Synthesis, Molecular Docking, And Antitumor Evaluation of Some New Pyrazole, Pyridine, And Thiazole Derivatives Incorporating Sulfonamide Residue

Cancer is the second leading cause of death worldwide. There is always a huge demand for novel anticancer. New series of pyrazole, pyridine, thiazole derivatives containing sulfonamide moiety were prepared and screened for their antitumor activity against breast cancer cell line (MCF-7). The results of this investigation revealed that compounds 3 and 8 had signicant anticancer activity against the MCF-7 cancer cell line with IC50 values 19.2 and 14.2 μM, respectively, in relation to the standard drug, doxorubicin.


Introduction
Cancer is one of the most dreadful diseases in the world and despite immense advances in the eld of basic and clinical research, which have resulted in higher cure rates for a number of malignancies 1,2 . Tumor growth and metastasis depend on various factors, including the physiological process of angiogenesis. For example, elevated expression of vascular endothelial growth factor, epidermal growth factor, broblast growth factor, and platelet-derived growth factor are associated with tumor angiogenesis, metastases, survival, and resistance to apoptosis. Therefore, these growth factors have represented potential molecular targets for inhibition of tumor growth and progression [3][4][5][6] . Presently, a large number of anti-cancer drugs in clinical practice have shown pervasive side effects, low speci city, and multidrug resistance, so, there is always an urgent demand to develop novel anticancer drugs, and diverse new natural or synthetic compounds are developed continuously by scientists 7 . Sulfa drugs were the rst broadly effective antibacterial to be used systemically and paved the way for the antibiotic revolution in medicine (see Fig. 1). In addition, Sulfonyl or sulfonamide hybrids were broadly explored for their anticancer activities via different mechanisms 8- 17 and it was found that they possess minimum side effects along with multi-drug resistance activity. Therefore, and in continuation of our interest to develop a series of novel small molecules as potential antitumor agents that can block biologically relevant molecular targets 18-22 , we prepared certain pyrazoles, pyridines, thiazoles derivatives containing sulfonamide pharmacophores and studied their activity against breast cancer cell line (MCF-7).

Chemistry Section
Compounds 1a and 1b were prepared according to literature procedure 23 . Condensation of 1a and 1b and phenylhydrazine provided the hydrazones derivatives 2a and 2b. The Vilsmeier-Haack reaction using 2.5 equiv of phosphoryl chloride performed a double addition of reagent to afford, ultimately after hydrolysis, the desired pyrazole 4-carbaldehyde derivative 3 (Scheme 1). The structure of 3 was established using both spectral and analytical analyses, in addition to X-ray analysis (Fig. 2). The IR spectrum of 3 showed absorption bands at ν = 1672 (CHO), 1633 (N = CH-), and 1342, 1153 (SO2); while 1HNMR showed signals at 10.52 (s, 1H, CHO) and 9.28 (s, 1H, pyrazole H-5) besides the rest of protons in its expected locations.
Treatment of the pyrazole carbaldehyde derivative 3 with some selected nitrogenous active reagents, namely (thiosemicarbazide, carbothidihydrazide, and cyanoacetohydrazide), afforded the condensed adduct 4, 5, and 6 respectively without any observation of cyclized products. The versatile α, βunsaturated nitrile 7 is obtained upon treatment of 3 with ethyl cyanoacetate in the presence of few drops of triethylamine which was directly subjected to react with acetophenone in boiling ethanol containing a catalytic amount of ammonium acetate to afford 1,2 dihydropyridine derivatives 8. The structure of 8 was established by both spectral and analytical analysis, the infra-red spectrum of compound 8 showed absorption peaks ν = 3360( NH), 2218 (CN), 1655 (CO), in addition, the 1H NMR exhibited signals at δ = 12.54 (NH), 9.28 (s, 1H, pyrazole H-5), and 7.74 (pyridine H-5), supported the suggested structure.

Molecular Modeling
Molecular docking for synthesized compounds was performed to explore their biological pro le due to their binding with CDK2. Subsequently, their bound conformations and binding a nities were estimated. The structure preparation of the receptor was done by the removal of co-crystallized ligands and water molecules before energy minimization using the CHARMM force eld. Hydrogens were added to the protein followed by adding the charges. The CDK2 coordinates were gained from the protein data bank (PDB 6GUH) and the structure was adjusted by Accelrys Discovery Studio 2.5. The missing residues were added as well as the structure was relaxed to correct the protein errors. Finally, the active site of CDK2 was distinct and all the synthesized compounds were docked and their binding energies were calculated.
The synthesized derivatives were docked utilizing the default parameters of the C-Docker protocol (see Figures. 3 and 4), (Table 1).

Materials And Methods
A digital Gallen-KampMFB-595 instrument was used to measure melting points using open capillary tubes. The results were not corrected. KBr pellets were used to measure IR spectra using a Shimadzu FTIR-440 spectrometer. The MS-50 Kratos (A.E.I.) spectrometer, equipped with a data system, was used to obtain mass spectra at 70 eV. Chemical shifts in ppm units for 1 H NMR (500 MHz) and 13 CNMR (125 MHz) were measured using a Bruker model Ultra Shield NMR spectrumrometer in DMSO-d6 using tetramethyl silane (TMS) as an internal reference. Cairo University's Microanalytical Center conducted the elemental studies (percent C, H, and N). The appropriate precautions in handling moisture-sensitive compounds were taken. Solvents were dried by standard techniques. The monitoring of the progress of all reactions and homogeneity of the synthesized compounds were carried out and were run using thinlayer chromatography (TLC) aluminum sheets silica gel 60 F254 (Merck).
General procedure for synthesizing of hydrazones derivatives 2a and 2b: To 1a and 1b (10 mmol) in acetic acid (100 mL) and water (10 mL) phenylhydrazine (11 mmol) was added. The reaction mixture was stirred at room temperature until completion (TLC, 5 h). The solid which was formed was ltered off, washed with water, dried and crystallized from acetic acid to give: General procedure for the synthesis of compounds 4, 5, and 6. A solution of 3 (4.63 g, 10 mmol) and thiosemecarbazide or carbothidihydrazide or cyanacetic acid hydrazide (11 mmol ) in a mixture of acetic acid/ ethanol ( 1:1) was re uxed for 6 h. The solid formed on boiling was ltered of, dried and crystallized from acetic acid to afford:  Three-dimensional structure of human cycline-dependantkinase CDK2, PDB:2FVD, was obtained from protein data bank. Energy minimization of newly designed compounds was done by employing discovery studio 2.5 for structure re nement. The geometry of all designed analogues is typed with CHARMm 25 force eld; then partial charges are calculated by Momany Rone method 26 . Further, they are optimized through a smart minimizer algorithm, which performs 1000 steps of steepest descent with a root meansquare (RMS) gradient tolerance of 0.1. Same as the preparation of ligands for the target, its active site was also passed with the energy minimization process and it was done using charm force eld which is de ned by the equation given below:
CDOCKER (CHARMm-based DOCKER), a docking program provided by discovery studio 2.5, uses a CHARMm based molecular dynamics (MD) scheme to dock ligands into a receptor binding site, and then random conformations will be produced using high-temperature molecular dynamics. When these conformations are translated to the active site, candidate poses are then generated using random rigid body rotations followed by simulated annealing. CDOCKER offers all the advantages of full ligand exibility (including bonds, angles, and dihedrals) and reasonable computation times. CDOCKER uses soft core potentials, which are found to be effective in exploring the conformational space of macromolecules used in various docking studies. The nonbonded interactions which involve van der Waals (vdW) and electrostatics are softened at different levels, except during the nal minimization step 27 . Initially, ten conformations for each inhibitor are generated in the active site of the target enzyme, which is created as a spherical region with a diameter of 10 Ǻ. Simulated annealing is performed using a exible ligand and a rigid protein.
Receptor-ligand interactions are calculated from grid extension 8.0, random conformations are generated using speci c molecular dynamics steps, and the system is heated to 700 K in 2000 steps, cooling steps to 5000, and cooling temperature to 300 K. The nal re nement step of minimization is performed using full potential. Minimized docking poses are then clustered, based on a heavy atom RMSD approach. The ranking is based on the total docking energy, which is composed of the ligand's intramolecular energy and the ligand-receptor interaction. Figure 1 Chemical structure of sulfonamides (SAs).