Synthesis, Characterization, Molecular Docking, Cytotoxic and Antioxidant Activities of Di(indolyl)thiazolylpyrazoles

Some new di(indolyl)thiazolylpyrazoles were prepared from the synthetic intermediate E-1,3-di(1H-indol-3-yl) prop-2-en-1-one under ultrasonication and studied their cytotoxic and antioxidant activities. All the compounds were screened for in vitro cytotoxic activity on three cancer cell lines. The compound 7e exhibited appreciable anticancer activity on NCI-H1299, HCT-166 p53 and PC-3 cancer cell lines. The binding conformation of the target molecules was predicted by docking methodology to explain the biological activities. In fact, the docking studies indicated that could be used as possible leads for therapies against cancers. Amongst all the tested compounds dimethoxy substituted di(indolyl)thiazolylpyrazole (7i) displayed significant radical scavenging activity.


VEGA-QSAR for toxicity prediction
We used VEGA-QSAR model platform (http://www.vega-qsar.eu) for toxicity prediction of synthesized compounds. It includes one or more QSAR models for different end points [43]. Here we accessed CAESAR models for prediction of carcinogenicity, mutagenicity and skin sensitization of synthesized compounds.

Molecular docking
Several reports suggest that molecules with indole nucleus have promising antiproliferative activity by inhibiting tubulin polymerization [44]. We obtained the structural information of tubulin in complex with compound CN2 from protein Data Bank (PDB ID: 1SA0) [45]. Protein and ligands for docking were prepared in Chimera 1.10.2 [46] by removing water molecules, adding hydrogen atoms and Gesteiger partial charges. Molecular docking simulation was performed with AutoDock 4.2 [47] using empirical free energy force field and Lamarckian genetic algorithm conformational search with the default parameters. The grid box was set around the cochicine-binding site in αβ tubulin hetero dimer. Previous reports show and that Cys-β241, Lys-β254, Asn-α101, Thr-α224, Gln-α176 are the key interacting residues for anti-tubulin agents in colchicines binding pocket with grid centre X 115.57 A 0 , Y 89.1142 A 0 , Z 6.0915 A 0 and grid point spacing 0.375 A 0 .

Measurement of cancer viability:
Cell viability and proliferation were determined with EZ-cytox cell viability assay kit based on the cleavage of the tetrazolium salt to water-soluble formazan by succinatetetrazolium reductase system, which belongs to the respiratory chain of the mitochondria and is active only in the viable cells. Therefore the amount of formazan dye increased with an increase in cell viability [48]. Initially, the cells were seeded into 96-well culture plates at 1 × 10 4 cells/ml and NCI-H1299 and HCT-166 p53 cells were cultured in DMEM and PC-3 cells were cultured in RPMI-1640 media containing 10% FBS at 37 o C. When cells reached 70% confluence, the medium was replaced with DMEM or RPMI-1640 containing 10% FBS and each 100 µM of compounds for 24 h. EZ-cytox cell viability kit reagents were added to the medium, and the cells were incubated for 1 h. The index of cell viability was determined by measuring formazan production with a microplate reader at an absorbance of 450 nm. Cells in fresh medium without any test compound were used as the control. The % cell viability was calculated by the formula:

%Cell viability=(Mean absorbance in test wells/Mean absorbance in control wells) × 100
As the % cell viability decreases the % inhibition increases.      The % inhibition was calculated by the formula: % Inhibition=100-% Cell viability.

In vitro antioxidant activity
The compounds 7a-7i were tested for antioxidant activity by DPPH, NO and H 2 O 2 methods.
DPPH radical scavenging activity: The hydrogen atom or electron donation ability of the compounds was measured from the bleaching of the purple colored methanol solution of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). This property makes it suitable for spectrophotometric studies. 1 mL of various concentrations of the test compounds (25,50,75 and 100 μg/mL) was added to 4 mL of 0.004% (w/v) methanol solution of DPPH. After a 30 min incubation period at room temperature, the absorbance was read against blank at 517 nm. Ascorbic acid was used as the standard. Tests were carried out in triplicate. The percent inhibition (I%) of free radical production from DPPH was calculated by the following equation.

I%=[(A control -A sample )/A control ] × 100
Where A control was the absorbance of the control reaction (containing all reagents except the test compound), A sample was the absorbance of the test compound (containing methanolic DPPH and test compound). IC 50 value of each compound was considered as the concentration (μg/ mL) of the compound at which 50% of DPPH reduction was observed [49,50].

I%=[(A control -A sample )/A control ] × 100
Where A control was the absorbance of the control reaction (containing all reagents except the test compound), A sample was the absorbance of the test compound (containing all reagents and test compound).
Nitric oxide (NO) scavenging activity: Nitric oxide scavenging activity was measured by slightly modified methods [51][52][53]. Nitric oxide radicals (NO) were generated from sodium nitroprusside. 1 mL of sodium nitroprusside (10 mM) and 1.5 mL of phosphate buffer saline (0.2 M, pH 7.4) were added to different concentrations (25, 50, 75 and 100 μg/mL) of the test compounds and incubated for 150 min at 25 o C. After incubation 1 mL of the reaction mixture was treated with 1mL of Griess reagent (1% sulfanilamide, 2% H 3 PO 4 and 0.1% naphthylethylenediaminedihydrochloride). The absorbance of the chromatophore was measured at 546 nm. Ascorbic acid was used as the standard. Tests were carried out in triplicate. Nitric oxide scavenging activity was calculated by the following equation.

I%=[(A control -A sample )/A control ] × 100
Where A control was the absorbance of the control reaction (containing all reagents except the test compound), A sample was the absorbance of the test compound (containing all reagents and test compound).
In the 1 H NMR spectrum of 7a the absence of signal due to NH 2 and presence of a singlet due to C 5"' -H at downfield region confirms its formation. Besides, a broad singlet at δ 10.37 ppm was attributed to NH which disappeared on deuteration. The structures of all the compounds were further established by IR, 13 C NMR, mass spectra and microanalyses.

VEGA-QSAR for toxicity prediction:
The toxicity of compounds 7a-7i predicted for selected endpoints are shown in Table 1 and the results revealed that all the tested compounds are non-mutagens, noncarcinogens and non-skin sensitizers.

Molecular docking:
The compounds 7a-7i were subjected to energy minimization using open Babel module in Pyrx program [54]. The docking protocol was validated using redocking experiment by removing CN2 from the co-crystal structure and allowed it for docking into the same binding pocket with specified docking parameters in AutoDock 4.2. CN2 interacted with the same residues that are involved in interaction with CN2 in co-crystallized structure and the RMSD value obtained from redocking experiment for the top ranked pose was 1.56 A 0 . It indicated that these parameters are good enough for docking process. Molecular docking results revealed that the compounds 7a-7i tend to bind with colchicine binding site with good binding free energies ranging from -9.66 Kcal/mol to -12.21 Kcal/mol. Docking results are summarized in Table 2. Figure 1

b)
Br c) OMe -NaOH / EtOH / )))) )))) )))) Scheme 2: Nucleophilic reaction of (5) with phenacyl bromide (6) followed by intramolecular cyclization and elimination of water.   The best binding free energies (∆G b ) and inhibition constants (K i ) among the docked poses of compounds 7a-7i. colchicine binding site. All the compounds showed hydrogen bonding interaction with amino acids in the colchicine binding pocket in addition to hydrophobic interactions except 7d. The most common hydrogen bonding interactions observed in all docked compounds formed between indole NH groups and Asn249(β) and Lys352(β). Compound 7d exhibited only hydrophobic interactions whereas 7b displayed hydrogen bonding with Lys352 only. It can also be inferred that compound 7e in which Br substitution at R, R' positions of phenyl ring has lowest binding energy and good inhibition constants followed by compounds 7f, 7h for colchicine binding site in tubulin.
In vitro cytotoxic activity: The compounds 7b, 7e, 7f and 7h were screened for in vitro anticancer activity against lung (NCI-H1299), colon (HCT-166 P 53 ) and prostate (PC-3) cancer cell lines by EZ-cytox cell viability assay kit. However, the remaining compounds are inactive at 100 µM. To determine the anticancer activity of the target compounds 7b, 7e, 7f and 7h the cancer cells were treated at a concentration of 100 µM for 24h and measured the cell viability using the EZ-cytox cell viability kit. The inhibition percentage of compound 7e was 82.42 (NCI-H1299), 65.30 (HCT-166 P 53 ), 73.09 (PC-3) ( Table 3). Figure 2 evidenced the anticancer effect of compound 7e on NCI-H1299, HCT-166 P 53 and PC-3 cancer cell lines. Further it was observed that the anticancer activity of compound 7e (0-200 µM) stimulation for 24h, cancer cells decreased in a dose dependent manner (Figures 3-5  infers that the compound 7e pre-treatment was clearly shown to modulate the anticancer activity. Statistical analysis: Experiments were performed in triplicate (n=3) and results are expressed as mean ± standard deviation (SD). Two-way ANOVA (MS-Excel) was used for multiple comparisons and it showed that P<0.01 which represent statistically significant differences.
In vitro antioxidant activity: The compounds 7a-7i were tested for antioxidant activity by 2,2-diphenylpicrylhydrazyl (DPPH), hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) methods. The experimental data on the antioxidant activity of the compounds 7a-7i and control drug are presented in Tables 4-6 ( Figures 6-8). The results revealed that compounds 7a, 7c, 7g and 7i showed good radical scavenging activity in all the three methods when compared with the standard drug Ascorbic acid. On the other hand, the compounds 7b, 7f and 7h     displayed moderate activity while 7d and 7e exhibited least activity. It was observed that the compounds containing electron donating substituent (OCH 3 ) on the phenyl ring enhances the activity when compared with those having electron withdrawing substituent (Br). Moreover it was noticed that compounds with more number of electron donating groups displayed higher radical scavenging activity. This was exemplified that 7i exhibited excellent radical scavenging activity.

Conclusion
Some new di(indolyl)thiazolylpyrazoles were prepared from the synthetic intermediate E-1,3-di(1H-indol-3-yl)prop-2-en-1-one under ultrasonication and studied their cytotoxic and antioxidant activities. All the compounds were screened for in vitro cytotoxic activity on three cancer cell lines. However, the compound 7e exhibited appreciable anticancer activity on NCI-H1299, HCT-166 p53 and PC-3 cancer cell lines with IC 50 values of 15.74, 26.95 and 19.02 µM respectively. The binding conformation of the target molecules was predicted by docking methodology to explain the biological activities. In fact, the docking studies indicated that bromo, dibromo, bromomethoxy and methoxybromo substituted di(indolyl)thiazolylpyrazoles (7b, 7e, 7f and 7h) could be used as possible leads for therapies against cancers. Amongst all the tested compounds dimethoxy substituted di(indolyl) thiazolylpyrazole (7i) displayed significant radical scavenging activity.