Synthesis, Spectroscopic Properties and Antipathogenic Activity of New Thiourea Derivatives

A number of acylthioureas, 2-((4-methylphenoxy)methyl)-N-(aryl-carbamothioyl)benzamides (aryl = 3,5-dichlorophenyl, 2,3-dichlorophenyl, 3,4-dichloro-phenyl, 2,4,5-trichlorophenyl, 3,4,5-trichlorophenyl, 2-bromophenyl, 2,4-dibromophenyl, 2,5-dibromophenyl, 2-iodophenyl, 3-fluorophenyl, 2,3,4-trifluorophenyl, 2,4,5-trifluoro-phenyl, 2,4,6-trifluorophenyl) have been synthesized, characterized by elemental analysis, IR and NMR spectroscopy and tested for their interaction with bacterial cells in free and adherent state. The anti-pathogenic activity was correlated with the presence of one iodine, bromide or fluorine, and two or three chloride atoms on the N-phenyl substituent of the thiourea moiety, being significant especially on Pseudomonas aeruginosa and Staphylococcus aureus strains, known for their ability to grow in biofilms. Our results demonstrate the potential of these derivatives for further development of novel anti-microbial agents with antibiofilm properties.


Introduction
Nowadays antibiotic resistance has become a serious public health problem. As a result of the rapid development of resistance to the existing portfolio of antimicrobial drugs, there is an increasing need to design new antibacterial and antifungal agents with better activity profiles and lower toxicity.

thiazol-2-ylamino)carbonothioyl]thiophene-2-carboxamide and N-[(1,3-
thiazol-2-ylamino)carbonothioyl]morpholine-4-carboxamide exhibit broad spectrum antifungal activity [6]. For example, the 1-(isomeric fluorobenzoyl)-3-(isomeric fluorophenyl)thioureas were proven to exhibit better antifungal than antibacterial activity, probably due to the inclusion of fluorine that may increase the lipophilicity and enhance the rate of cell penetration and transport of the drug to an active site. The higher polarizability due to the C-F bond may provide new possibilities for binding to the receptor [7].
Taking into account the improvement of the antimicrobial activity by the inclusion of fluorine in the organic molecule of thioureas [7], we synthesized, characterized and studied the antimicrobial activity of the new 2-((4-methylphenoxy)methyl)-N-(arylcarbamothioyl)benzamides, substituted with phenyl groups carrying one, two or three atoms of F, Cl, Br and I.

Chemistry
Our decision to synthesize and evaluate this kind of substances for their antimicrobial activity was influenced by several reports disclosing the antimicrobial activities of thiourea-based compounds. We have previously synthesized and demonstrated the microbicidal activities of 2-(4-methoxyphenoxymethyl)benzoic acid [23], 2-(4-chlorophenoxymethyl)benzoic acid [24], 2-(4-fluorophenoxymethyl)benzoic acid [25], 2-(4-ethylphenoxymethyl)benzoic acid [26,27], and 2-(4-methylphenoxymethyl)benzoic acid thioureides [28].   The new derivatives 1a-m were synthesized in good yields following the method described by us (Scheme 1). 2-(4-Methylphenoxymethyl)benzoic acid (2) was converted into the corresponding acid chloride 3 using thionyl chloride and anhydrous 1,2-dichloroethane as reaction medium, then treated with a solution of ammonium thiocyanate in acetone to afford arylisothiocyanate 4 in situ, followed by refluxing with amines to provide acylthioureas 1a-m in good yields. The compounds were purified by recrystallization from isopropanol. The acid 2 was synthesized with the best yield by using phtalide 5, which was treated with potassium para-cresolate in xylene under reflux. The obtained potassium salt 6 of 2-(4-methylphenoxymethyl)benzoic acid, due to its good solubility in 10% aqueous potassium hydroxide solution allowed its facile separation from xylene. The acid 2 was then precipitated using a mineral acid solution. The necessary potassium para-cresolate was obtained from the corresponding phenol and potassium hydroxide in xylene. The resulting water was removed by azeotropic distillation.
The new compounds have all been characterized by their melting point, elemental analysis, infrared and NMR spectral studies. All spectroscopic and elemental analyses data confirm the proposed structures of the new compounds.

Antimicrobial Activity
The emergence of bacterial resistance and multiresistance to antibiotics has been declared by the European Center for Disease Control (ECDC) as one of the major public health problems, besides HIV infection, tuberculosis and influenza. The increasing occurrence of multiresistant pathogenic bacterial strains has gradually rendered traditional antimicrobial treatment ineffective. Consequently, there is a pressing need to develop new antibiotics in order to overcome the bacterial resistance [29].
The qualitative screening of the susceptibility spectra of various microbial strains to the newly synthesized compounds showed that only five of the tested compounds (1c, 1d, 1f, 1i and 1j) exhibited an antimicrobial effect quantified by the occurrence of a growth inhibition zone ( Table 1). The compounds 1c (substituted with two chloride atoms in positions 3 and 4) and 1j (containing one fluorine atom in position 3) exhibited activity against S. aureus ATCC 25923 strain, while 1i (containing one iodine atom in position 2) exhibited activity against S. aureus 1694 clinical strain. The compound 1d (substituted with three chloride atoms in positions 2, 4 and 5) exhibited anti-Pseudomonas activity, and 1f (containing one bromide atom in position 2) was active on P. aeruginosa and K. pneumoniae strains. The compound 1j was the only one active both on Gram negative P. aeruginosa and the Gram positive S. aureus.
The results of the quantitative assay revealed that the serial dilutions of the compounds that proved to be active on different microbial strains in the qualitative screening assay, exhibited a low inhibitory activity on the bacterial growth, with high MIC values of 1,000-500 μg/mL (Table 2). Despite the high MIC values obtained for the tested compounds, we further tested them for their anti-pathogenic features, taking into account that, in many cases, the sub-inhibitory concentrations of the antimicrobial substances interfere with the expression of different virulence features, such are adhesins or toxins [30].  The genetic resistance of different microbial strains to numerous antimicrobial agents is amplified when one organism is found growing in a biofilm. Antimicrobial resistance is a trait typical of most biofilm organisms and it has been speculated that biofilms are the causative agent of up to 65% of bacterial infections [31]. Biofilms are thought to become recalcitrant to antimicrobial assault through a number of different mechanisms.
Giving the increasing risk of antimicrobial resistance, research is focusing on the discovery of anti-pathogenic drugs that block the coordinated expression of virulence factors, such as adherence capacity or production of toxins, thus rendering the bacteria harmless. Contrary to antibiotics, these new anti-pathogenic drugs do not kill the bacteria, and since the survival of the bacteria is not threatened by this approach, the development of resistance, like that to antibiotics, is not anticipated to be a serious problem.
The inert substrata including the prosthetic medical devices represent risk factors for the occurrence of biofilm associated infections. Taking into account the differences in physiology and susceptibility to antibiotics of biofilm-embedded bacteria [32], the compounds active against different bacterial strains in free state were further chosen and investigated concerning their efficiency against the adherent cells grown in biofilms developed in plastic wells. The experimental model uses mini volumes and multiple well plastic plates, allowing the simultaneous testing of a large spectrum of antibiotic concentrations.
Our results showed that the five compounds selected for the quantitative MIC assay proved to be extremely active, inhibiting the ability of the S. aureus, Ps. aeruginosa and K. pneumoniae strains to adhere and colonize the inert substratum at very low concentrations (with one exception, ranging between 13 µg/mL and 27 µg/mL), probably due to their interference with adhesins expression (Table 3). Thus, despite their high MICs, the tested substances seem to be promising as antipathogenic compounds, which may be used in low concentrations for prophylaxis or therapy by themselves or in combination with an antibiotic.

General
All chemicals used for the preparation of the compounds were of reagent grade quality and were obtained from Merck, Fluka or Sigma-Aldrich. p-Methylphenol was used freshly distilled. Acetone was dried over K 2 CO 3 and then distilled and ammonium thiocyanate was treated by heating at 100 °C before use. The necessary liquid amines were dried with potassium hydroxide and afterwards distilled.
Melting points were determined in open capillary tubes on an Electrothermal 9100 apparatus and uncorrected. C, H, N and S analysis were carried out on a Perkin Elmer CHNS/O Analyzer Series II 2400 elemental analyzer. The room temperature attenuated total reflection Fourier transform infrared (FT-IR ATR) spectra of the all synthesized compounds were registered using a Bruker Vertex 70 spectrophotometer. The IR bands are given as w -weak, m -medium, s -intense, vs -very intense. 1 H-NMR and 13 C-NMR spectra were recorded in DMSO-d 6 on a Gemini 300BB instrument, at room temperature, operating at 300 MHz for 1 H and 75 MHz for 13 C and a Unity Inova 400 instrument, operating at 400 MHz for 1 H and 100 MHz for 13 C. Chemical shifts were given as δ values in parts per million (ppm) relative to tetramethylsilane as internal standard. Coupling constants J were given in Hz. Spin multiplets are given as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet), td (triple doublet) and br (broad). The chemical shifts for hydrogen and carbon atoms were established also by G-COSY, G-HSQC, G-HMBC experiments. The 2-(4-methylphenoxymethyl)benzoic acid and 2-(4-methyl-phenoxymethyl)benzoyl chloride derivatives were obtained in good yields similar to those reported in the previous article [33].

Study of the Influence of the Selected Compounds on the Ability of P. aeruginosa, S. aureus and K. pneumoniae to Colonize the Inert Substratum and Form Biofilms
The anti-biofilm activity of the tested compounds was tested by the microtiter method. For this purpose, the microbial strains have been grown in the presence of two-fold serial dilutions of the tested compounds performed in liquid nutrient broth/YPG, distributed in 96-well plates and incubated for 24 hours at 37 °C for bacterial strains, respectively for 48 hours at 28 °C for fungal strains. At the end of the incubation period, the plastic wells were emptied, washed three times with phosphate buffered saline (PBS), fixed with cold methanol and stained with 1% violet crystal solution for 30 minutes. The biofilm formed on plastic wells was resuspended in 30% acetic acid. The intensity of the colored suspensions was assessed by measuring the absorbance at 490 nm. The last concentration of the tested compound that inhibited the development of microbial biofilm on the plastic wells was considered the minimum inhibitory concentration of the biofilm development and was also expressed in μg/mL [36,37].

Conclusions
New 2-((4-methylphenoxy)methyl)-N-(arylcarbamothioyl)benzamides were prepared from 2-(4methylphenoxymethyl)benzoyl chloride via isothiocyanate formation, followed by treatment with various substituted amines. Our results showed that some of the newly synthesized thiourea derivatives carrying aryl groups substituted with one iodine, bromide, fluorine or with two or three chloride atoms could be used for the further development of novel antimicrobial agents with anti-biofilm properties.