Molecular Modeling and Synthesis of New Heterocyclic Compounds Containing Pyrazole as Anticancer Drugs

Several modifications in CA-4 were reported in literature for the development of various tubulin inhibitors. In our study, twenty-two newly synthesized heterocyclic derivatives of Combretastatin A-4 (CA-4) have been tested for their cytotoxic effect on four different types of cells with malignant behavior using CA-4 as a positive control. Compounds 5b, 15 and 16 showed the foremost potent antiproliferative activities as compared to CA-4 with IC50 starting from 6.9 to 13.7 μM. Molecular docking was performed with the crystal structure of tubulin employing a potent tubulin inhibitor CA-4 as a parent molecule. Molecular study advised that 5b, 15, 16 and 17 are promising tubulin inhibitors.


Introduction
Microtubule-targeting agents are used clinically as antitumor drugs.Their anticancer activity is commonly due to tubulin inhibition.They prevent the formation of microtubule, thus affecting cellular replication and shape [1].In general, antitubulin agents bind with five totally different binding pockets on the β-tubulin subunit [2].Agents that strongly react with colchicine binding site induce depolymerization of tubulin, therefore they inhibit tubulin action [3] (Figure 1(a)).This study will focus on agents that target colchicine-binding site [4].
The natural product CA-4 that is under investigation as it has a strong affinity to colchicine binding site, and therefore a strong tubulin inhibitor.Thus, we use CA-4 as a lead molecule for synthesis of new derivatives and more studied as tubulin inhibitors [5].It is formed of three parts (2 carbons bridge, ring A and ring B) [6] (Figure 1(b)).Various changes to CA-4 structure have been performed in literature.The changes to the central bridge affect the cytotoxic activity the most [7]- [15].Ring A has received little or no attention while ring B has received larger attention.Impressed by these finding, the double bond of CA-4 is substituted by α, β-unsaturated ketone, pyrazole, quinoline-3-carbonitriles, fused pyrimidine, fused quinazoline moieties, imine derivatives and 2-amino-4H-chromenes. Besides, ring A and B are replaced by 1, 5-diphenylpyrazole, naphthalene and pyrazole moieties.
Those were evaluated in vitro for their anticancer tendency toward four cancer cell types.Additionally, molecular docking studies are performed and compared to biological results.
The MTT assay was applied to assess cytotoxic activity using the cell lines mentioned above.This type of colorimetrical chemical assay relies on the modification of the yellow tetrazolium bromide (MTT) to a color of formazan derivative by mitochondrial succinate dehydrogenase in viable cells.Cell lines were cultured in RPMI-1640 medium with ten percent fetal bovine blood serum.
Then, antibiotics added were one-hundred units/ml penicillin and one-hundred µg/ml streptomycin at 37˚C in a five percent CO 2 incubator.Then, drugs were prepared in different concentrations and added to the cells, incubated for 24 hrs.
After the period was completed, MTT solution (20 µl) with concentration 5 mg/ml was added to the mixture and incubated for 4 hrs.Then, DMSO (100 µl) is sided to every single plate to remove purple color of formazan.The colorimetrical chemical assay is measured and recorded at an absorbance of 570 nm employing a plate reader (EXL 800, USA) [28] [29].

Molecular Modeling
The docking studies and modeling calculations were done using MOE version 2008.10 release of Chemical Computing Groups using Windows XP software.
The 3D structures of the synthesized molecules were built using Chem Biooffice suite then saved as Mol 2 format with the help of MOE software, then subjected to docking simulation.The three dimensional X-ray structure of protein (PDB code: 1SA1) was obtained from the Protein Data Bank.

Antitumor Activity
All the synthesized compounds were evaluated for their cytotoxic activity toward a panel of 4 human neoplastic cell lines using the MTT assay.The results, in Table 1 showed that every compound have antitumor activities with noticeable differences due to structural variations.shows wide spectrum cytotoxic activity with IC50 starting from 6.9 to 12.7 μM.

As shown in
Replacement of phenyl ring with acetyl group as in compound 5c or unsubstituted indazole in compound 5a and uncyclized chalcone as in compound 4a exhibited a decrease within the activity against the four cell lines.Additionally, the cytotoxic activities of recent synthesized quinoline-3-carbonitrile 6-

Molecular Modeling
Molecular docking was performed for compounds that inhibited tubulin polymerization so as to explore attainable binding modes.The tubulin protein structure in complex with podophyllotoxin (PDB 1SA1) was used for the study [30] [31] [32].In molecular docking, MOE programme (molecular operating environment) was used in our study.CA-4 is employed as lead compound due to its excellent affinity to colchicine binding site (Figure 2).Comparing to the lead molecule CA-4 and biological activity, we have a tendency to found that compounds 5b, 15, 16 and 17 have the lowest energy and give the most stable complexes with the receptor and are the most biological active against cell lines so we concluded that these new compounds have very good affinity binding with the protein.During this study, we observed that basic amino acid Lys (B254) is essential as it is bound with 15 via arene cation interaction and with 16 via hydrogen bond (angle NH…N = 101.9,distance = 2.9 A 0 ) (Figure 4, Figure 5).

Experimental
Melting points (˚C) were recorded using a Fisher-Johns melting point apparatus and are uncorrected.Microanalyses (C, H, and N) were performed in Cairo University Microanalytical unit, and all the results within ±0.4.IR spectra (KBr) were recorded on Thermo Fisher Scientific Nicolet IS10.Spectrometer (ν in International Journal of Organic Chemistry     were prepared as previously discussed in literature [16] [33].

General Method for Preparation of Chalcones 4a, b
Ethanolic solution (20 ml) of NaOH (1 g, 0.025 mol) was mixed with a solution of 3-methyl-

General Procedure for Synthesis of 5a, c
Hydrazine hydrate (98%) (0.3 ml, 0.05 mol) introduced to a solution of 4a in ethyl alcohol (99%) (20 ml) 5a or ethanoic acid (10 ml) 5c and the mixture was refluxed for 5 h.The solvent was decreased using evaporator.After cooling the separated product was filtered, dried and finally recrystallized from ethyl alcohol.

General Procedure for Synthesis of 5b
A mixture of chalcone 4a (0.7 g, 0.005 mol), phenyl hydrazine (0.12 g, 0.005 mol) in glacial ethanoic acid (15 ml) was heated for 8 h.Then the mixture was diluted with ice-cold water.The precipitate was collected, washed with water, dried and purified by SiO 2 (preparative TLC in petroleum ether: ethyl acetate

Preparation of 10a, b
The α, β-unsaturated ketone 4a or 4b (0.01 mol) was mixed with thiourea (0.8 g, 0.01 mol) and Sodium metal (0.5 g) in butyl alcohol (50 mL).The mixture was refluxed for 10 h.The extra of solvent became concentrated in vacuo and the residue changed into pH 6.The separated stable turned into filteration, washed with water, dried and purified through SiO 2 (preparative TLC petroleum ether: ethyl acetate 3:1).
Solvent was dried under reduced pressure; then washed with water.The obtained product was recrystallized from ethyl alcohol.

Preparation of 17
A solution of compound 16 (4.6g, 2 mol) in15 ml of ethanol was added to 80% hydrazine hydrate (0.6 ml, 1 mol).The mixture was stirred at 25˚C for 4 hrs, the solvent was evaporated under reduced pressure.The residue was washed with ethyl alcohol, recrystallized from the water.

Conclusion
A series of new compounds have been synthesized using efficient methods and tested for their biological activities.There is a strong correlation between molecular modeling and biological screening results which suppose that the structural modification of the lead structure affects the activity in a predictable manner; as shown in (Figures 2-6), CA-4 shows highest binding affinity with its methoxy F. N.

Figure 1 .
Figure 1.(a) Tubulin inhibitors that bind to colchicine binding site; (b) Common structure characteristic of CA-4.

Table 1 ,
compound 5b with 2-phenyl substituted indazole International Journal of Organic Chemistry
Compounds 8 and 9shows low activity within the four cell lines.By replacing CH 2 of cyclohexanone chalcone 4a by N-CH 3 chalcone 4b, the cytotoxic activity is almost doubled against the four human cell lines.While the cytotoxic activity of 10a and 11b is ranged from moderate to weak activity parison to compound 6c, replacing propoxy group with ethoxy group in compound 6b resulting in 1.5 times decrease in cytotoxic activity.Also replacing propoxy group with amino group in compound 7 resulting in reduce in activity International Journal of Organic Chemistry by almost 4 times.(11.4 ± 0.98 for HCT-116, 14.4 ± 1.05 for Hela, 15.6 ± 1.20 for PC3, 9.1 ± 1.10 for MCF-7) and (17.4 ± 1.32 for HCT-116, 13.1 ± 1.10 for Hela, 16.3 ± 1.36 for PC3, 10.7 ± 1.23 for MCF-7) respectively.