-2,3-dihydro quinazolin-4(1H)-one 1b and 2-(4-chlo-
rophenyl)-2,3-dihydroquinazolin-4(1H)-one 1e showed maximum
inhibition on cathepsin B activity. Compound 1f (K
has been found to inhibit cathepsin B to the extent of leupeptin.
Here, K value of compound is slightly lesser than that of K of leu-
i
ꢁ 0.0064
lM)
i
i
peptin for brain enzyme, 12.5 nM [59] but is comparable to liver
cathepsin B and was reported to be 7.0 nM [60]. Another com-
However in cathepsin H, the decrease in total energy for the ref-
erence inhibitor Leu-CH
designed compounds. Here, it can be seen that though Leu-CH
2
Cl was less as compared to all the
Cl
pound 1e also inhibited the enzyme effectively (K
Compound 1b has been found to inhibit cathepsin B with K
of 1.57 M.
-(4-Methylphenyl)-2,3-dihydroquinazolin-4(1H)-one 1c showed
i
ꢁ 0.041
l
M).
2
i
values
is specific inhibitor for cathepsin H [61,63], but possess only one
amino acid residue as compared to leupeptin–cathepsin B. There-
fore, the Leu-CH Cl–cathepsin H interaction cause a decrease in
2
l
2
maximum inhibition on activity of cathepsin H. Spiro compounds
did not show much inhibition on both cathepsins B and H activity.
energy of ꢀ59.3601 of which ꢀ42.9381 is the Van der Waal inter-
action and ꢀ16.4221 is due to hydrogen bonds. As listed in Table 4,
all the designed compounds have been found to show
more decrease in ligand–cathepsin H interaction energy than
2
Leu-CH -Cl, a potential inhibitor to cathepsin H showed ꢁ93.5%
inhibition at 10
lM concentration which is in accordance with
the previously reported results [61]. When the inhibition
2
Leu-CH Cl–cathepsin H. The compound, 1c, found to be most
pattern of the compounds under consideration is compared with
inhibitory to cathepsin H show a total decrease in energy as

M. Singh, N. Raghav / Bioorganic Chemistry 59 (2015) 12–22
21
ꢀ
69.234 of which ꢀ61.1921 is assigned to Van der waal interac-
Appendix A. Supplementary material
tions whereas ꢀ8.0424 is of H-bond.
Fig. 5 shows the docked view of compounds, 1e and 1f, respec-
tively along with the substrate BANA in the active site of cathepsin
B. The active site consisting of Cys-29 which interacts with the sub-
strate cf Fig. 5 interacts with the designed compounds under con-
sideration. In addition, Trp-30 and Gly-198 amino acids residues of
cathepsin B also interact with substrate as well as with the inhib-
itor. It can be observed that all 2,3-dihydroquinazoline-4(1H)-ones
are in good alignment with the substrate BANA. Like leupeptin,
which has been reported as competitive inhibitor to cathepsin B,
the designed compounds also show competitive inhibition. The
competitive inhibition of 2,3-dihydroquinazoline-4(1H)-ones, as
established by in vitro studies is also supported by in silico studies
where the docked poses of the inhibitory compounds are in good
alignment with the substrate in the active site.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2015.01.
005.
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[
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Fig. 6 shows the docked view of compounds, 1c and 1f, respec-
tively alongwith Leu-bNA in the aminoacyl binding site of cathep-
sin H. Here, the compound seems to interact with the enzyme at a
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proposed for cathepsin B might hold equally good for cathepsin H,
where the ASH group present at the active site of the enzyme is
involved in the nucleophilic attack resulting in enzyme inhibition.
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these two but in-solution studies are equally important.
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[
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