Efficient synthesis, biological evaluation, and docking study of isatin based derivatives as caspase inhibitors

ABTRACT In this paper, a new series of isatin-sulphonamide based derivatives were designed, synthesised and evaluated as caspase inhibitors. The compounds containing 1-(pyrrolidinyl)sulphonyl and 2-(phenoxymethyl)pyrrolidin-1-yl)sulphonyl substitution at C5 position of isatin core exhibited better results compared to unsubstituted derivatives. According to the results of caspase inhibitory activity, compound 20d showed moderate inhibitory activity against caspase-3 and −7 in vitro compared to Ac-DEVD-CHO (IC50 = 0.016 ± 0.002 μM). Among the studied compounds, some active inhibitors with IC50s in the range of 2.33–116.91 μM were identified. The activity of compound 20d was rationalised by the molecular modelling studies exhibiting the additional van der Waals interaction of N-phenylacetamide substitution along with efficacious T-shaped π-π and pi-cation interactions. The introduction of compound 20d with good caspase inhibitory activity will help researchers to find more potent agents.


Introduction
Caspases, cysteinyl aspartate-specific proteases, are a family of signalling molecules playing a key role in apoptosis. Apoptosis is a physiological suicide process which gives an opportunity to dismantle unwanted cells population during animal development and tissue homeostasis 1 . Morphological changes such as DNA strand breaks along with nuclear membrane damage occur as a result of some biochemical events during apoptosis 2 . Two intrinsic and extrinsic pathways are responsible for initiating the apoptosis process. Binding of certain protein to the death receptor activates caspase-8 and subsequently triggers apoptosis by promoting effector caspases (-3, À6, À7). It should be noted that caspase enzymes are classified as initiator (caspase-2, caspase-8, caspase-9, and caspase-10) and effector (caspase-3, caspase-6, and caspase-7) which are exploited in re sponse to proapoptotic signals 3,4 . Caspase-3, activated by the upstream caspase-8 and caspase-9, is considered as a crucial mediator of apoptotic cell death in mammals by which more than 500 cellular substrates are cleaved to execute the apoptosis programme 5,6 . Regarding the close relationship between apoptosis and the wide range of disease, caspase inhibitors are capable of opening new paths to treat several diseases involving immunodeficiency, Alzheimer's, Parkinson's, Huntington's diseases, ischaemia, brain trauma, and amyotrophic lateral sclerosis 7 . Taking the obtained data from the X-ray structure of caspase-3 into account, four main binding sites (S1-S4) are determined in which the binding to the S2 and S3-pockets are responsible for inhibitory activity and selectivity of caspase-3, respectively [8][9][10] . This knowledge along with the importance of this family clearly helps medicinal chemists to design new specific inhibitors of caspase enzymes [11][12][13][14][15][16][17][18][19] .
Isatin sulphonamides are introduced as a new class of potent and selective non-peptide caspase-3 and À7 inhibitors. Previously, various isatin sulphonamide derivatives were prepared and evaluated as caspase-3 inhibitors [20][21][22][23][24][25] . The studies indicated the connection between carbonyl group of isatin ring and cysteine thiol in the binding site of the enzyme. 5-Pyrrolidinyl sulphonyl isatins are evidently found effective in inhibition of the caspase-3 and À7 in vitro. The selectivity of 5-pyrrolidinyl sulphonyl isatins is referred to the interaction of pyrrolidine ring with S2 subsite of enzyme without the interaction with S1 subsite of caspase-3 26 . The sidechains, attached to pyrrolidine meaning methoxymethyl or phenoxymethyl groups, occupy the S3 pocket. In this regard, many studies have been focussed on the synthesis of several modified isatin derivatives (1), relying on the structure-activity relationship (SAR) studies ( Figure 1). Interestingly, it was observed that good IC 50 values in nanomolar ranges are obtained when hydrophobic groups are attached to the N-1 position of structure 1 ( Figure 1). Furthermore, the amide moiety is also found necessary in producing various potent inhibitors [27][28][29] . Considering the above mentioned findings about the importance of isatin sulphonamide derivatives, especially as caspase-3 inhibitors and following our ongoing projects on the design and synthesis of biologically active agents [30][31][32][33][34][35][36][37] , we synthesised isatin based compounds containing N-aryl acetamide and N-prop-2-yn-1-yl as caspase-3 and À7 inhibitors through the structural modification of compound 1.
In this paper, 33 compounds are synthesised and their structures are deduced by IR, 1 H, 13 C NMR, mass spectroscopy, and elemental analysis. For example, the IR spectrum of these three series showed the stretching bands, related to C¼O bonds of ketone and amide functional groups at nearly 1700 and 1670 cm À1 , respectively. The mass spectrum of each compound displayed the molecular ion (M þ ) peak, which is consistent with a 1:1 adduct, formed by the substitution at NH of isatin and loss of chlorine and bromine atom of propargyl bromide or 2-chloro-Narylacetamide derivatives. The 1 H-NMR spectrum of compounds exhibited the characteristic signals at d 4. 3-4.6 and 8.2-8.8 ppm related to NCH 2 and NH, respectively. The characteristic signals related to pyrrolidine and isatin moiety at aliphatic and aromatic region confirmed the structures of final compounds. The 1 H-decoupled 13 C-NMR spectrum of compounds showed characteristic signals at related aliphatic and aromatic regions which are in agreement with the proposed structure.
As can be seen in Table 1, those compounds containing no substituent at C 5 position of isatin core (R 2 ¼ H) are weak inhibitors compared to the positive control. All amounts are provided as inhibition percentage at 20 lg/ml. Among this series, the best and weakest activity was observed in 11c and 11f with inhibition percentage of 71% and 5%, respectively. The presence of 2-(phenoxymethyl)pyrrolidine functionality on isatin core led to the more active compounds against caspase-3 and À7 than that of substituted ones with pyrrolidin-1-yl sulphonyl moiety.
In compounds 20a-k, the comparison between the para substituted derivatives revealed that the electron-donating substituents (20j, 20k) exhibited the lowest enzymatic inhibition. The most active compound was the 4-chlorophenylacetamide containing derivative, meaning 20d against caspase-3 and À7. Compounds 20a-c and 20f have also appreciable IC 50 values and can be regarded as moderated caspase-3 and À7 inhibitors in comparison to Ac-DEVD-CHO (IC 50 ¼ 0.016 ± 0.002 lM). In compounds 19a-k and 20 a-k, the least electronegative and more bulky atom, bromine, had clear negative effect on inhibitory potency of the compound compared to fluorine and chlorine containing derivatives. As previously reported, compounds with a selectivity index greater than 1.5 are considered as selective inhibitors of caspase -3, so, compounds 19a, 19d, 19e, 20c, 20d, and 20e exhibited this selectivity. Regarding the significant activity and selectivity of compound 20d, this compound could be studied for further modification to develop novel hit compounds.

Docking study
To investigate the binding mode of these potent inhibitors, molecular docking computations were performed using Autodock Tools (ver.1.5.6) programme 43 . Compound 20d was docked into the active site of caspase-3 crystallographic structure (PDB ID: 1GFW), retrieved from protein data bank (http://www.rcsb.org/ pdb/home/home.do) ( Figure 2). The phenyl ring of phenoxymethyl group formed pi-cation interaction with HIS:121. The isatin core formed T-shaped p-p interactions with His 121 and Tyr 204. His 121 formed a carbon hydrogen bond in isatin sulphonamide crystal ligand. A Pi-alkyl interaction is formed between the oxygen of sulphonyl group and Trp 206 and Tyr 204. The carbonyl moiety interacted through p-sulfur with Cys 163 in compound 20d and through p-hydrogen bond in isatin sulphonamide. The p-p stacked interaction is formed between isatin core and Phe 256 in isatin sulphonamide and compound 20d. Moreover, N-phenylacetamide substitution provided enough length for more efficient interactions, like an additional van der Waals interaction between LeuA 168 and ThrA 166 and phenyl moiety. Table 2, presentesd the comparision between the type of interaction and involved amino acid residues of the most active compound, 20d, and isatin sulphonamide. These interactions along with distances are schematically presented in Figure 3. Superimposition of the binding pose of 20d and natural ligand at the 1GFW active site is shown in Figure 4. The binding interaction energy of compound 20d is À4.04 kcal/mol, which stated that this compound is less potent than statin sulphonamide (-5.44 kcal/mol) towards caspase-3.

Conclusion
A series of novel isatin-sulphonamide derivatives were designed, synthesised and evaluated for their caspase-3 and À7 inhibitory activity. The results showed that most of the synthesised compounds exhibited moderate inhibitory activity against caspase-3 and À7. The results revealed that 4-chloro phenylacetamide derivative 20d exhibited the best profile of inhibitory activity on caspase-3 with IC 50 value of 2.33 mM. The docking studies showed the perfect binding of compound 20d to the active site of caspase-3 enzyme. The prepared product 20d in the present study may be subjected to further optimisation to find more effective agent as caspase-3 inhibitor.
Other starting materials, chemical reagents, and solvents used in this study were commercially available (from Merck and Aldrich Chemicals) and were used without further purification. TLC was conducted on silica gel 250 micron. Melting points were determined on a Kofler hot stage apparatus and are uncorrected. The IR spectra were run on a Shimadzu 470 spectrophotometer (potassium bromide disks). Mass spectra were recorded on an Agilent Technologies (HP) 5973 mass spectrometer operating at an ionisation potential of 70 eV. The NMR spectra were recorded on a Varian unity 500 spectrometer, and the chemical shifts (d) are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard.

Caspase-3 and -7 inhibition assay
The activity assay of caspase-3 was performed in a system of 50 mL containing 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 50 mM HEPES pH 7.4, 10 mM Ac-DEVD-AMC (Bachem Bioscience, Philadelphia, PA, USA) and 2 nM caspase-3 in the 1 mL DMSO. Caspase-3 was incubated with synthesised compounds in 384-well plates for 10 min. The %inhibition of target compounds was measured at 20 mg/ml. The enzymatic activity of the caspase-3 was measured based on production of a fluorogenic substrate, 7amino-4-methyl coumarin, which was monitored for 10 min and detected using an EnVision (PerkinElmer, Wellesley, MA, USA) at k ex ¼ 360 nm and k em ¼ 460 nm. The initial rate of hydrolysis was determined using the early linear region of the enzymatic reaction curve. For IC 50 determination, about 8 concentrations of the synthesised compounds were freshly prepared by three-fold serial dilutions DMSO and the assay buffer such that following the addition of the inhibitors, DMSO concentration would equal to 0.2%. and GraphPad Prism 5 software was used to calculate the IC 50 values.