SAR studies of some acetophenone phenylhydrazone based pyrazole derivatives as anticathepsin agents

Bioorganic Chemistry 75 (2017) 38–49

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SAR studies of some acetophenone phenylhydrazone based pyrazole derivatives as anticathepsin agents Neera Raghav ⇑, Mamta Singh Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India

a r t i c l e

i n f o

Article history: Received 4 May 2017 Revised 18 August 2017 Accepted 19 August 2017 Available online 31 August 2017 Keywords: Cathepsin B Cathepsin H Cathepsin L Non peptidyl inhibitors Pyrazole based compounds

a b s t r a c t Cathepsins have emerged as promising molecular targets in a number of diseases such as Alzeimer’s, inflammation and cancer. Elevated cathepsin’s levels and decreased cellular inhibitor concentrations have emphasized the search for novel inhibitors of cathepsins. The present work is focused on the design and synthesis of some acetophenone phenylhydrazone based pyrazole derivatives as novel non peptidyl inhibitors of cathepsins B, H and L. The synthesized compounds after characterization have been explored for their inhibitory potency against cathepsins B, H and L. The results show that some of the synthesized compounds exhibit anti-catheptic activity with Ki value of the order of 10 10 M. Differential inhibitory effects have been observed for cathepsins B, H and L. Cathepsin L is inhibited more pronounced than cathepsin B and cathepsin H in that order. Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction Over expression of cathepsins in different cancerous processes has gained attention of researchers to understand their precise role in tumor cell proliferation and metastasis [1], angiogenesis and invasion [2]. In addition, role of cathepsins in the degradation of extracellular matrix has been an important factor in establishing these as important targets in various conditions related to Alzeimer’s disease, inflammation etc. Elevated levels of cathepsins B, H and L [3–5] signify the contribution of inhibitors in control of these diseased states [6–7] to an extent that anti-cathepsin activities are now the focus for the development of novel therapeutic possibilities. Based on the peptidyl nature of cellular catheptic inhibitors initial research was focused on peptide based warheads [8–12], where peptidyl backbone contributed toward specificity and active group resulted in inhibition generating diverse variety of inhibitors usually irreversible in nature. Gastric instability and immunological problems associated with these inhibitors, envisaged the importance of low molecular weight inhibitors which can be easily synthesized. With this background we explored simple compounds like semicarbazones and thiosemicarbazones carbonyl compounds and chalcones, derivatives of chalcones as inhibitors of cathepsins [13–16]. Some benzofuran derivatives [17], acylhydrazides and triazolesi [18] have also been found to be potential inhibitors of ⇑ Corresponding author. E-mail address: [email protected] (N. Raghav). http://dx.doi.org/10.1016/j.bioorg.2017.08.006 0045-2068/Ó 2017 Elsevier Inc. All rights reserved.

cathepsins. Identification of semicarbazones and thiosemicarbazones [13] and pyrazolines [14] as potential inhibitors of cathepsins B, H and L motivated us to explore the designed molecules having two different pharmacophores i.e., pyrazole and different side chain azomethine groups as a novel class of cathepsins B, H and L inhibitors which may provide new therapeutic opportunities in diseased states caused by imbalanced activities of these cathepsins. In the present work, we synthesized differently functionalized pyrazole-4-carbaldehydes from hydrazones of aryl methyl ketones. Semicarbazones, thiosemicarbazones and phenyl hydrazones of pyrazole-4-carbaldehydes were also synthesized. All the synthesized compounds were evaluated as inhibitors to cathepsin B, H and L. Detailed analysis of effectiveness of these derivatives may provide a platform for development of the potent enzyme inhibitors. When these analogs were screened against cathepsin B, H and L in enzyme assays, two promising inhibitors were identified. The results are compared with in silico studies which support the postulation that synthesized compounds may act as enzyme inhibitors. 2. Experimental protocols 2.1. Materials All the chemicals were of analytical grade. Fast Garnet GBC (o-aminoazotoluene diazonium salt), Various substrates used i.e. a-N-benzoyl-D, l-arginine-b-naphthylamide (BANA) for cathepsin

N. Raghav, M. Singh / Bioorganic Chemistry 75 (2017) 38–49

B, Z-phenylalanylarginyl-b-naphthylamide (Z-Phe-Arg-bNA) for cathespsin L and Leucyl-b-naphthylamide (Leu-bNA) for cathepsin H 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. 2.2. Methods 2.2.1. Purification of cathepsins Β, H and L All the purification steps were carried out at 4 °C. Cathepsin B and H were isolated, separated and purified from goat liver by established procedure [19]. The specific activities of the cathepsin B, H and L thus obtained, were 10.38, 22.56 and 17.48 nmol/ min/mg respectively. 2.2.2. Enzyme assays Stock solutions of the compounds (5 mM) were prepared in DMSO. The purified cathepsin B, H and L were first activated in presence of thiol activators at pH 6.0, 7.0 and 6.0, respectively. Then, 100 ml of the enzyme solution was mixed with 855 ml of 0.1 M phosphate buffer containing 1 mM EDTA separately for 10 min at 37 °C. Then, 20 ll of stock solution of different compounds under study were added separately to the activated enzyme assay mixtures to effect final drug concentrations as 1  10 4 M in 1 ml assay. After 30 min, 25 ml of 100 mM substrate stock solution was added to start the reaction. The released bnaphthylamine was quantitated colorimetrically at 520 nm by the usual assay procedure [19–21]. In control experiments, an equivalent amount of DMSO was added and percent residual activities were calculated with reference to control. The compounds exhibiting complete inhibition at 1  10 4 M concentration were further studied for their inhibitory effect at their lower concentrations i.e. 10 5 M, 10 6 M, 10 7 M and so on till some activity was observed for cathepsins B, H and L. Table 1 displays the effect of individual compounds on cathepsin B, H and L activities at a particular concentration (10 5 M as shown in parenthesis in respective positions). Once the inhibitory potential was established experiments were conducted to study the effect of individual compound at varying concentration. The results are shown in Figs. 1, 2 and 3 (I, II, III, IV, V, VI). 2.2.3. Enzyme kinetic studies After establishing the inhibitory action of synthesized compounds on cathepsins B, H and L, Lineweaver-Burk plots were drawn to evaluate the type of inhibition and to determine their Ki values. For that, enzyme activity was evaluated at different substrate concentrations (2.5  10 4 M, 2.0  10 4 M, 1.5  10 4 M, 1.0  10 4 M, 0.50  10 4 M, 0.30  10 4 M, 0.25  10 4 M and 0.20  10 4 M) in presence and absence of a particular concentration of inhibitor (the concentration of each inhibitor at which the experiment was conducted is given in parenthesis in Table 1 as Z  10 5 M concentration). The enzyme concentration was kept constant in all the experiments as detailed previously. The values represent Mean ± S.M.D. of at least three individual experiments. The Ki values of compounds were calculated using the Lineweaver-Burk equation Km = Km (1 + [I]/Ki) for competitive inhibition and Vmax = Vmax (1 + [I]/Ki) for non-competitive inhibition. The mode of inhibition was investigated and Ki values obtained are tabulated in Table 2. The Km values for cathepsin B, H and L were found to be 4.0  10 4 M, 5.0  10 4 M and 7.6  10 5 M, respectively, whereas, the 1/Vmax values were found to be 0.120, 0.170 and 0.130, respectively.

39

2.2.4. Drug modeling studies All docking studies were performed using iGemdock. For these studies, the structures of cathepsin B and cathepsin H were retrieved from Protein Data Bank (http://www.rcsb.org/) as 2IPP B [22], 8PCH H [23] and 3BC3L [24], respectively. The bound ligands PYS, NAG and CSW were removed prior to docking. The cavity structure selected was 8 Ǻ. Structures of the ligands were prepared in Marvin sketch and were minimized before saving as MDL Mol File. The ligand structures and enzyme active sites were loaded and docking was run where GA parameters were defined according to drug screening setting for population size, number of generation and solutions. The stabilization energies calculated as Etotal as a consequence of enzyme ligand interaction for cathepsin B, H and L and are presented in Tables 3, 4 and 5, respectively. The docked poses of most inhibitory compounds, 4d and 4e for cathepsin B and H are revealed in Figs. 4 and 5, respectively. The most inhibitory compounds 1b and 2b for cathepsin L are depicted in Fig. 6. 2.2.5. General procedure for synthesis Melting points were determined in open capillary tubes and are uncorrected. All the chemicals and solvents used were of laboratory grade. IR spectra (KBr, cm 1) were recorded on a PerkinElmer spectrometer. 1H NMR spectra was recorded on Brucker 300 MHz 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 aluminium plates percolated 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. 2.2.6. General procedure for the synthesis of 1H-pyrazole-4carbaldehydes (2) At 0 °C POCl3 (0.98 ml) was added dropwise with stirring to ice cold DMF solution over a period of 30 min. Then, pbromoacetophenone phenylhydrazone 1a (1.0 g, 0.0036 mol) in N,N-dimethylformamide (3 ml) was added drop wise. Stirring was continued under ice cold condition for another half an hour. The reaction mixture was brought to room temperature and refluxed at 60–70 °C for 4–5 h. The reaction mixture was then cooled and poured into crushed ice with stirring and neutralized with aq. NaHCO3 solution. The solid obtained was filtered and recrystallized from ethanol. The structure elucidation of compound, 3-(4-bromophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 2a was based on the spectral data (IR, 1H NMR & 13C NMR). 2.2.6.1. 3-(4-Bromophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (2a). Yield 67.85%; m.p.160–162 °C[25]; IR (KBr, cm 1): 1674 (AC@O str), 1597(AC@N str), 1450, 1566(AC@CAstr), 825(ACABr str); 1H NMR (300 MHz, CDCl3, d ppm): 7.38–7.40 (3H, m, ArAH), 7.64(2H, d, J = 8.1 Hz, ArAH), 7.77–7.82(4H, m, ArAH), 9.03 (1H, s, AC5H), 10.06 (1H, s, CHO); 13C NMR (75 MHz, CDCl3, d ppm): 184.52, 152.93, 139.72, 133.08, 132.95, 131.84, 129.99, 129.38, 126.11, 122.44, 120.45, 110.72. Following exactly the same procedure as detailed above for compound 2a, the other substituted (1H)-pyrazole-4carbaldehydes (2b–2f) were prepared from the corresponding acetophenone and substituted benzaldehydes. The physical and spectral data of synthesized compounds are given below. 2.2.6.2. 3-(4-Nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (2b). Yield 75.63%; m.p.165-167 °C[25]; IR (KBr, cm 1): 1682 (AC@O str), 1597(AC@N str), 1450, 1585(AC@CAstr), 1342, 1520 (ANO2 str); 1H NMR (300 MHz, CDCl3, d ppm): 7.44–7.60 (3 H, m, ArAH), 8.02(2H, d, ArAH), 8.26–8.37(4 H, m, ArAH), 9.43 (1H, s,

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N. Raghav, M. Singh / Bioorganic Chemistry 75 (2017) 38–49

Table 1 Effect of some hydrazones of aryl methyl ketone (1), pyrazole-4-carbaldehydes (2) and semicarbazones (3), thiosemicarbazones (4) and phenyl hydrazone derivatives (5) on hydrolysis of b-naphthylamide derivatives as substrates for Cathepsin B, H and L activities.

Code No.

Control 1a 1b 1c 1d 1e 1f 2a 2b 2c 2d 2e 2f 3a 3b 3c 3d 3e 3f 4a 4b 4c 4d 4e 4f 5a 5b 5c 5d 5e 5f Leupeptin LeuCH2Cl

Cathepsin B

Cathepsin H

Cathepsin L

Mean ± SMD

% Residual activity

Mean ± SMD

% Residual activity

Mean ± SMD

% Residual activity

5.65 ± 0.06(1) 0.57 ± 0.01(1) 1.51 ± 0.01(0.1) 2.60 ± 0.04(1) 0.469 ± 0.04(0.1) 1.35 ± 0.09(0.1) 0.71 ± 0.02(1) 5.03 ± 0.13(1) 1.79 ± 0.08(1) 3.67 ± 0.17(1) 1.20 ± 0.19(1) 1.61 ± 0.06(1) 2.58 ± 0.03(1) 3.07 ± 0.27(1) 1.14 ± 0.08(1) 2.85 ± 0.20(1) 0.90 ± 0.09(0.1) 1.53 ± 0.05(0.1) 1.86 ± 0.10(1) 3.76 ± 0.24(0.01) 3.28 ± 0.10(0.01) 2.55 ± 0.08(1) 0.21 ± 0.02(0.01) 0.63 ± 0.06(0.01) 1.47 ± 0.02(0.1) 4.52 ± 0.05(1) 4.12 ± 0.29(1) 4.86 ± 0.39(1) 2.71 ± 0.13(1) 3.96 ± 0.24(1) 2.94 ± 0.06(1) 0.067 ± 0.0012(0.1) –

100 10.09 26.73 46.02 08.30 23.89 12.56 89.02 31.68 64.95 21.24 28.42 45.66 54.34 20.18 50.44 15.95 27.08 32.92 66.55 58.05 45.13 03.77 11.17 26.02 80.00 72.92 86.02 47.96 70.09 52.03 1.20 –

3.70 ± 0.25(1) 0.89 ± 0.02(1) 1.26 ± 0.08(1) 1.78 ± 0.02(1) 0.93 ± 0.05(1) 0.67 ± 0.06(1) 1.48 ± 0.02(1) 1.26 ± 0.11(1) 1.61 ± 0.03(1) 2.52 ± 0.06(1) 1.07 ± 0.11(1) 0.82 ± 0.08(1) 1.78 ± 0.05(1) 1.32 ± 0.10(1) 1.99 ± 0.14(1) 2.26 ± 0.12(1) 1.81 ± 0.08(1) 0.87 ± 0.05(1) 2.29 ± 0.08(1) 1.44 ± 0.05(1) 0.48 ± 0.09(1) 2.03 ± 0.12(1) 0.37 ± 0.03(1) 0.16 ± 0.09(1) 1.29 ± 0.08(1) 3.26 ± 0.14(1) 3.16 ± 0.21(1) 3.33 ± 0.15(1) 3.03 ± 0.07(1) 1.85 ± 0.08(1) 2.52 ± 0.10(1) – 0.241 ± 0.015(1)

100 24.05 34.05 48.12 25.13 18.11 40.00 34.05 43.51 68.11 28.91 22.13 48.11 35.67 53.78 61.08 48.92 23.57 61.89 38.92 12.89 54.86 9.94 4.22 34.86 88.11 85.40 90.00 81.89 50.00 68.11 – 6.50

7.10 ± 0.25(0.01) 1.49 ± 0.03(0.01) 0.71 ± 0.04(0.001) 3.95 ± 0.12(0.01) 1.06 ± 0.01(0.001) 3.70 ± 0.07(0.001) 2.84 ± 0.08(0.01) 1.91 ± 0.95(0.01) 1.28 ± 0.13(0.001) 5.32 ± 0.03(0.01) 1.42 ± 0.09(0.001) 3.19 ± 0.02(0.01) 3.55 ± 0.04(0.01) 2.70 ± 0.05(0.01) 0.568 ± 0.03(0.01) 1.49 ± 0.08(0.1) 3.55 ± 0.12(0.001) 1.92 ± 0.13(0.1) 2.27 ± 0.02(0.1) 3.28 ± 0.06(0.01) 1.77 ± 0.09(0.001) 4.69 ± 0.07(0.01) 1.42 ± 0.04(0.001) 4.26 ± 0.03(0.01) 2.13 ± 0.14(0.01) 6.03 ± 0.09(0.001) 2.48 ± 0.16(0.01) 4.40 ± 0.03(0.01) 2.98 ± 0.02(0.01) 4.26 ± 0.11(0.01) 4.97 ± 0.05(0.01) 90.98 ± 0.89 (0.01) –

100 20.98 10.00 55.63 14.93 52.11 40.00 26.90 18.03 74.93 20.00 44.93 50.00 38.03 08.00 20.98 50.00 27.04 31.97 46.20 25.00 66.06 20.00 60.00 30.00 88.73 34.93 61.97 41.97 60.00 70.00 09.12

The results are presented in nmoles of 2-naphthylamine released per min under the assay conditions as Mean ± S.M.D. of the experiment conducted in triplicate at a concentration (Z)  10 5 M. The % residual activity is calculated w.r.t. control.

AC5H), 10.03 (1H, s, CHO); 13C NMR (75 MHz, CDCl3, d ppm): 184.45, 152.92, 148.65, 139.82, 139.64, 130.82, 129.89, 128.78, 126.28, 121.38, 120.34, 110.23.

10.06 (1H, s, CHO); 13C NMR (75 MHz, CDCl3, d ppm): 184.48, 160.78, 152.92, 139.82, 130.82, 129.42, 128.39, 126.78, 120.45, 114.65, 108.34, 55.89.

2.2.6.3. 3-(4-Methylphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (2c). Yield 72.43%; m.p. 98–100 °C[25]; IR (KBr, cm 1): 1682 (AC@O str), 1597(AC@N str), 1481, 1627(AC@CAstr), 2924 (ACHAstr); 1H NMR (300 MHz, CDCl3, d ppm): 2.36 (3 H, s, ACH3), 7.24(2H, d, J = 7.5 Hz, ArAH), 7.15(2H, d, J = 7.5 Hz, ArAH), 7.36–7.85 (5H, m, ArAH), 9.45 (1H, s, AC5H), 10.04 (1H, s, CHO); 13 C NMR (75 MHz, CDCl3, d ppm): 184.41, 152.9, 139.65, 138.82, 130.83, 130.04, 129.91, 129.37, 127.45, 126.80, 120.65, 108.96, 24.38.

2.2.6.5. 1-Phenyl-3-(thiophen-2-yl)-1H-pyrazole-4-carbaldehyde (2e). Yield 62.54%; m.p. 120–122 °C; IR (KBr, cm 1): 1682 (AC@O str), 1597(AC@N str), 1600, 1481 (AC@CAstr); 1H NMR (300 MHz, CDCl3, d ppm): 6.92–7.28(5H, m, ArAH), 7.36–7.42 (3 H, m, ArAH), 9.46 (1H, s, AC5H), 10.03 (1H, s, CHO); 13C NMR (75 MHz, CDCl3, d ppm): 184.03, 142.32, 140.08, 139.75, 129.67, 128.92, 127.95, 127.62, 126.18, 125.92, 120.62, 109.71.

2.2.6.4. 3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (2d). Yield 71.93%; m.p. 135–137 °C; IR (KBr, cm 1): 1666 (AC@O str), 1605 (AC@N str), 1450, 1620(AC@CAstr), 3124(ACHAstr), 1026(ACAOCH3 str); 1H NMR (300 MHz, CDCl3, d ppm): 3.82 (3 H, s, ACOCH3), 7.03(2H, d, J = 6.0 Hz, ArAH), 7.33(2H, d, J = 6.0 Hz, ArAH), 7.80–7.82(5H, m, ArAH), 8.54 (1H, s, AC5H),

2.2.6.6. 1-Phenyl-3-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde (2f). Yield 79.14%; m.p. 130–132 °C; IR (KBr, cm 1): 1674 (AC@O str), 1597(AC@N str), 1458–1605(AC@CAstr); 1H NMR (300 MHz, CDCl3, d ppm): 7.37–7.96(5H, m, ArAH), 8.03(2H, t, J = 8.1 Hz, ArAH), 8.15(1H, d, J = 8.4 Hz, ArAH), 8.72(1H, d, J = 8.4 Hz, ArAH), 9.21(1H, s, AC5H), 10.68 (1H, s, CHO); 13C NMR (75 MHz, CDCl3, d ppm): 184.08, 155.38, 149.75, 142.94, 139.79, 137.43, 129.79, 128.92, 126.11, 124.98, 120.62, 120.24, 109.78.

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N. Raghav, M. Singh / Bioorganic Chemistry 75 (2017) 38–49

(I)

(VI)

(II)

(VII)

(III)

(VIII)

Fig. 1. Effect of varying concentrations of hydrazones of aryl methyl ketone (I), pyrazole-4-carbaldehydes (II) and semicarbazones (III), thiosemicarbazones (IV) and phenyl hydrazone derivatives (V) at pH 6.0 on cathepsin B activity. Results are the mean of the experiment conducted in triplicates taking different concentrations of compounds. Activities are expressed as percent of control. Lineweaver-Burk plots for cathepsin B activity in presence and absence of hydrazones of aryl methyl ketone (VI), pyrazole-4carbaldehydes (VII) and semicarbazones (VIII), thiosemicarbazones (IX) and phenyl hydrazone derivatives (X) at a concentration reported in Table 1.

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N. Raghav, M. Singh / Bioorganic Chemistry 75 (2017) 38–49

(IX)

(IV)

(X)

(V) Fig. 1 (continued)

Table 2 Ki values of substituted hydrazones of aryl methyl ketone (1), pyrazole-4-carbaldehydes (2) and semicarbazones (3), thiosemicarbazones (4) and phenyl hydrazone derivatives (5) on cathepsin B, H and L. Code

Ki (nM) Hydrazones of arylmethylketones (1)

a b c d e f

1H-pyrazole-4-carbaldehydes (2)

Semicarbazones of 1Hpyrazole-4-carbaldehyde (3)

Thiosemicarbazones of 1H-pyrazole-4carbaldehyde (4)

Hydrazones of 1H-pyrazole4-carbaldehyde (5)

B

H

L

B

H*

L

B

H

L

B

H

L

B

H

L

420 150 3880 33 130 870

1490 2500 3906 1430 810 2910

5.61 0.24 769.2 0.395 17.24 33.4

5618 1960 4700 1110 1360 3150

5682 7570 56800 4030 2780 9090

6.5 0.314 141 0.391 12.8 20.3

4700 870 3510 64 150 1790

4808 4270 5494 3330 869 6672

11.92 2.73 47.7 3.22 56.15 64.72

76 49 2820 3.3 5.5 140

240 640 5380 53 42 1110

1.39 0.561 65.53 0.48 30.86 8.224

15000 10000 28490 2890 6670 3510

23200 19400 29400 15100 6700 7353

11.61 9.12 30.89 10.96 27.05 42.23

The results are presented as Mean ± S.M.D. of the experiment conducted in triplicate in presence and absence of a fixed concentration of different compounds (mentioned in Table 1 in parenthesis of each compound  10 5 M), separately. The results were then plotted between 1/V and 1/S to obtain Lineweaver–Burk plots and then the Ki values were calculated using Lineweaver–Burk equations for competitive and non-competitive inhibition depending upon the results (Figures of cathepsins H and L in the supplementary file). All the compounds exhibited competitive inhibition except marked * for cathepsin H only where non-competitive inhibition was observed.

2.2.7. General procedure for the synthesis of semicarbazones of 1Hpyrazole-4-carbaldehydes (3) Semicarbazide hydrochloride (0.07 g, 0.0006 mol) and sodium acetate (0.05 g, 0.0006 mol) was dissolved in 20 ml ethanol. To this solution, compound 2a (0.20 g, 0.0006 mol) was added. Then few

drops of concentrated hydrochloric acid were added and heated to reflux for 4 h. Reaction was monitored on TLC. The reaction mixture was cooled. The separated solid was filtered and recrystallized from ethanol to give desired semicarbazone. The structure elucidation of compound, 1-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4

N. Raghav, M. Singh / Bioorganic Chemistry 75 (2017) 38–49

(I)

(II)

(III)

(IV)

43

Fig. 4. Docking results (I), (II), (III) and (IV) showing the alignment of BANA substrate, reference inhibitor, leupeptin, most inhibitory compounds 4d and 4e, respectively in the active site of cathepsin B, respectively.

-yl)methylene)semicarbazide 3a was based on the spectral data (IR, 1H NMR & 13C NMR). 2.2.7.1. 1-((3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) semicarbazide (3a). Yield 77.1%; m.p. 192–194 °C[26]; IR (KBr, cm 1): 3379, 3202 (ANH str), 3094(@CHAstr), 1666(AC@O str), 1589(AC@N str), 1600, 1435 (AC@CAstr), 825(ACABr str); 1H NMR (300 MHz, CDCl3, d ppm): 7.24 (s, 2H, ANH2), 7.34–7.40(5H, m, ArAH), 7.47(2H, d, J = 8.1 Hz, ArAH), 7.65–7.67(2H, d, J = 8.1 Hz, ArAH), 7.80 (1H, s, AC5H), 8.26 (1H, s, ACH@N), 9.42 (1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm):165.12, 149.82, 143.58, 139.76, 132.64, 132.45, 130.18, 129.72, 129.56, 129.48, 123.42, 120.18, 107.82. Following exactly the same procedure as detailed above, other semicarbazone derivatives of (1H)-pyrazole-4-carbaldehyde (3b– 3f) were prepared from corresponding (1H)-pyrazole-4carbaldehydes (2b–2f). The physical and spectral data of synthesized compounds are given below. 2.2.7.2. 1-((3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) semicarbazide (3b). Yield 76.85%; m.p. 195–196 °C [26]; IR (KBr, cm 1): 3392, 3217 (ANH str), 2914(@CHAstr), 1690(AC@O str), 1589(AC@N str), 1600, 1427(AC@CAstr), 1342, 1512(ANO2 str); 1 H NMR (300 MHz, CDCl3, d ppm): 6.42 (s, 2H, ANH2), 7.40–7.87 (5H, m, ArAH), 7.93 (1H, s, AC5H), 7.96 (2H, d, J = 8.4 Hz, ArAH), 8.34 (2H, d, J = 8.4 Hz, ArAH), 9.15 (1H, s, ACH@N), 10.16 (1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 165.78, 150.15, 148.82, 143.56, 139.88, 139.22, 138.71, 130.22, 129.42, 128.52, 126.12, 120.38, 107.57.

2.2.7.3. 1-((3-(4-Methylphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)semicarbazide (3c). Yield 79.22%; m.p. 193–194 °C [26]; IR (KBr, cm 1): 3302, 3215 (ANH str), 3024(@CHAstr), 1682(AC@O str), 1597(AC@N str), 1627, 1441 (AC@CAstr); 1H NMR (300 MHz, CDCl3, d ppm): 2.34 (3H, s, ACH3), 6.32 (2H, s, ANH2), 7.01(2H, d, J = 7.5 Hz, ArAH), 7.32(1H, m, ArAH), 7.48(2H, d, J = 7.8 Hz, ArAH), 7.56(2H, d, J = 7.8 Hz, ArAH), 7.80(2H, d, J = 7.5 Hz, ArAH), 7.89 (1H, s, AC5H), 9.02 (1H, s, ACH@N), 10.05 (1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm):165.48, 150.45, 143.56, 139.28, 138.52, 132.22, 130.08, 129.65, 129.48, 127.59, 126.44, 120.26, 107.86, 22.62. 2.2.7.4. 1-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)semicarbazide (3d). Yield 75.21%; m.p. 194–195 °C [26]; IR (KBr, cm 1): 3347, 3132 (ANH str), 3024(@CHAstr), 1682(AC@O str), 1597(AC@N str), 1600, 1443 (AC@CAstr), 1057(ACAOCH3 str); 1H NMR (300 MHz, CDCl3, d ppm): 3.82 (3H, s, AOCH3), 6.41 (2H, s, ANH2), 7.05 (2H, d, J = 7.8 Hz, ArAH), 7.32–7.61 (5H, m, ArAH), 7.88 (2H, d, J = 7.8 Hz, ArAH), 7.94(1H, s, AC5H), 9.03 (1H, s, ACH@N), 10.06 (s, 1H, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 165.52, 160.98, 150.25, 143.05, 139.21, 130.06, 129.42, 128.56, 126.72, 125.58, 120.45, 115.12, 107.15, 55.25. 2.2.7.5. 1-((1-Phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-yl)methylene) semicarbazide (3e). Yield 88.96%; m.p. 240–242 °C; IR (KBr, cm 1): 3394, 3202 (ANH str), 3055(ACHAstr), 1690(AC@O str), 1589(AC@N str), 1600, 1450(AC@CAstr); 1H NMR (300 MHz, CDCl3, d ppm): 6.41(2H, s, ANH2), 7.16–7.34 (3H, m, ArAH), 7.45–7.70 (5H, m, ArAH), 8.20 (1H, s, AC5H), 9.09 (1H, s, ACH@N),

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10.20 (1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 165.25, 143.52, 141.45, 140.58, 139.92, 129.84, 128.52, 127.95, 127.83, 126.88, 125.68, 108.12. 2.2.7.6. 1-((1-Phenyl-3-(pyridin-2-yl)-1HApyrazol-4-yl)methylene) semicarbazide (3f). Yield 72.41%; m.p. 210–212 °C; IR (KBr, cm 1): 3349, 3232 (ANH str), 1690 (AC@O str), 3094(@CHAstr), 1597(AC@N str), 1600, 1435(AC@CAstr); 1H NMR (300 MHz, CDCl3, d ppm): 7.27 (2H, s, ACONH2), 7.28–7.48 (5H, m, ArAH), 7.61(1H, d, J = 6.9 Hz, ArAH), 7.40–7.85 (2H, m, ArAH), 8.65 (1H, d, J = 6.9 Hz, ArAH), 8.55 (1H, s, AC5H), 8.90 (1H, s, ACH@N), 10.34 (1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 165.21, 155.64, 149.72, 143.82, 142.28, 139.29, 137.52, 129.95, 128.96, 126.72, 124.48, 120.82, 120.24, 108.02. 2.2.8. General procedure for the synthesis of thiosemicarbazones of 1H-pyrazole-4-carbaldehydes (4) A mixture of compound 2a (0.20 g, 0.0006 mol) and thiosemicarbazide (0.056 g, 0.0006 mol) was taken in 20 ml ethanol. Very few drops of acetic acid were added in the reaction mixture to catalyze the reaction in the forward direction. The reaction mixture was refluxed for 4 h. The reaction mixture was cooled. The separated solid was filtered and recrystallized from ethanol to give desired thiosemicarbazone derivative. The structure elucidation of compound, 1-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)thiosemicarbazide 4a was based on the spectral data (IR, 1H NMR & 13C NMR). 2.2.8.1. 1-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) thiosemicarbazide (4a). Yield 77.32%; m.p. 123–125 °C; IR (KBr, cm 1): 3340, 3148 (ANH str), 3016 (@CHAstr), 1597 (AC@N str), 1543, 1443(AC@CAstr), 1281(AC@S str), 825(AC-Br str); 1H NMR (300 MHz, CDCl3, d ppm): 6.67 (1H, s, ANH/ASH), 6.95 (1H, s, ANH/ASH), 7.36–7.41 (2H, d, J = 8.1 Hz, ArAH), 7.49–7.66(5H, m, ArAH), 7.69 (2H, d, J = 8.1 Hz, ArAH), 8.06(1H, s, AC5H), 8.33 (1H, s, ACH@N), 10.95 (s, 1H, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 177.15, 149.54, 143.46, 139.34, 132.95, 132.48, 130.04, 129.79, 129.43, 126.37, 123.57, 120.22, 107.45. Following exactly the same procedure as detailed above, the other thiosemicarbazones of 1H-pyrazole-4-carbaldehyde (4b–4f) were prepared. The physical and spectral data of synthesized compounds are given below. 2.2.8.2. 1-((3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) thiosemicarbazide (4b). Yield 67.87%; m.p. 198–200 °C; IR (KBr, cm 1): 3364, 3178 (ANH str), 3024 (@CHAstr), 1597 (AC@N str), 1551, 1458(AC@CAstr), 1342, 1504(ANO2 str), 1288 (AC@S str); 1 H NMR (300 MHz, CDCl3, d ppm): 6.75(1H, s, ANH/ASH), 6.95 (1H, s, ANH/ASH), 8.03 (2H, d, J = 8.4 Hz, ArAH), 8.13 (1H, s, AC5H), 8.25–8.62 (5H, m, ArAH), 8.74–8.77 (2H, d, J = 8.4 Hz, ArAH), 9.55 (1H, s, ACH@N), 10.56 (1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 177.65, 150.89, 148.46, 143.14, 139.78, 139.25, 130.03, 129.45, 128.43, 126.45, 121.60, 120.93, 107.46. 2.2.8.3. 1-((3-(4-Methylphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiosemicarbazide (4c). Yield 88.76%; m.p. 110–112 °C; IR (KBr, cm 1): 3302, 3215 (ANH str), 3014 (@CHAstr), 1597 (AC@N str), 1627, 1441(AC@CAstr), 1285(AC@S str); 1H NMR (300 MHz, CDCl3, d ppm): 2.34 (3 H, s, ACH3), 6.64(1H, s, ANH/ASH), 6.82 (1H, s, ANH/ASH), 7.56–7.86 (5H, m, ArAH), 7.14 (2H, d, J = 6.9 Hz, ArAH), 7.42 (2H, d, J = 6.9 Hz, ArAH), 7.84 (1H, s, AC5H), 8.30 (1H, s, ACH@N), 9.34(1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 177.34, 150.32, 143.82, 139.72, 138.42, 130.10, 130.08, 129.48, 129.12, 127.44, 126.42, 120.34, 107.19, 22.61.

2.2.8.4. 1-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiosemicarbazide (4d). Yield 82.43%; m.p. 175–176 °C [26]; IR (KBr, cm 1): 3342, 3112 (ANH str), 3014(@CHAstr), 1597(AC@N str), 1600,1453 (AC@CAstr), 1288(AC@S str), 1036(-CAOCH3 str); 1 H NMR (300 MHz, CDCl3, d ppm): 3.42 (3 H, s, AOCH3), 6.31(1H, s, ANH/ASH), 7.28(1H, s, ANH/ASH), 7.01 (2H, d, J = 6.9 Hz, ArAH), 7.48 (2H, d, J = 6.9 Hz, ArAH), 7.62–7.85 (5H, m, ArAH), 7.77 (2H, d, ArAH), 7.93 (1H, s, AC5H), 8.34 (1H, s, ACH@N), 9.41 (1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm):178.53, 160.17, 151.54, 143.55, 139.93, 130.54, 129.58, 128.40, 126.77, 125.48, 120.42, 114.53, 107.92, 55.21. 2.2.8.5. 1-((1-Phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-yl)methylene) thiosemicarbazide (4e). Yield 68.56%; m.p. 220–222 °C; IR (KBr, cm 1): 3333, 3256 (ANH str), 3044 (@CHAstr), 1597 (AC@N str), 1600, 1497(AC@CAstr), 1242(AC@S str); 1H NMR (300 MHz, CDCl3, d ppm): 6.63 (1H, s, ANH/ASH), 7.08 (1H, s, ANH/ASH), 7.06–7.34 (5H, m, ArAH), 7.39–7.45 (3H, m, ArAH), 8.26 (1H, s, AC5H), 8.34 (1H, s, ACH@N), 10.94 (1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 178.12, 144.53, 141.78, 140.15, 139.58, 129.63, 128.92, 127.84, 127.65, 126.38, 125.54, 120.87, 108.12. 2.2.8.6. (1-((1-Phenyl-3-(pyridin-2-yl)-1H-pyrazol-4-yl)methylene) thiosemicarbazide (4f). Yield 52.42%; m.p. 186–188 °C; IR (KBr, cm 1): 3378, 3146 (ANH str), 3016 (@CHAstr), 1597 (AC@N str), 1600, 1443 (AC@CAstr), 1242(AC@S str); 1H NMR (300 MHz, CDCl3, d ppm): 6.92 (1H, s, ANH/ASH), 7.28 (1H, s, ANH/ASH), 7.39–7.71 (5H, m, ArAH), 8.04 (1H, d, J = 6.9 Hz, ArAH), 8.41 (2H, m, ArAH), 8.54 (1H, d, J = 6.9 Hz, ArAH), 8.65 (1H, s, AC5H), 8.78 (s, 1H, ACH@N), 10.64 (1H, s, ANH); 13C NMR (75 MHz, CDCl3, d ppm):177.98, 155.43, 149.72, 143.23, 142.28, 139.75, 137.51, 129.53, 128.91, 126.76, 124.98, 120.42, 120.12, 108.06. 2.2.9. General procedure for the hydrazones derivatives of 1Hpyrazole-4-carbaldehydes (5) A mixture of compound 2a (0.2 g, 0.0006 mol) and phenyl hydrazine (0.065 g, 0.0006 mol) was taken in 20 ml ethanol. Thereafter, a catalytic amount of acetic acid was added and the reaction mixture was heated to reflux for 4 h. The progress of the reaction was monitored by TLC examination. On completion of reaction, the reaction mixture was cooled at room temperature. The separated solid was filtered and recrystallized from ethanol to give desired phenylhydrazone derivative. The structure elucidation of compound, 1-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)met hylene)-2-phenylhydrazine 5a was based on the spectral data (IR, 1 H NMR & 13C NMR). 2.2.9.1. 1-((3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-phenylhydrazine (5a). Yield 76.88%; m.p. 172–174 °C; IR (KBr, cm 1): 3214(ANH str), 1605 (AC@N str), 1558, 1454 (AC@CAstr), 825(ACABr str); 1H NMR (300 MHz, CDCl3, d ppm): 6.42–7.09 (5H, m, ArAH), 7.24–7.38 (5H, m, ArAH), 7.42 (2H, d, J = 7.5 Hz, ArAH), 7.54 (2H, d, J = 7.5 Hz, ArAH), 8.12 (1H, s, AC5H), 8.93 (1H, s, ACH@N), 10.68 (1H, br s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 150.33, 143.48, 143.12, 139.78, 132.25, 131.13, 130.84, 129.87, 129.78, 129.49, 126.38, 123.34, 120.22, 118.04, 116.36, 107.32. Following exactly the same procedure as detailed above, the other hydrazones derivatives of (1H)-pyrazole-4-carbaldehyde (5b–5f) were prepared. The physical and spectral data of synthesized compound are given below2.2.9.2. 1-((3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)2-phenylhydrazine (5b). Yield 85.65%; m.p. 228–230 °C; IR (KBr, cm 1): 3248(ANH str), 1597(AC@N str), 1543, 1443 (AC@CAstr), 1342, 1512(ANO2 str); 1H NMR (300 MHz, CDCl3, d ppm): 6.75–

N. Raghav, M. Singh / Bioorganic Chemistry 75 (2017) 38–49

7.43 (5H, m, ArAH), 7.55–7.68 (5H, m, ArAH), 7.80 (2H, d, J = 8.4 Hz, ArAH), 8.27 (2H, d, J = 8.4 Hz, ArAH), 8.94(1H, s, AC5H), 9.01(1H, s, ACH@N), 10.29(1H, Br s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 150.38, 148.32, 143.24, 143.13, 139.87, 139.72, 130.09, 129.66, 129.59, 128.38, 126.29, 121.86, 120.21, 118.72, 116.24, 107.82. 2.2.9.3. 1-((3-(4-Methylphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-phenylhydrazine (5c). Yield 84.43%; m.p. 122–124 °C; IR (KBr, cm 1): 3225(ANH str), 1605(AC@N str), 1585, 1481 (AC@CAstr), 3024(@CHAstr); 1H NMR (300 MHz, CDCl3, d ppm): 2.40 (3H, s, CH3), 6.43–7.08 (5H, m, ArAH), 7.24 (2H, d, J = 7.5 Hz, ArAH), 7.45 (2H, d, J = 7.5 Hz, ArAH), 7.48–7.68 (5H, m, ArAH), 8.15(1H, s, AC5H), 8.94 (1H, s, ACH@N), 10.65(1H, br s, ANH); 13 C NMR (75 MHz, CDCl3, d ppm): 150.57, 143.23, 144.01, 139.86, 138.42, 130.12, 130.09, 129.69, 129.62, 129.56, 127.47, 126.21, 120.41, 118.72, 116.46, 106.29, 24.28.

45

phenyl hydrazone 5 derivatives, respectively. The structures of the synthesized compounds were confirmed by spectral data. The synthesized compounds 2a–2f, 3a–3f, 4a–4f and 5a–5f exhibited characteristic peaks in the IR as well as in 1H NMR spectra (the details are provided in the Section 2). In 1H NMR spectra, the singlet peak observed in the region of d 10.03–10.11 for ACHO protons in compounds, 2a–2f were not observed in succeeding compounds 3a–3f, 4a–4f and 5a–5f, instead peaks at 8.24–9.15 (ACH@N) were observed. In addition, singlets in the region of d 9.42–10.95 were obtained for ACONH, ACSNH, ANHPh in compounds 3a–3f, 4a–4f and 5a–5f, respectively. Simultaneously in the IR spectra also AC@O str of 1H-Pyrazol-4-carbaldehydes 2a– 2f at 1680–1690 cm 1 were not observed in 3a–3f, 4a–4f and 5a–5f. However these derivatives clearly exhibited ANH str absorptions at 3115–3374 cm 1. 3.2. Pharmacological evaluation

2.2.9.4. 1-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2-phenylhydrazine (5d). Yield 76.54%; m.p. 152 °C [26]; IR (KBr, cm 1): 3185(ANH str), 1597(AC@N str), 1585, 1485 (AC@CAstr), 3054(@CHAstr); 1H NMR (300 MHz, CDCl3, d ppm): 3.84 (3H, s, CH3), 6.48–7.02 (7H, m, ArAH), 7.39 (2H, d, J = 7.8 Hz, ArAH), 7.45–7.85 (5H, m, ArAH), 8.14 (1H, s, AC5H), 8.91(1H, s, ACH@N), 10.68 (1H, br s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 160.62, 150.25, 143.86, 143.14, 139.69, 129.71, 129.58, 128.59, 126.30, 125.54, 120.17, 119.02, 116.67, 116.98, 114.87, 106.78, 55.81. 2.2.9.5. 2-Phenyl-1-((1-phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-yl) methylene)hydrazine (5e). Yield 78.96%; m.p. 138–140 °C; IR (KBr, cm 1): 3215(ANH str), 1597(AC@N str), 1582, 1480 (AC@CAstr); 1 H NMR (300 MHz, CDCl3, d ppm): 6.76–7.07 (5H, m, ArAH), 7.29–7.38 (5H, m, ArAH), 7.46–7.89 (3H, m, ArAH), 8.14 (1H, s, AC5H), 8.91 (1H, s, ACH@N), 10.68 (1H, br s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 143.18, 143.08, 141.71, 140.54, 139.64, 129.69, 129.62, 128.94, 127.89, 127.61, 126.43, 125.68, 120.32, 118.87, 116.28, 106.23.

Active participation of cathepsins in tumor-promoting and cancer progression by digestion of extracellular matrix at invasion and metastasis stage has emphasized the revelation of anticathepsin therapy as novel concept of cancer treatment. Presence of elevated levels of cathepsins and decreased levels of cytosolic inhibitors in variety of –itis condition [27–28], also signifies role of anticathepsin therapy in disease management. In recent years molecules belonging to different class have been reported as cathepsins B, H and L [29–36] inhibitors. Pyrazoles (I and II) [37–38], thiosemicarbazones (III and IV) [39–41] as individual entities have already been reported as cathepsin inhibitors. Here, we have evaluated the synergistic effect of two pharmacophores, pyrazoles and their imine functionalities, toward anticathepsin activity.

2.2.9.6. 2-Phenyl-1-((1-phenyl-3-(pyridin-2-yl)-1H-pyrazol-4-yl) methylene)hydrazine (5f). Yield 78.96%; m.p. 153–155 °C; IR (KBr, cm 1): 3152(ANH str), 1597(AC@N str), 1545, 1454 (AC@CAstr); 1 H NMR (300 MHz, CDCl3, d ppm): 6.74–6.97(5H, m, ArAH), 7.34 (1H, d, J = 8.1 Hz, ArAH), 7.38–7.64 (5H, m, ArAH), 7.68 (1H, m, ArAH), 7.86 (1H, m, ArAH), 8.89 (1H, d, J = 8.1 Hz, ArAH), 9.14 (1H, s, AC5H), 9.45 (1H, s, ACH@N), 10.68 (1H, br s, ANH); 13C NMR (75 MHz, CDCl3, d ppm): 155.45, 149.26, 143.32, 143.15, 142.34, 139.62, 129.69, 129.44, 128.83, 126.41, 124.31, 120.81, 120.12, 118.65, 116.47, 116.34, 107.23. 3. Result and discussion 3.1. Chemistry The synthesis of various structural analogs i.e., hydrazones of aryl methyl ketone (1), pyrazole-4-carbaldehydes (2), and their semicarbazones (3), thiosemicarbazones (4) and phenyl hydrazone derivatives (5) was carried out as detailed in Scheme 1. Phenyl hydrazones 1 were synthesized by condensation of the respective acetophenones and phenyl hydrazine. 4Formylpyrazolic precursors 2 were initially synthesized by Vilsmeier-Haak formylation from phenyl hydrazones. These pyrazole-4-carbaldehyde derivatives on refluxing with semicarbazide hydrochloride, thiosemicarbazide, phenyl hydrazine in ethanol gave desired semicarbazone 3, thiosemicarbazone 4,

Reports by Abadi et al.[42] on 1,3,4-trisubstituted pyrazole derivatives (V-VII) as antitumor and antiangiogenic agents and presence of pyrazoles [43], hydrazones [44], semicarbazones [45– 46] and thiosemicarbazones [47–50] as key functional groups in variety of anticancer agents also provided the foundational support for the current study involving structural diversity within the aryl portion of the target compounds.

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Scheme 1.

Pyrazole moiety has already been a part of various existing drug such as celecoxib, lonazolac, crizotinib, tolpiprazole and cefoselis [51]. Based on these observations in the present work we synthesized some acetophenone phenylhydrazone based pyrazole carbaldehydes, their semicarbazones, thiosemicarbazones and phenylhydrazones and screened their inhibitory potential on cathepsins B, H and L. The compounds 1b, 2b, 4d and 4e have been

identified as potential inhibitors of the target enzymes with Ki values equivalent to the potential inhibitors of peptidyl nature taken as positive control in experimental studies. 3.2.1. Enzyme inhibition studies Table 1 shows the effect of individual compound on cathepsins B, H and L activities at a particular concentration given in parenthesis 10 5 M. The potential of inhibition shown by the

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synthesized compounds is related to electronic effects exerted by the substituent present on benzene ring as well as with the type of side chain present in the molecule. The thought to combine two pharmacophores has resulted in enhanced inhibition. It can be observed from Table 1 that cathepsin L is inhibited at a lower concentration in each compound as compared to cathepsin B. Cathepsin H is inhibited least in that order. Once the comparative inhibitory potential of the compounds were established, effect of different concentration of individual compounds was observed on the activity of each enzyme. Effect of varying concentrations of hydrazones of aryl methyl ketone, 1a–1f; pyrazole-4-carbaldehydes, 2a–2f; semicarbazones, 3a–3f; thiosemicarbazones, 4a–4f; phenyl hydrazone derivatives, 5a–5f on cathepsin B activity are presented in Fig. 1 (I, II, III, IV and V respectively). Similar figs. pertaining to cathepsins H and L inhibiting activities by individual compound of the series 1, 2, 3, 4 and 5 are provided in ‘supplementary data’ as Figs. 2 and 3. Based on these results it can be deduced that active site of cathepsin L is more susceptible to inhibition as compared to cathepsins B and H. It was found that most effective inhibition was observed for p-nitrophenyl and p-methoxyphenyl residues. Electronic effects seem to be more prominent over mesomeric effect. p-Bromophenyl, thiophenyl- and pyridyl- substituents inhibited to a lesser extent. p-toluyl group was least inhibitory. After establishing the inhibitory potential of the compounds, Lineweaver-Burk plots were drawn studies were 1/V versus 1/S in presence and absence of fixed amount of inhibitor were drawn to evaluate the type of inhibition and to calculate the Ki values. All the compounds inhibited these enzymes in a competitive manner except for pyrazole-4-carbaldehydes which inhibited cathepsin H in a non-competitive manner. The Ki values calculated for cathepsins B, H and L have been presented in Table 2. For cathepsins B and H, thiosemicarbazones (4) exhibited maximum inhibition followed by hydrazones (1). In the related series maximum inhibition for cathepsin B was observed for methoxy substituted compounds, e.g. 4d (Ki  3.3 nM) followed by 1d (Ki  33 nM), 3d (Ki  64 nM), 2d (Ki  1110 nM) and 5d (Ki  2890 nM), respectively. However, in cathepsin H, the maximum inhibition was observed for compounds having thienyl ring substitution, e.g. 4e (Ki  42 nM) followed by 1e (Ki  810 nM), 3e (Ki  869.6 nM), 2e (Ki  2780 nM) and 5e (Ki  6700 nM), respectively. Structural analogs bearing substituents like methyl (1c), (2c), (3c), (4c) and (5c) were not effective inhibitors for both enzymes. Among the series (3), (4) and (5) for cathepsins B and H; thiosemicarbazones were most inhibitory followed by semicarbazones and phenyl hydrazones. The difference in inhibiting potency can be explained on the basis of hard and soft nucleophilic interaction between enzyme active site group (ACH2SH) and the two isosteres, C@O and C@S, present in the target molecules. The structural analogs of series (3) and (4) were better inhibitors than phenyl hydrazones (5) indicating that a polar side chain is more acceptable at this position. Among various phenyl hydrazones, series (1) has been found to more inhibitory than (5). In cathepsin L, the maximum inhibition was observed for compounds having nitro and methoxy substitution on benzene ring, e.g. Ki values for compounds having nitro substitution; 1b (0.24 nM) followed by 2b (0.314 nM), 4b (0.561 nM), 3b (2.73 nM) and 5b (9.12 nM). The Ki values for methoxy substituted compounds were, e.g. 2d (0.391 nM) followed by 1d (0.395 nM), 4d (0.48 nM), 3d (3.22 nM) and 5d (10.96 nM), respectively. For cathepsin L, the most potent compound were the hydrazones 1b and 1d, the aldehydes 2b and 2d and the thiosemicarbazones 4b and 4d, all of hich with either 4-nitrophenyl or 4-methoxyphenyl residues.

47

The Ki values have been presented in Table 2. Lineweaver Burk plots for cathepsins H and L to ascertain the type of inhibition exerted by individual compound of the series 1, 2, 3, 4 and 5 are depicted as Figs. 2 and 3 (VI, VII, VIII, IX and X) and are supplied in supplementary data. As cathepsins B, H and L contain cysteine at their active site therefore, inspired by the mechanism proposed for inhibition of cathepsin L with thiosemicarbazones analogs [41], a mechanism for inhibition by the designed compounds citing active site amino acids of cathepsin B has been proposed (Scheme 2). It can be observed that under the experimental conditions, the reference inhibitor leupeptin showed 98.8% inhibition at 10 6 M concentration for cathepsin B and whereas Leu-CH2-Cl showed 93.5% inhibition at 10 5 M concentration for cathepsin H as positive controls for respective enzymes, which is in accordance with the previously reported results. The results obtained are comparable as reported for brain cathepsin B [20] and cathepsin H [21]. Some of the compounds exhibited inhibition comparable to reference peptidic inhibitors e.g. leupeptin for cathepsin B and Leu-CH2Cl for cathepsin H. As reported in literature, leupeptin is a potential peptide inhibitor of cathepsin B [52], inhibited the goat brain cathepsin B competitively with Ki value of 12
5  l0 9 M [20] whereas Ki value for human liver cathepsin Β [53] was reported to be 7
0  10 9 M. In contrast, Ki value for human liver cathepsin H was reported to be 9.2  10 6 M [54]. It has been reported that cathepsin L is inhibited by leupeptin with a Ki value of 1.45  10 9 M [54].

Within the semicarbazones derivatives, the most potent inhibitors, 4d demonstrated an impressive Ki value of 3.3 nM and 53 nM and 4e showed a Ki value of 5.5 nM and 42 nM for cathepsin B and cathepsin H. The extent of inhibition observed is similar to those reported previously for pyrazoline derivatives where nitro substituted N-formylpyrazolines and N-benzoylpyrazolines showed Ki values of 1.1  10 9 M and 19.5  10 8 M for cathepsin B and Ki values of 5.19  10 8 M and 9.8  10 7 M for cathepsin H, respectively [36]. In case of cathepsin B, the Ki value for 4d and 4e in the order of 10 8-10 9 M is quite comparable to Leupeptin although the latter is peptidyl in nature and provide complementary binding with the enzyme active site. The inhibitory potential of title compounds and the reference inhibitor, Leu-CH2-Cl, for cathepsin H is comparable. For cathepsin L also the synthesized compounds have proved to be effective inhibitors as compared to peptidyl inhibitor, leupeptin. Nitro substituted N-formylpyrazolines and Nbenzoylpyrazolines exhibited Ki values of 6.4  10 10 and 5.7  10 9 M for cathepsin L [32]. In an attempt to obtain new cathepsin inhibitors with higher potency, we have identified small molecules as inhibitors of cathepsin B, H and L with equivalent potency compared to peptidyl inhibitors. 3.3. Molecular docking experiment The docking approach used in this study was aimed to observe the binding of compounds to the active site of cathepsins B, H and

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N. Raghav, M. Singh / Bioorganic Chemistry 75 (2017) 38–49

Scheme 2.

L. Etotal of iGemDOCK, the empirical scoring function is the estimated as sum total of van der Waal, H-bonding and electrostatic interaction between enzyme and the ligand. In case of cathepsin B Etotal of leupeptin, a peptidyl inhibitor taken as the reference is greater than the designed compounds (Table 3, supplementary data). A higher value of leupeptin-cathepsin B binding energy is due to peptide protein interaction. iGemDOCK provide algorithms for flexible docking approach for both ligands and proteins [55]. Leupeptin, a peptide based inhibitor of cathepsin B, provides a complementary binding with the enzyme and results in higher binding energies in comparison to the molecules under study. The synthesized compounds are smaller in structure as well as less flexibile than leupeptin, therefore exhibit lower binding energies. Fig. 4, I and II represent the docking poses of BANA and reference inhibitor, leupeptin, respectively for cathepsin B. The compounds are displayed as wire frame and enzyme backbone as ribbon and interacting amino acid residues are displayed as sticks; the hydrogen bonding residues are in green and the van der Waal interactions are in shown in grey color. The active site amino acid residues Cys-29 and His-199 of catalytic triad of cathepsin B and Trp-30, Gly-27, Gly-73 and Gly-198 has been found to interact with the BANA substrate as well as with the reference inhibitor, Leupeptin. We can see that most of the amino acids residues interact with the best inhibitors 4d and 4e (Fig. 4, III and IV) suggesting a competitive nature of the compounds and supports the in vitro studies carried out on these compounds. The compounds have been evaluated as inhibitors with potency comparable to reference inhibitor, leupeptin. Like leupeptin, the designed compounds also demonstrated competitive inhibition. The proposed mechanism is shown in Scheme 2. Fig. 5 I and II, (supplementary data) show docking results of Leu-bNA and reference inhibitor, Leu-CH2Cl, respectively in the

active site of cathepsin H. The amino acids Asn-79, Asn-112, Gln70, Glu-73 and Pro-77 are involved in the stabilization of the Leu-bNA and Leu-CH2Cl inhibitor. 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 H [21,56] but possess only one amino acid residue as compared to leupeptincathepsin B. Therefore, the Leu-CH2Cl-cathepsin H interaction cause a decrease in energy only of 70.33 kcal/mol. As listed in Table 4 (supplementary data), all the designed compounds have been found to show more decrease in ligand-cathepsin H interaction energy than Leu-CH2Cl-cathepsin H. The docking poses of the best inhibitors 4d and 4e for cathepsin H are shown as Fig. 5, III and IV, (supplementary data). As we can see that most of the amino acids residues of cathepsin H which interact with the substrate (I) and the reference inhibitors (II) also interacts with the designed compounds, therefore the compounds competes with the substrate and inhibits the enzyme in a competitive manner. Docking figures of Z-Phe-Arg-4 mbNA and reference inhibitor, leupeptin in the active site of cathepsin L (Fig. 6, I and II) are provided in supplementary data. The amino acids Gly-68, Asp-162 and His 163 are involved in their stabilization. Compounds 1b and 2b interact with the active site Gly-68 and Asp-162 and 2b with His 163 (Fig. 6, III and IV; supplementary data). The active site amino acids that interact with the substrate and the inhibitor partially are involved in the binding of compounds. This explains the competitive inhibition exerted by the compounds. In-silico studies have been used as a supporting tool for enzyme inhibition studies. After applying the consensus scoring criteria [57], the scoring function showing Etotal are presented in Table 5 (supplementary data). Here, we have used decrease in total energy of the enzyme–compound complex as a key to measure of the binding affinity between

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compound and the enzyme site. However, approach of free energy to understand such interactions [58] has also been used to describe such interactions. The behaviour of the enzyme–ligand interaction predicted by in silico studies can give an idea about the interaction between these two. 4. Conclusion In the present work, we have evaluated each compound of structurally related five series for its inhibitory activity against cathepsin B, H and L and identified synthetic non-peptidyl inhibitors for cathepsin B, H and L having comparable inhibitory potency with known peptidyl inhibitors. It was observed that for cathepsin B and H linear, series (1) and polar side chain, series (3) and (4) compounds resulted in better inhibition than carbaldehydes, (2) and their phenylhydrazones, (5). Representatives, (5) were least inhibitory to cathepsin L also. Preliminary profiling indicated these derivatives are potent inhibitors for cathepsins B, H and L and can lead to the development of improved inhibitors of cathepsins B, H and L. Acknowledgement One of the authors, Mamta Singh is thankful to CSIR New Delhi, India for award of SRF and also to Kurukshetra University, Kurukshetra for providing necessary research laboratory facilities. The authors have declared no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bioorg.2017.08. 006. References [1] T. Nomura, N. Katunuma, J. Med. Invest. 52 (2005) 1–9. [2] J.A. Joyce, A. Baruch, K. Chehade, N. Meyer-Morse, E. Giraudo, F.Y. Tsai, D.C. Greenbaum, J.H. Hager, M. Bogyo, D. Hanahan, Cancer Cell 5 (2004) 443–453. [3] A. Schweiger, A. Staib, B. Werle, M. Krasovec, T.T. Lah, W. Ebert, V. Turk, J. Kos, Br. J. Cancer 82 (2000) 782–788. [4] I.T. Lim, S.O. Meroueh, M. Lee, M.J. Heeg, S. Mobashery, J. Am. Chem. Soc. 126 (2004) 10271–10277. [5] V. Gocheva, X. Chen, C. Peters, T. Reinhecke, J.A. Joyce, Biol. Chem. 391 (2010) 937–945. [6] K.M. Bell-McGuinn, A.L. Garfall, M. Bogyo, D. Hanahan, J.A. Joyce, Cancer Res. 67 (2007) 7378–7385. [7] M. Sever, J. Filipic, T.T Lah Brzin, Biol. Chem. 383 (2002) 839–842. [8] P.D. Greenspan, K.L. Clark, R.A. Tommasi, S.D. Cowen, L.W. McQuire, D.L. Farley, J.H. Van Duzer, R.L. Goldberg, H. Zhou, Z. Du, J.J. Fitt, D.E. Coppa, Z. Fang, W. Macchia, L. Zhu, M.P. Capparelli, R. Goldstein, A.M. Wigg, J.R. Doughty, R.S. Bohacek, A.K. Knap, J. Med. Chem. 44 (2001) 4524–4534. [9] M. Frizler, F. Lohr, M. Lulsdorff, M. Gutschow, Chemistry 17 (2011) 11419– 11423. [10] P.E. Edem, S. Czorny, J.F. Valliant, J. Med. Chem. 57 (2014) 9564–9577. [11] M.D. Mertens, J. Schmitz, M. Horn, N. Furtmann, J. Bajorath, M. Mares, M. Gutschow, Chem. Biol. Chem. 15 (2014) 955–959. [12] J. Borisek, M. Vizovisek, P. Sosnowski, B. Turk, D. Turk, B. Mohar, M. Novic, J. Med. Chem. 58 (2015) 6928–6937. [13] N. Raghav, R. Kaur, Med. Chem. Res. 23 (2014) 4669–4679. [14] N. Raghav, R. Kaur, Int. J. Biol. Macromol. 80 (2015) 710–724.

49

[15] S. Garg, N. Raghav, Bioorg. Chem. 67 (2016) 64–74. [16] M. Singh, N. Raghav, Bioorg. Chem. 59 (2015) 12–22. [17] N. Raghav, S. Jangra, A. Kumar, S. Bhattacharya, D. Wadhwa, J. Sindhu, RSC Adv. 6 (2016) 34588–34599. [18] M. Singh, N. Raghav, Eur. J. Med. Chem. 77 (2014) 231–242. [19] N. Raghav, M. Singh, S. Garg, R. Kaur, S. Jangra, I. Ravish, Int J. Pharmaceut. Sci. Res 6 (7) (2015) 2944–2949. [20] R.C. Kamboj, S. Pal, H. Singh, J. Biosci. 15 (1990) 397–408. [21] N. Raghav, R.C. Kamboj, S. Parnami, H. Singh, Indian J. Biochem. Biophys. 32 (1995) 279–285. [22] C.P. Huber, R.L. Campbell, S. Hasnain, T. Hirama, R. To, Crystal Structure of the Tetragonal Form of Human Liver Cathepsin B, 2013. (http://www.ebi.ac.uk/ pdbe-srv/view/entry/2ipp/citation.html). [23] G. Guncar, M. Podobnik, J. Pungercar, B. Strukelj, V. Turk, D. Turk, Structure 6 (1998) 51–61. [24] S.F. Chowdhary, L. Joseph, S. Kumar, S.R. Tulsidas, S. Bhat, E. Ziomek, R.M. Menard, J. Sivaraman, E.O. Purisima, J. Med. Chem. 51 (2008) 1361–1368. [25] S.C. Shetty, V.C. Bhagat, Asian J. Chem. 20 (2008) 5037–5045. [26] R. Pundeer, P. Ranjan, K. Pannu, Syn. Commun. 39 (2009) 316–324. [27] Y. Ikeda, T. Ikata, T. Mishiro, S. Nakano, M. Ikebe, S. Yasuoka, J. Med. Invest. 47 (2000) 61–75. [28] T. Shikimi, D. Yamamoto, M. Hanada, J. Pharmacobiol. Dyn. (Japan) 10 (1987) 750–751. [29] N. Raghav, M. Singh, Bioorg. Med. Chem. 22 (2014) 4233–4245. [30] I. Ravish, N. Raghav, RSC Adv. 5 (2015) 50440–50453. [31] N. Raghav, S. Garg, RSC Adv. 5 (2015) 72937–72949. [32] S. Garg, N. Raghav, AIMS Mol. Sci. 3 (2016) 454–465. [33] M. Singh, N. Raghav, Eur. J. Pharm. Sci. 54 (2014) 28–39. [34] N. Raghav, S. Garg, BOAJ Cancer Res. Ther. 2 (2016) 1–6. [35] N. Raghav, S. Garg, Eur. J. Pharm. Sci. 60 (2014) 55–63. [36] N. Raghav, S. Garg, Bioorg. Chem. 57 (2014) 43–50. [37] N. Asaad, P.A. Bethel, M.D. Coulson, J.E. Dawson, S.J. Ford, S. Gerhardt, M. Grist, G.A. Hamlin, M.J. James, E.V. Jones, G.I. Karoutchi, P.W. Kenny, A.D. Morley, K. Oldham, N. Rankine, D. Ryan, S.L. Wells, L. Wood, M. Augustin, S. Krapp, H. Simader, S. Steinbacher, Bioorg. Med. Chem. Lett. 19 (2009) 4280–4383. [38] D.M. Huryn, A.B. Smith III, Curr. Top. Med. Chem. 9 (2009) 1206–1216. [39] J.P. Mallari, A. Shelat, A. Kosinski, C.R. Caffrey, M. Connelly, F. Zhu, J.H. McKerrow, R.K. Guya, Bioorg. Med Chem. Lett. 18 (2008) 2883–2885. [40] G.D.K. Kumar, G.E. Chavarria, A.K. Charlton-Sevcik, W.M. Arispe, M.T. MacDonough, T.E. Streckera, Shen-En Chen, Siim, B.G.D.J. Chaplin, M.L. Trawick, K.G. Pinney, Des. Bioorg. Med. Chem. Lett. 20 (2010) 1415–1419. [41] G.D.K. Kumar, G.E. Chavarria, A.K. Charlton-Sevcik, G.K. Yoo, J. Song, T.E. Strecker, B.G. Siim, D.J. Chaplin, M.L. Trawick, K.G. Pinney, Bioorg. Med. Chem. Lett. 20 (2010) 6610–6615. [42] A.H. Abadi, A.A.H. Eissa, G.S. Hassan, Chem. Pharm. Bull. 51 (2003) 838–844. [43] M.I. El-Zahar, S.S. Adb El-Karim, M.E. Haiba, M.A. Khedr, Acta. Pol. Pharm. 68 (2011) 357–373. [44] J. Easmon, G. Purstinger, K.S. Thies, G. Heinisch, J. Hofmann, J. Med. Chem. 49 (2006) 6343–6350. [45] K. Islam, S.M.M. Ali, M. Jesmin, J.A. Khanam, Cancer Biol. Med. 9 (2012) 242– 247. [46] S.N. Pandeya, P. Yogeeswari, E.A. Sausville, A.B. Mauger, V.L. Narayanan, Arzneimittelforschung 52 (2002) 103–108. [47] S.N. Pandeya, J.R. Dimmock, Die Pharmazie 48 (1993) 659–666. [48] J. Patole, S. Padhye, S. Padhye, C.J. Newtona, C. Ansonb, A.K. Powell, Indian J. Chem.-A 43A (2004) 1654–1658. [49] R.W. Brockman, J.R. Thomson, M.J. Bell, H.E. Skipper, Cancer Res. 16 (1956) 167–170. [50] J. Easmon, G. Pürstinger, G. Heinisch, T. Roth, H.H. Fiebig, W. Holzer, W. Jäger, M. Jenny, J. Hofmann, J. Med. Chem. 44 (2001) 2164–2171. [51] S.G. Kucukguzel, S. Senkardes, Eur. J. Med. Chem. 97 (2015) 786–815. [52] A. Baici, M. Gyger-Marazzi, Eur. J. Biochem. 129 (1982) 33–41. [53] C.G. Knight, Biochem. J. 189 (1980) 447–453. [54] A. Azaryan, A. Galoyan, Neurochem. Res. 12 (1987) 207–213. [55] J.-M. Yang, C.-C. Chen, GEMDCK: a generic evolutionary method for molecular docking, Prot.: Struct. Funct. Bioinform. 55 (2004) 288–304. [56] W.N. Schwartz, A.J. Barrett, Biochem. J. 191 (1980) 487–497. [57] J.-M. Yang, Y.-F. Chen, T.-W. Shen, B.S. Kristal, D.F. Hsu, J. Chem. Inf. Model. 45 (2005) 1134–1146. [58] Z. Zhang, V. Martiny, D. Lagorce, Y. Ikeguchi, E. Alexov, M.A. Miteva, PLoS One 9 (2014) 1108884.