Design, synthesis and anticancer activity of some novel thioureido-benzenesulfonamides incorporated biologically active moieties

Background Many thiourea derivatives have exhibited biological activities including anticancer activity through several mechanisms. On the other hand, benzenesulfonamide derivatives have proven to be good anticancer agents. Hybrids of both moieties could be further developed to explore their biological activity as anticancer. Results Novel series of thioureidobenzenesulfonamides incorporating miscellaneous biologically active moieties 3–17 were designed and synthesized utilizing 4-isothiocyanatobenzenesulfonamide 2 as strategic starting material. The structures of the newly synthesized compounds were established on the basis of elemental analyses, IR, 1H-NMR, 13C-NMR and mass spectral data. All the newly synthesized compounds were evaluated for their in vitro anticancer activity against various cancer cell lines. Most of the synthesized compounds showed good activity, especially compounds 3, 6, 8, 9, 10, 15 and 16 which exhibited good activity higher than or comparable to the reference drugs, DCF and Doxorubicin, except breast cancer line. As a trial to suggest the mechanism of action of the active compounds, molecular docking on the active site of mitogen kinase enzyme (MK-2) was performed and good results were obtained especially for compound 3. Conclusion Compounds 3, 6, 8, 9, 10, 15 and 16 may represent good candidates for further biological investigations as anticancer agents. Their cytotoxic activity could be due to their action as MK-2 enzyme inhibitors.Graphical abstract Compound 3 on the active site of MK-2 enzyme


Background
Various types of cancer are now considered as the second cause of death after cardiovascular disorders [1]. The inability of the present anticancer chemotherapeutic agents to discriminate between normal cells and cancer cells comprises the biggest challenge for successful cancer treatment [2]. Serious side effects of anticancer chemotherapeutic agents limit their usage and in many cases surgery or radiotherapy replace them [3]. The continuous seek for safer and more effective anticancer agents is still a major goal for medicinal chemists.
Thiourea is a versatile synthetic block for the synthesis of a wide variety of new organic compounds with biological activity including antimicrobial, antifungal, antidiabetic, antimalarial, anti HIV and CNS active drugs [4][5][6][7][8][9][10][11]. Many of aryl thiourea derivatives have applications in medicine, industry and agriculture [12][13][14][15]. Thiourea was incorporated in many tyrosine kinase inhibitors because of its ability to form powerful hydrogen bonds in the ATP binding pocket of the enzymes [16]. The thiourea derivative YH345 A has shown strong protein farnesyl transferase inhibition activity [17]. Also several heterocyclic thiourea derivatives have shown strong DNA topoisomerase inhibitory activity [18].
Based on the previous facts and as a continuation of our previous work in the seek of novel anticancer agents [32][33][34][35][36][37][38], we herein report the synthesis and biological evaluation of new sulfonamide thiourea derivatives 3-17 presented by general structure C as hybrid molecules of benzensulfonamide and thiourea moieties as anticancer agents. Molecular docking of the active newly synthesized compounds was performed on the active site of mitogen activated kinase enzyme (MK-2) in a trial to suggest a mechanism of action for their cytotoxic activity.

In-vitro anticancer evaluation
The synthesized compounds were evaluated for their in vitro anticancer activity against human lung cancer cell line (A549-Raw), cervical (Hela) cancer cell line, colorectal cell line (Lovo) and breast cancer cell line (MDA-MB231) using 2′7′dichlorofluorescein (DCF) and Doxorubicin as reference drugs in this study. The relationship between surviving fraction and drug concentration was plotted to obtain the survival curve of cancer cell lines. The response parameter calculated was the IC 50 value, which corresponds to the concentration required for 50 % inhibition of cell viability. The results are presented in Table 1.

Structure activity relationship
In a closer look to the biological results we can see that: the thiourea derivatives 3, 6, 8, 9, 10, 15 and 16 were the active compounds on most of the cell lines while the rest of the compounds were inactive. It was obvious that incorporating an n-heptane aliphatic substitution as in compound 3 gave the most activity on all cell line. This activity was reduced upon replacing this substituent with another tricyclic aliphatic one as in compound 9. In case of aromatic substitution the activity was retained but markedly decreased as in the 2-methyl-6-nitrophenyl thiourea derivative 6, the 3-benzo[d] [1,3]dioxol-5-ylmethyl thiourea derivative 8, the 3-(5,6-dimethylbenzo[d]thiazol-2-yl)thiourea derivative 10, the tetrahydronaphthalen derivative 15 and the quinoline derivative 16.
Comparing compound 3 which was the most active compound among the newly synthesized compounds with the reference drug Doxorubicin we can see that: compound 3 was more active that Doxorubicin as cytotoxic agents on lung cancer cell line, Hella cells and colorectal cancer cells with IC 50 value of 29.12, 35.63 and 39.83 µg ml −1 , respectively. However, in case of breast cancer cell line compound 3 was less active than Doxorubicin with IC 50 value of 26.28 µg ml −1 .

Molecular docking
Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK-2 or MK-2) is an important enzyme in signal transduction pathway controlling several pathways in cell proliferation [39]. MK-2 inhibition is one of the strategies of discovering new anticancer agents [40]. Recently, several urea and thiourea derivatives have shown good inhibitory activity on MK-2 [41]. Based on the thiourea scaffold of our newly synthesized compounds and as a trial to suggest a mechanism of action for their cytotoxic activity, molecular docking was performed on the active site of MK-2 for the most active compound. The protein data bank file (PDB:3WI6). The file contains MK-2 enzyme co-crystalized with an inhibitor. All docking procedures were achieved by MOE (Molecular Operating Environment) software 10.2008 provided by chemical computing group, Canada. The inhibitor interacts with MK-2 active site with four hydrogen bonds involving Glu 190, Leu 141, Asn 191 ans Asp 207 (Fig. 1). The docking protocol was validated by redocking of the ligand on the active site of MK-2 enzyme with energy score (S) = −15.4978 kcal mol −1 and root mean square deviation (RMSD) = 1.1457.
The active compounds were docked on the active site of MK-2 using the same docking protocol. Energy scores and amino acid interactions were displayed in Table 2.
All the docked compounds were fit on the active site of MK-2 with energy scores ranging between −10.2371 and −20.1443 kcal mol −1 . Best docking score was exhibited by compound 16 which interacted with Lys 188 with one hydrogen bond (Fig. 2) while the best amino acid interaction was exhibited by compound 3 which interacted with Leu 141 by two hydrogen bonds and Asp 207 with one hydrogen bond (Figs. 3, 4). The same previous two amino acids interacted also with the co-crystalized inhibitor in a comparable manner.

General chemistry
Melting points (uncorrected) were and determined in open capillary on a Gallen Kamp melting point apparatus (Sanyo Gallen Kamp, UK). Precoated silica gel plates (Kieselgel 0.25 mm, 60 F254, Merck, Germany) were used for thin layer chromatography. A developing solvent system of chloroform/methanol (8:2) was used and the spots were detected by ultraviolet light. IR spectra (KBr disc) were recorded using an FT-IR spectrophotometer (Perkin Elmer, USA). 1 H-NMR spectra were scanned on an NMR spectrophotometer (Bruker AXS Inc., Switzerland), operating at 500 MHz for 1 H-and 125.76 MHz for

General procedure
A mixture of 4-isothiocyanatobenzenesulfonamide 2 (2.14 g, 0.01 mol) and amines (0.012 mol) in dry dimethylformamide (15 ml) containing three drops of  triethylamine was refluxed for 24 h, then left to cool. The solid product formed upon pouring onto ice/water was collected by filtration and recrystallized from ethanoldimethylformamide to give 3-17, respectively.

Cytotoxicity assay
The in vitro anticancer screening was done at pharmacognosy Department, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Cells were seeded (10 5 cells/well) in 96-well flat-bottom plates (Becton-Dickinson Labware) a day before treatment and grown overnight. Compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma) and finally prepared as 1.0 mg ml −1 stocks, respectively in the culture media. The final concentration of DMSO never exceeded 0.1 % in the treatment doses. Four different doses of compounds (50, 25, 12.5 and 6.25 µg ml −1 ) were further prepared by diluting the stocks in culture media, and cells were treated (in triplicate/dose). 2′7′ dichlorofluorescein (DCF) was included as standard reference drug (positive control) and untreated culture was considered as negative control. The treated cultures were further incubated for 48 h. At 48 h post-treatment, cell viability test was performed using TACS MTT Cell Proliferation and Viability Assay Kit (TACS) as per manufacturer's instructions. The optical density (OD) was recorded at 570 nm in a microplate reader (BioTek, ELx800) and cell survival fraction was determined. The cell survival fraction was calculated as [(A − B)/A], where A and B are the OD of untreated and of treated cells, respectively [42]. The IC 50 values of the tested compound were estimated using the best fit regression curve method in Excel.

Microscopy
A direct visual investigation was made under an inverted microscope (Optica, 40× and 100×) to observe any morphological changes in the cells cultured with different treatment doses at 24 and 48 h.

Molecular docking
All the molecular modeling studies were carried out on an Intel Pentium 1.6 GHz processor, 512 MB memory with Windows XP operating system using Molecular Operating Environment (MOE, 10.2008) software. All the minimizations were performed with MOE until a RMSD gradient of 0.05 kcal mol −1 Å with MMFF94X force field and the partial charges were automatically calculated.
The protein data bank file (PDB: 3WI6) was selected for this purpose. The file contains MK-2 enzyme co-crystalized with a ligand obtained from protein data bank. The enzyme was prepared for docking studies where: (1) Ligand molecule was removed from the enzyme active site.
(2) Hydrogen atoms were added to the structure with their standard geometry. (3) MOE Alpha Site Finder was used for the active sites search in the enzyme structure and dummy atoms were created from the obtained alpha spheres. (4). The obtained model was then used in predicting the ligand enzymes interactions at the active site.

Conclusions
In summary, we had synthesized a novel series of sulfonamide thiourea derivatives. Seven compounds 3, 6, 8, 9, 10, 15 and 16 showed good anticancer activity against lung (A594 Raw), Hela, and Colorectal (Lovo) cancer cell lines with better or comparable activity to DCF. Moreover, molecular docking for these active compounds showed proper fitting on the active site of MK-2 enzyme suggesting their action as inhibitors for this enzyme but more investigation should be carried out in the future to explore precisely the mechanism of the action of the synthesized derivatives.