N. Raghav, M. Singh / European Journal of Medicinal Chemistry 77 (2014) 231e242
235
Table 3
presented in Fig. 5. These studies suggested that a non-competitive
type of inhibition is exhibited by these compounds (Fig. 5). Using
the Line-weaver Burk equation of non-competitive inhibition the Ki
values were calculated, which has been presented in Table 3.
Among the different triazoles, maximum inhibition has shown to
Type of inhibition exerted by acyl hydrazides and N4-amino 1,2,4-triazoles on
cathepsin B and cathepsin H.
S. No.
Compound name
Ki values (10ꢀ4 M)a, type
of inhibition
be for 2i with the Ki value of 9.5 ꢂ 10ꢀ7
.
Cathepsin Bb
Cathepsin Hc
1.
2.
3.
4.
5.
6.
2-Methoxybenzohydrazide (1a)
4-Methoxybenzohydrazide (1b)
4-Hydroxybenzohydrazide (1c)
Phenylacetyl hydrazine (1d)
2-Chlorobenzohydrazide (1e)
3,5-diphenyl-4-amino-1,2,4-
triazole(2a)
0.127, C
0.0064, C
0.019, C
0.0087, C
0.0136, C
1.205, NC
5.88, NC
4.2.2. Cathepsin H inhibition studies in presence of acyl hydrazides
and triazoles
0.082, NC
1.123, NC
0.425, NC
0.901, NC
0.232, NC
Similarly, the activities of cathepsin H were estimated at
varying concentrations of synthesized acyl hydrazides Fig. 6 shows
the relationship between the enzyme activity and concentration
of substituted acyl hydrazides. Among the various compounds
tested, 4-Methoxybenzohydrazide (1b) was found to be most
inhibitory followed by phenylacetyl hydrazine (1d), 2-
chlorobenzohydrazide (1e), 4-hydroxybenzohydrazide (1c) and
2-methoxybenzohydrazide (1a) in that order, which is similar to
that of cathepsin B.
The inhibition type was studied at varying concentration of
substrate in presence and absence of a fixed concentration of in-
hibitor acyl hydrazides and Line-weaver Burk plots were drawn. It
was found that the plots of 1/V and 1/S were straight lines inter-
secting at the Y-axis and showed that value of Km remained con-
7.
8.
9.
3-phenyl-5-(40-nitrophenyl)-
4-amino-1,2,4-triazole (2b)
3-phenyl-5-(30-nitrophenyl)-
4-amino-1,2,4-triazole (2c)
3-(20-Methylphenyl)-5-(20-
methylphenyl)-4-amino-1,2,
4-triazole (2d)
0.048, NC
0.0143, NC
0.149, NC
0.364, NC
0.086, NC
0.532, NC
10.
11.
12.
3-(30-Methylphenyl)-5-(30-
methylphenyl)-4-amino-1,2,
4-triazole (2e)
0.385, NC
0.080, NC
0.032, NC
0.387, NC
0.901, NC
0.781, NC
3-(40-Methylphenyl)-5-(40-
methylphenyl)-4-amino-1,2,
4-triazole (2f)
3-(20-Hydroxy-30-
0
stant in all the compounds whereas the value of Vmax changes with
methylphenyl)-5-(20-hydroxy-
30-methylphenyl)-4-amino-1,
2,4-triazole (2g)
each compound. These studies suggested a non-competitive type of
inhibition exhibited by these compounds (Fig. 7). Using the Line-
weaver Burk equation of non-competitive inhibition, the Ki values
were calculated, which has been presented in Table 3.
13.
14.
3-(40-Chlorophenyl)-5-(40-
chlorophenyl)-4-amino-1,2,
4-triazole (2h)
0.024, NC
0.033, NC
0.020, NC
The activities of cathepsin H were estimated at varying con-
centrations of synthesized triazoles. Fig. 8 shows the relationship
between the enzyme activity and concentration of substituted tri-
azoles. The most potent inhibitors have been found to be 3-(30-
Aminophenyl)-5-(30-aminophenyl)-4-amino-1,2,4-triazole (2i), 3-
(40-chlorophenyl)-5-(40-chlorophenyl)-4-amino-1,2,4-triazole (2h)
and 3-(30-Nitrophenyl)-5-(30-Nitrophenyl)-4-amino-1,2,4-triazole
(2c). Further evaluation on the type of inhibition when Line-
weaver Burk plots were drawn in presence and absence of a fixed
concentration of individual triazoles at varying substrate concen-
trations, the graphs obtained showed a non-competitive type of
inhibition exhibited by these compounds on cathepsin H activity
(Fig. 9). Using the Line-weaver Burk equation of non-competitive
inhibition the Ki values were calculated, which has been pre-
sented in Table 3.
The inhibitory activities of synthesized compounds were
assessed for two related cysteine proteases, cathepsin B and
cathepsin H. The detailed kinetic analysis have shown that though
the order of inhibition of both cathepsin B and cathepsin H by acyl
hydrazides (1ae1e) is same but cathepsin B is inhibited more than
cathepsin H and at the same time when we studied the kinetics of
inhibition, cathepsin B was inhibited in a competitive manner
whereas cathepsin H was inhibited in a non-competitive by acyl
hydrazides. In case of triazoles (2ae2i) also, more of less similar
trend is obtained but again cathepsin B is inhibited to a greater
extent than cathepsin H. But here the type of inhibition exhibited
by triazoles for cathepsin B and cathepsin H is non-competitive. The
Ki values obtained are presented in Table 3 which is in the low
micro molar range.
3-(30-Aminophenyl)-5-(30-
aminophenyl)-4-amino-1,
2,4-triazole (2i)
0.0095, NC
C ¼ Competitive, NC ¼ Non-competitive.
a
Represents Mean ꢃ S.M.D. of the experiment conducted in triplicate 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-weavereBurk plots and then
the Ki values were calculated using Line-weavereBurk equations for competitive
and non-competitive inhibition depending upon the results.
b
Enzyme assays were conducted using BANA as substrate for Cathepsin B having
specific activity was found to be w10.38 nmol/min/mg.
c
Enzyme assays were conducted using Leu-bNA as substrate for Cathepsin H
having specific activity was found to be w22.56 nmol/min/mg.
acyl hydrazides. Thereafter, enzyme kinetics were carried out to
further evaluate the type of inhibition and to determine the Ki
value. Line-weaver Burk plots were drawn in presence and
absence of a fixed concentration of inhibitor. The results (Fig. 3)
show that acyl hydrazides inhibit cathepsin B activity in
a
competitive manner. Using the Line-weaver Burk equation of
competitive inhibition the Ki values were calculated, which has
been presented in Table 3.
The activities of cathepsin B were estimated at varying con-
centrations of synthesized triazoles. Fig. 4 shows the relationship
between the enzyme activity and concentration of substituted
triazoles.
3-(30-Aminophenyl)-5-(30-aminophenyl)-4-amino-
1,2,4-triazole (2i) has been found to be most inhibitory to
cathepsin B activity. It was found that at 0.1 mM concentration,
the compound inhibited cathepsin B activity completely and half
maximal activity was lost at less than 0.05 mM concentration.
Another compound
amino-1,2,4-triazole (2c), also inhibited cathepsin B to almost
similar extent.
To evaluate the type of inhibition caused by these compounds,
the activity of cathepsin B was measured in presence and absence
of a fixed concentration of individual triazoles at varying substrate
concentrations. The Line-weaver Burk plots thus obtained are
In order to ascertain inhibition ability of the studied com-
pounds, results were compared with specific inhibitors of cathepsin
B and cathepsin H e.g., Leupeptin and LeueCH2eCl. As reported in
literature, Leupeptin being a specific peptide inhibitor of cathepsin
B, inhibited the goat brain cathepsin B competitively with Ki value
of 12$5 ꢂ 10ꢀ9 M [60] whereas Ki value for human liver cathepsin B
[63] was reported to be 7$0 ꢂ 10ꢀ9 M. In contrast, Ki value for
human liver cathepsin H was reported to be 9.2 ꢂ 10ꢀ6 M [64].
3-(30-nitrophenyl)-5-(30-nitrophenyl)-4-