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Chalcones, semicarbazones and pyrazolines as inhibitors of cathepsins B, H and L.

Highlights • Three bio macromolecules cathepsins B, H and L of physiological and pathological significance have been selected for the study.• The molecules have been designed by combining two important pharmacophores their cyclized analogues and were studied for their inhibitory effects on selected enzymes.• Two isomeric forms of chalconesemicarbazones are reported for the first time.• The synthesized compounds showed a competitive inhibition towards cathepsins B, H and L.• Docking experiments were run along with to relate with in vitro studies.


Introduction
Semicarbazones are important bioactive compounds possessing different biological activities such as anticonvulsant [1], antimicrobial [2], antiviral [3], antitumoral [4], antihypertensive [5] and antitubercular [6]. These compounds have also been found to be potent inhibitors to cruzain, cysteine protease of Trypanosoma cruzi and been shown to be trypanocidal [7]. The trypanocidal activity is related to cruzain inhibiting tendencies of the semicarbazones. Falcipain, another protozoal cysteine protease has also been reported to be inhibited by variety of semicarbazones. Therefore, semicarbazone inhibitors of cysteine proteases have potential use for prevention and treatment of protozoan infections such as trypanosomiasis, malaria and leishmaniasis. It has also been reported that semicarbazones of aryl and alkyl carbonyl compounds inhibit cysteine proteases of parasites more as compared to mammalian proteases and therefore indicate the possibility of their therapeutic use. The compounds also find use in inhibiting cysteine proteases associated with carcinogenesis, including cathepsins B, H and L [8].
We are working in the identification of small molecular weight compounds as inhibitors to endogenous proteolytic activities [47][48][49][50][51][52]. In quest for the identification of some novel inhibitors to cathepsin B and cathepsin H, we have recently reported various non-peptidyl inhibitors such as bischalcones based quinazoline-2(1H)-ones, quinazoline-2(1H)-thiones (iv-vi) [53] and acyl hydrazides, triazoles (i-iii) [ Effects of hydrazones [55], hydroxyl chalcones [56] and their cyclized derivatives, formyl and benzoyl pyrazolines [57] have also been established on these enzymes. To explore further in this direction the present work is focused on chalcones, chalconesemicarbazones and their cyclized derivatives as inhibitors to cathepsins B, H and L. The intention to synthesize semicarbazones of chalcones was undertaken keeping in view that the semicarbazones possessing azomethine group in conjunction with ␣,␤-unsaturation will have extended pharmacological activities. The compounds were further cyclized to pyrazolines which are also reported to possess diverse biological activities such as antimicrobial [58], antiamoebic [59], anti-inflammatory [60], anticancer [61], antidepressant [62] and antitubercular [63] activities. Effect of synthesized compounds was observed on cathepsins B, H and L to establish a structure activity relationship between the 1,3diphenylprop-2-en-1-ones, substituted chalconesemicarbazones and substituted 3,5-diphenyl-2-pyrazoline-1-carboxamide derivatives. Structure activity relationship (SAR) studies of open chain and cyclized derivatives can result in identification of potential inhibitors among related class of compounds [55,56]. A comparative account of different moieties introduced in the synthesized compounds on cathepsin B, H and L, three lysosomal cysteine proteases inhibiting tendency has also been evaluated. The studies can lead to the development of new inhibitors of these enzymes.

Materials
All the chemicals were of analytical grade. Fast Garnet GBC (o-aminoazotoluene diazonium salt), ␣-N-benzoyl-d,l-arginine-2-naphthylamide (BANA), Leu-␤NA and ZPheArg-4m␤NA were purchased from Bachern Feinchemikalien AG, Switzerland. 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.

Methods
Melting points were taken in open capillaries and are uncorrected. The progress of the reactions was monitored on silica gel G plates using iodine vapor as visualizing agent. Elisa plate reader was used for measuring absorbance in the visible range. The spectrofuge was used for centrifugation purpose. IR spectra were recorded on Horizon 300 MHz spectrometer. NMR spectra were recorded on Bruker 300 MHz instrument. The chemical shifts are expressed in ppm units from an internal TMS standard. All commercially available reagents were used as-received.

Synthesis
The title compounds were synthesized according to Scheme 1.

Synthesis of substituted 1,3-diphenylprop-2-en-1-ones (1a-1j)
Acetophenone (0.01 mol) was added to equimolar quantity of substituted benzaldehyde (0.01 mol) dissolved in methanol (25 ml). To this solution equimolar NaOH pellets (0.01 mol) were added at once and the reaction mixture was stirred for 40 min at room temperature. Excess of methanol was again added and then again stirred for next 40 min at room temperature. It was then diluted with cold water. The product was separated, filtered and washed with water until neutral. The resulting chalcones were purified by recrystallization with methanol. The structures were characterized using melting point compared with literature melting point [19,64], IR and NMR.

Synthesis of substituted chalconesemicarbazones (2a-2j, 2a -2j )
To an aqueous semicarbazide hydrochloride (0.02 mol), sodium acetate (0.03 mol) was dissolved. This solution was added to the ethanolic solution of substituted 1,3-diphenyl-2-en-1-ones (0.01 mol) (1a-1j). Then, few drops of concentrated hydrochloric acid were added. Reaction mixture was stirred at 40 • C and was monitored on TLC. After completion, the reaction mixture was poured into ice and precipitate, so obtained was filtered, washed with water, dried and recrystallized with ethanol. The product thus obtained was found to possess two stereoisomeric forms ( Fig. 1) which were separated on column. The two isomers have been characterized using 1 H NMR. To an aqueous sodium hydroxide (0.017 mol), semicarbazide hydrochloride (0.0085 mol) was dissolved. This solution was added to the solution of substituted 1,3-diphenyl-2-en-1-ones (0.005 mol) (1a-1j) in ethanol (25 ml) and then, refluxed for 6-8 h. TLC monitoring was extensively done. The solvent was evaporated and the solid so obtained was filtered, washed with water to neutral reaction, dried and recrystallized from ethanol. The structure was characterized using melting point compared  with literature melting point [65], IR and NMR spectroscopic techniques.

Purification of cathepsins B, H and L
All the purification steps were carried out at 4 • C. Cathepsins B, H and L were extracted and purified from goat liver by the established procedure reported previously [53] including the steps of acetone powder preparation, homogenization, acid autolysis, 30-70% (NH 4 ) 2 SO 4 fractionation, molecular sieve chromatography on sephadex G-100 and ion exchange chromatographies on CM Sephadex C-50 and DEAE A-50 sephadex. The specific activities of the cathepsin B, H and L were equal to ∼11.28 nmol/min/mg, ∼24.01 nmol/min/mg and ∼16.78 nmol/min/mg, respectively.

Enzyme inhibition studies
Cathepsin B and L activity was determined using BANA [66] and ZPheArg-4m␤NA [67] substrate at pH 6.0, respectively whereas cathepsin H activity was determined using Leu-␤NA [68]   assay using the respective substrates. The experiments were performed in triplicate for each concentration and % activity has been calculated with respect to the control where no compound was added but an equivalent amount of solvent was present. Enzyme assays were similarly conducted at lower concentrations of each compound to observe the inhibitory effect of compounds at varying concentrations. The results are presented in Figs. 2-4 for cathepsins B, H and L, respectively.

Drug modelling studies
Docking studies were performed using iGemdock software. To conduct these, small molecular weight ligands were prepared using marvin sketch and were saved as MDL Mol File. Enzyme structure active site was retrieved from the Protein Data Bank (http://www.rcsb.org/) as cav2IPP B PYS.pdb, cav8PCH H NAG.pdb and cav3BC3L CSW [69][70][71]. The prepared ligands and the binding site was loaded in the iGemdock programme and docking was run by setting GA parameters for Standard Docking Accuracy Settings, Docking experiments show a decrease in energy when enzyme and ligands interact. The E total resulting after H-bonding and van der Waals interactions are presented in Tables S1-S3 (Supplementary data
The synthesis of chalconesemicarbazones resulted in two isomeric form 2a-2j and 2a -2j . These two isomeric forms were separated by column chromatography on silica gel using petroleum ether and ethyl acetate as eluent. The separated isomers were characterized by 1 H NMR data. The isomers obtained were characterized on the basis of interaction of lone pair on nitrogen with C H proton as explained by Curphy-Morrison chemical shift [72]. When lone pairs on nitrogen are syn to C H bonds (2a-2j), the proton is shifted upfield showing the lower ı value. When lone pairs on nitrogen are anti to C H bonds (2a -2j ), the proton is shifted downfield. The conclusion was arrived on the basis of 3D molecular modelling studies. In 2i where lone pair on nitrogen is syn to C 2 -H (Scheme 1) on minimization of energy in 3D molecular modelling ( Fig. 1) [73] clearly indicate that lone pair on nitrogen is in optimal position to affect shielding of C 2 -H and also to C 3 -H to some extent because of extension of conjugation. In 2i the lone pair on nitrogen is anti to C 2 -H and after 3D molecular modelling in minimized energy structure shown in Figure 1, it is clear that these can't have any effect on CH CH protons. On comparing the 1 H NMR, peaks for C 2 -H and C 3 -H for chalcones and chalconesemicarbazones, distinctive shielding effect is observed due to substitution of adjacent C O group with C N NHCONH 2 . These isomeric forms are reported here for the first time, although a large number of reports are available in literature on the synthesis of these compounds [74][75][76]. In case of p-OCH 3 and p-NO 2 substituted isomers only one isomer was formed i.e. 2f and 2h, respectively.
On cyclization of chalconesemicarbazones, diarylpyrazolines are obtained and each of the two isomeric forms (2a-2j) and (2a -2j ) resulted in single product (3a-3j). In contrast, two isomeric forms of pyrazolines have been reported by Shekarchi et al. [77]. In the 1 H NMR spectra of (3a-3j), the characteristic ABX pattern has been obtained.

Effect of synthesized compounds on in vitro endogenous proteolysis in liver homogenate
Proteolytic activity is inhibited appreciably in presence of these compounds. In some cases ∼100% inhibition is achieved at 1 × 10 −4 M concentration. Moreover the inhibition was more at 3.0 h and less at 24.0 h, emphasizing that the compounds inhibit the proteolytic activity reversibly. In case of chalcones, chloro and unsubstituted compounds showed 100% inhibition. For chalconesemicarbazones and diaryl pyrazolines, chloro and nitro substituted compounds showed 100% inhibition. In addition, o-and p-methoxy substituted chalconesemicarbazone and o-methoxy pyrazoline also exhibited 100% inhibition while unsubstituted and methyl substituted chalconesemicarbazones and pyrazolines exerted inhibition to lesser extent.
After establishing the inhibition of endogenous proteolytic activity in presence of chalcones and their derivatives at pH 5.0, where most of the proteolytic activity is attributed to cysteine proteases it was thought proper to study the effect of synthesized compounds on purified cathepsins B, H and L.

Effect of synthesized compounds on the activity of cathepsins B, H and L
The effect of differently substituted chalcones (1a-1j), chalconesemicarbazones (2a-2j, 2a -2j ) and diarylpyrazolines (3a-3j) on the activity of cathepsins B, H and L at varying concentrations is shown in Figs. 2-4. From these plots of % residual activities versus the concentrations of different compounds, it can be observed that at a particular concentration all the synthesized compounds inhibited cathepsin L activity more than cathepsin B and H.

The inhibition type and K i values
The type of inhibition caused by various compounds was determined through Lineweaver-Burk double reciprocal plot. In order to establish inhibition ability of the under consideration compounds results were compared with potent inhibitors of cathepsin B, e.g. Leupeptin and cathepsin H e.g. Leu-CH 2 -Cl, respectively. As reported in literature, Leupeptin being a potent peptide inhibitor of cathepsin B, inhibited the goat brain cathepsin B competitively with K i value of 12.5 × l0 −9 M [66] whereas K i value for human liver cathepsin B [78] was reported to be 7.0 × 10 −9 M. In contrast, K i value for human liver cathepsin H was reported to be 9.2 × 10 −6 M [79] and K i value for goat brain cathepsin L was reported to be 1.45 × 10 −9 M [67].
For evaluating the type of inhibition caused by different chalcones, chalconesemicarbazones and diarylpyrazolines, cathepsins B, H and L activity was measured at varying substrate i.e. BANA, Leu ␤NA and ZPheArg-4m␤NA concentration in presence and absence of a fixed concentration of compound. The plots of 1/V and 1/[S] were straight lines intersecting at the Y-axis and shows that value of V max remains constant in all the compounds whereas the value of K m change with each compound. These studies suggested that chalcones, chalconesemicarbazones and diarylpyrazolines are competitive inhibitors to cathepsins B, H and L. Using the Lineweaver-Burk equation of competitive inhibition the K i values were calculated, which has been presented in Table 1.

Structure activity relationship
Proteases contribute to tumour cell invasion and angiogenesis and are commonly associated with metastasis. It is now recognized that cysteine proteases play pivotal roles in cancer progression. Of all the cathepsins, studies have shown that cathepsins B, H and L are of significant importance as these are involved in various pathologies and oncogenic processes. Though these enzymes have been intensively studied as valuable targets for drug discovery and development, a number of peptide [80] and non-peptide [53,54] inhibitors have been described in literature. Towards this endevour we have now synthesized chalcones and their semicarbazone and pyrazoline derivatives with different functionalities in order to explore their inhibitory potential on these important enzymes keeping in view the inhibitory potential of semicarbazones and pyrazolines as well as chalcones on cysteine proteases. The work has been envisaged by combining the two active pharmacophores. Out

Small molecular weight representative molecules
Further it can be observed that chalconesemicarbazones showed maximum inhibition than their cyclized precursor pyrazolines followed by chalcones on the activity of cathepsin B and cathepsin H. Whereas, for cathespin L chalcones and chalconesemicarbazones were found to show more inhibition than pyrazolines. The results are discussed later in the next section along with the results obtained after molecular docking.
Among the substituted chalcones, chalconesemicarbazones and pyrazolines, the compounds bearing electron withdrawing moiety were found to be more inhibitory probably inducing more nucleophilic character at the site of attack shown in Scheme 2.
The results obtained were compared with potential inhibitors of cathepsin B and L e.g. Leupeptin and cathepsin H, e.g. Leu-CH 2 -Cl, respectively [54]. It can be observed that Leupeptin showed ∼98.8% inhibition at 10 −6 M concentration for cathepsin B whereas it showed ∼52.0% inhibition at 10 −5 M concentration for cathepsin H and 89.2% inhibition at 10 −9 M concentration for cathepsin L which is in accordance with the previously reported results. Similarly, Leu-CH 2 -Cl showed ∼9.5% inhibition at 10 −5 M concentration for cathepsin B whereas it showed ∼93.5% inhibition at 10 −5 M concentration for cathepsin H and 1.5% inhibition at 10 −5 M concentration for cathepsin L. The results obtained are comparable with earlier results reported for brain cathepsin H [68], cathepsin B and cathepsin L [81].

Molecular docking experiment
The docking approach was used to study the interaction of compounds with the active site of cathepsin B, H and L to observe binding poses of individual compounds. Individual binding poses of each compound was assessed and their interactions in the active site of the enzyme were analyzed. The empirical scoring function of iGemDOCK is the estimated sum total of van der Waals, H-bonding and electrostatic energy.
Table S1 (Supplementary data) presents the data of docking studies of different chalcones, their acyclic and cyclic semicarbazone derivatives in cathepsin B active site (cav2IPP B PYS.pdb). The results clearly indicate that binding energy of chalcones is less than pyrazoline derivatives which is less than the open chain semicarbazones. The binding energy of chalcones (1a-1j), The docking results explain that the title compounds may not be potent inhibitors to the protease activity to an extent of specific pepidyl inhibitors but certainly suggest a differential inhibitory pattern of these compounds on cathepsin B activity. However, maximum interaction is observed for BANA with a score of −129.83.  Decrease in total energy for leupeptin-cathepsin B has come out be −116.42 of which the contribution of van der Waals interactions are more with a score of −86.37 as compared to H-bonds with a score of −29.44. 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. iGem-DOCK provide algorithms for flexible docking approach for both ligands and proteins [82] therefore flexible ligands like leupeptin 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. In the quest of synthesizing potent inhibitors of enzymes a structure-activity relationship is required where cyclic and acyclic analogues are to be studied. There are various pharmaco-dynamic reactions which molecule acting as drug encounters in vivo such as cyclization, oxidation, reduction etc. so we have here, provided the comparative account of in vitro studies and in-silico study on the structurally related compounds or their isomers. These compounds are synthesized from the same starting material. The open chain analogues are the intermediates therefore there are a need to screen their inhibitory potential along with the products i.e. the cyclized derivatives, pyrazolines in order to establish structure activity relationship. In this direction the present study has been undertaken to evaluate the effect of 1, 3-diphenylprop-2-en-1ones (1a-1j), substituted chalconesemicarbazones (2a-2j, 2a -2j ) which are open chain compounds and their cyclic analogues i.e. substituted 3,5-diphenyl-2-pyrazoline-1-carboxamide derivatives (3a-3j) on the activity of cathepsin B, a protease of immense importance. During the synthetic studies of semicarbazones we came across the presence of two stereoisomers in the preparation. Docking experiments were conducted on these two isomers also and a differential effect was observed (Table S1-Supplementary data). The results of the docking studies support the in vitro experimental studies conducted on liver cathepsin B. It has been observed that chalcones were less inhibitory to the enzyme activity whereas semicarbazones and pyrazoline derivatives. This was quite surprising as chalcones having an ␣-␤ unsaturated carbonyl group seems to be more responsive towards thiol attack of cysteine present at the active site of enzyme but both the results after, in-silico docking experiments as well as in vitro solution studies clearly indicate that presence of azomethine carboxamide might have provided larger interaction to the binding of compound with the enzyme active site and there by exerting more inhibition than its precursor chalcones, which is further confirmed by the enzyme-ligand interaction study. Fig. 8 shows the binding of most inhibitory compound 1a, 2a, 2a and 3a in the active site of cathepsin B. The ligands are shown in pink, residues involved in H-bonding and van-der Waals interactions are shown as green and grey, respectively. It is clearly observed that cys-29 and His-199 residues present at the catalytic site of the enzyme are involved in the binding of compounds. Based on these interactions, a mechanism of inhibition (Scheme 2) by chalcones and chalconesemicarbazones is proposed on the basis of a previously reported inhibition of cathepsin L, another important cysteine protease by thiosemicarbazones [83]. The thiolate of cys-29 attacks on the nucleophilic site in chalcones and chalconesemicarbazones, where the later is more stabilized to the binding site. In addition, Gly-198, Ala-200 and Trp-30 amino acids residues are also involved in the stabilization of compounds in binding site. Pyrazolines are also better inhibitors to cathepsin B activity than chalcones again emphasizes on the importance of binding of compound with the active site. Though pyrazolines lack any nucleophilic site in their structure but still they show better inhibitory potential probably due to the presence of side chain carboxamide and nitrogen in the ring altogether providing a larger binding area to the enzyme site. Among themselves the cyclized pyrazolines were less inhibitory than open chain semicarbazones. It can be interpreted that acyclic compounds interact more with the active site being straight in nature not to the extent of peptidyl inhibitors but certainly greater than the cyclized pyrazoline derivatives. This gives an understanding of the inhibition caused by the target compounds on structural basis. Similar trends have been observed for cathepsin H. However, in cathepsin H, the most inhibitory compound in each series have been found to be 1g, 2g, 2c and 3b. Fig. 9 shows the binding of the most inhibitory compounds in the active site of cathepsin H. The binding energies of title compounds in the amino acyl  binding site of cathepsin H (cav8PCHH NAG) is presented in Table  S2 (Supplementary data). Experimental results obtained can be correlated with the ligand-binding interactions. It is observed that for 1g, 2g, 2c and 3b, the binding energies computed come out to be −82.21, −96.59, −93.63 and −84. 78. In each series, these most inhibitory compounds show a decrease in binding energy towards higher side. The binding energies show effective interaction between the enzyme binding site and inhibitory compounds may be responsible for these inhibition patterns. Fig. 10 shows the binding of most inhibitory compounds 1b, 2c, 2a and 3g in the active site of cathepsin L. The results of the docking studies support the in vitro experimental studies conducted on goat liver cathepsin L. The binding energies of title compounds in the amino acyl binding site of cathepsin L (cav3BC3L CSW) is presented in Table S3 (Supplementary data). The binding energies of 1b, 2c, 2a and 3g were found to be −77.07, −92.93, −104.04 and −114.94, respectively.