Synthesis Antimicrobial and Anticancer Evaluation of 1-Aryl-5-(o-methoxyphenyl)-2-S-benzyl Isothiobiurets

A series of S-benzyl aryl thiourea were condensed with o-Methoxy phenyl isocyanate to yield respective isothiobiuret derivatives. The newly synthesized compounds were characterized by 1H-NMR, IR, and Mass Spectral studies and tested for biological activities.


Introduction
Thiourea and its derivatives such as thioureides possess interesting biological properties such as antibacterial [1][2][3], herbicidal, and fungicidal [4]. Many thiourea show in-vivio and in-vitro activity against HIV [5]. An impressive number of currently used drugs can be regarded as thiourea derivatives for example, thyreostatic: carbimazole, propylthiouracil, methylthiouracil, and ultrashortnarcotic: thiamylal. Thiourea shows considerable toxicity towards higher organisms and is used as insecticide [6] and rodenticide [7]. Thiourea derivatives find widespread uses in mining industry as floating aid for sulfidic ores [8].
In quest for biologically more potent compounds, we envisioned synthesizing series of isothiobiuret compounds by reacting S-benzyl arylthiourea with o-methoxy phenyl isocyanate and studied their antibacterial and anticancer activities.

Synthetic Procedure
2.3.1. Synthesis of Aryl S-benzyl Thiourea. General synthetic procedure for preparation of Aryl thiourea exemplified by phenyl thiourea: Aniline (30 g, 0.32 mol) was taken in a round bottom flask and, to this, concentrated hydrochloric acid (32.19 mL, 0.32 mol) was added dropwise with continues stirring. After 20 min turbidity appeared and 100 mL of water was added followed by a solution of ammonium thiocyanate (29.42 g, 0.38 mol) in 50 mL of water. This reaction mixture was heated till the solution starts becoming turbid, heating discontinued, reaction mass was poured in ice cold water, and precipitate formed was filtered off and dried. Crude product was recrystallized by hot water, yield (39.22 g, 80%), m.p. 152 ∘ C.
General synthetic procedure for preparation of Aryl Sbenzyl thiourea exemplified by phenyl S-benzyl thiourea (II): phenyl thiourea (35 g, 0.23 mol) was dissolved in 75 mL of ethanol and, to this reaction, mass benzyl chloride (29.11 mL, 0.25 mol) was added; once the exotherm subsides, reaction mass was gently heated to reflux and refluxed for 90 min. This reaction mixture was cooled to room temperature and basified under cold condition with ammonia solution, and, on standing, it yields phenyl S-benzyl thiourea. Yield (29.2 g, 52.4%), m.p. 75 ∘ C.

1-Phenyl-5-(o-methoxyphenyl)-2-S-benzyl Isothiobiuret.
Isothiobiuret was synthesized by condensing Phenyl S-benzyl thiourea (0.70 g, 2.89 mmole) with o-Methoxy phenyl isocyanate (0.43 g, 2.89 mmole) at room temperature in benzene overnight. Solvent was removed by distillation. Solid mass obtained was triturated with pet ether, to afford off-white solid.  2.4. Antimicrobial Activity. All the compounds were screened for their antibacterial activity against pathogenic bacteria and fungi such as E. coli, S. aureus, P. aeruginosa, and Aspergillus fusarium by cup plate agar diffusion method at a concentration 100 g/mL in DMSO. The zone of inhibition was measured in mm and is average of three readings. The readings are shown in Table 1. Molecule 4 showed moderate antimicrobial activity against E. coli and S. aureus, and considerable antifungal activity, whereas molecule number 2 showed a reverse trend in activities; from this observation, it can be concluded that substitution at para position of phenyl ring plays a crucial role in deciding activity toward bacterial and fungal stains.

Anticancer
Activity. Molecule number 1 as representative molecule was studied for short term in vitro cytotoxicity using Dalton's ascites (DLA) cells and Ehrlich ascites Carcinoma (EAC) Cells.
The tumor cells aspirated from the peritoneal cavity of tumor bearing mice were washed thrice with phosphate buffered saline (PBS) or normal saline. Cell viability was determined by trypan blue exclusion method, viable cell suspension (1 × 10 6 cells in 0.1 mL) was added to tubes containing various concentrations of the test compounds, and the volume was made up to 1 mL using PBS. Control tube contained only cell suspension; these assay mixtures were incubated for 3 hours at 37 ∘ C. Further cell suspension  was mixed with 0.1 mL of 1% trypan blue and kept for 2-3 minutes and loaded on a haemocytometer. Dead cells take up the blue colour of trypan while live cells do not take up the dye. The numbers of stained and unstained cells were counted separately; drug concentration versus percentage of death cells was tabulated in Table 2: %Cytotoxicity =

Number of dead cells Number of live cells + Number of dead cells
× 100. (1)

Conclusions
From the observation, it can be concluded that substitution at para position of phenyl ring plays a crucial role in deciding activity toward bacterial and fungal stain; as these molecules are easy to synthesize and purify, these classes of molecules can be explored further to develop SAR against different microbial and fungal stains as well as a potent anticancer agent.