Disubstituted 4-Chloro-3-nitrophenylthiourea Derivatives: Antimicrobial and Cytotoxic Studies

4-Chloro-3-nitrophenylthioureas 1–30 were synthesized and tested for their antimicrobial and cytotoxic activities. Compounds exhibited high to moderate antistaphylococcal activity against both standard and clinical strains (MIC values 2–64 μg/mL). Among them derivatives with electron-donating alkyl substituents at the phenyl ring were the most promising. Moreover, compounds 1–6 and 8–19 were cytotoxic against MT-4 cells and various other cell lines derived from human hematological tumors (CC50 ≤ 10 μM). The influence of derivatives 11, 13 and 25 on viability, mortality and the growth rate of immortalized human keratinocytes (HaCaT) was observed.


Antimicrobial Studies
Newly obtained 4-chloro-3-nitrophenylthioureas 20-24 and 27-30 were evaluated for their in vitro antimicrobial activity against a set of bacterial and fungal strains. The initial testing by the disc diffusion method [39,40] chose derivatives with meaningful antimicrobial properties. Considering the fact that all compounds exhibited activity against Gram-positive strains (GIZ ≥ 13 mm), in the next step the minimal inhibitory concentrations (MIC) were assessed by the twofold serial

Antimicrobial Studies
Newly obtained 4-chloro-3-nitrophenylthioureas 20-24 and 27-30 were evaluated for their in vitro antimicrobial activity against a set of bacterial and fungal strains. The initial testing by the disc diffusion method [39,40] chose derivatives with meaningful antimicrobial properties. Considering the fact that all compounds exhibited activity against Gram-positive strains (GIZ ≥ 13 mm), in the next step the minimal inhibitory concentrations (MIC) were assessed by the twofold serial microdilution method [41,42]. The results showed that all investigated compounds (except 21) exhibited the potential to moderate antibacterial potency, mainly against standard staphylococcal and M. luteus strains ( Table 2).
The potency of thioureas 22 and 24 towards all hospital methicillin-resistant strains of S. epidermidis was 2-16 stronger than the reference Ciprofloxacin ( Table 3). The antistaphylococcal activity of other tested compounds was equal or 2-4 times higher than observed for the standard antibiotic. Selected strains (MRSE 405/11, 406/11, 409/11, 411/11, 422/11) were remarkably less susceptible for the presence of synthesized thiourea than for the reference drug.  It is worth noting that the factor influencing the biological activity could be a molecular flexibility. It is evident from the determined crystal structures of 20 and 24 that molecules of this group of derivatives are able to adopt different conformations ( Figure 1). Variable orientations of the thiourea C=S bond versus either 4-chloro-3-nitrophenyl or the second substituent could be described as cis-trans and trans-cis, respectively. Further, the patterns of intermolecular hydrogen bonding are specific for given conformers. It is worth noting that the factor influencing the biological activity could be a molecular flexibility. It is evident from the determined crystal structures of 20 and 24 that molecules of this group of derivatives are able to adopt different conformations ( Figure 1). Variable orientations of the thiourea C=S bond versus either 4-chloro-3-nitrophenyl or the second substituent could be described as cis-trans and trans-cis, respectively. Further, the patterns of intermolecular hydrogen bonding are specific for given conformers.
None of tested compounds showed selective antiviral activity against HIV-1. However, all compounds turned out interestingly cytotoxic for exponentially growing MT-4 cells, with many of them cytotoxic in the low micromolar range (CC50 < 10 µM). The antiproliferative activity against the CD4+ human T cell line derived from a hematological human tumor prompted us to evaluate the antiproliferative activity of a group of cytotoxic compounds (1, 4, 6, 9, 10, 11, 13, 14, 17, 19), also for a panel of other human and solid tumors, as well as for cell lines derived from normal human tissues (Table 4).
Interestingly, the tested compounds showed CC50 values comparable to that obtained with MT-4 cells, but they did not result highly selective, showing cytotoxicity also against the "normal" CRL7065 cell lines, even if with lower values. The highest selectivity versus CCRF-CEM and SK-MEL-28 cells was observed for 4-bromophenyl (6) and 4-iodophenylthiourea (17) derivatives. None of the tested compounds showed CC50 values comparable with Camptotecin, used as the reference compound.

Cytotoxicity and Anti-HIV Activity
Due to the previously reported anti-HIV activities of thiourea derivatives [11,43], both formerly (1-19, 25, 26) and newly synthesized compounds (20-24, 27-30) were tested in cell-based assay against the human immunodeficiency virus type-1 (HIV-1), using Efavirenz as the reference inhibitor. The cytotoxicity against MT-4 cells was evaluated in parallel with the antiviral activity ( Table 1).
None of tested compounds showed selective antiviral activity against HIV-1. However, all compounds turned out interestingly cytotoxic for exponentially growing MT-4 cells, with many of them cytotoxic in the low micromolar range (CC 50 < 10 µM). The antiproliferative activity against the CD4+ human T cell line derived from a hematological human tumor prompted us to evaluate the antiproliferative activity of a group of cytotoxic compounds (1, 4, 6, 9, 10, 11, 13, 14, 17, 19), also for a panel of other human and solid tumors, as well as for cell lines derived from normal human tissues (Table 4).
Interestingly, the tested compounds showed CC 50 values comparable to that obtained with MT-4 cells, but they did not result highly selective, showing cytotoxicity also against the "normal" CRL7065 cell lines, even if with lower values. The highest selectivity versus CCRF-CEM and SK-MEL-28 cells was observed for 4-bromophenyl (6) and 4-iodophenylthiourea (17) derivatives. None of the tested compounds showed CC 50 values comparable with Camptotecin, used as the reference compound.

Cytotoxic Activity in HaCaT Cells
The most promising antistaphylococcal (11,13) and antimycobacterial (25) agents among derivatives presented in our previous study [2] were now screened with respect to their toxicity against HaCaT cell line (health cell line from adult human skin). The HaCaT cell line was chosen, because it exhibits normal differentiation capacity, and its DNA fingerprint pattern is unaffected by long-term cultivations, transformations, and multiple chromosomal alterations [44].
Human keratinocyte cell lines were exposed to different concentrations of synthesized compounds for 24 h. Derivatives were tested in concentrations equal to their cytotoxic concentrations against MT-4 cells (i.e., 8 µM for 11 and 13, and 37 µM for 25), as well as in concentrations 2 and 3 times higher. At the concentration 8 µM, the thiourea compound 11 caused approximately 20% decrease in cell viability, in comparison with controlled and untreated cells (Figure 2a), that indicated its weak cytotoxic influence against normal cells. On the other hand, cytotoxicity of antibacterial agents 13 and 25 observed at the level of 50%, was found to be moderate according to ISO 10993-5 standards [45].
Results of the LDH (lactate dehydrogenase release) assay indicated that cell mortality in the presence of compounds 11 and 13 increased to 20% and 30%, respectively ( Figure 2b). The LD 10 value (concentration at which 10% of cells were dead) was determined at the lowest investigated concentration of the compound 11 (8 µM). Substance 13 at concentrations 8 and 16 µM was found to be lowly cytotoxic, resulting in an approximate 10% release of LDH. Compound 25 had the strongest impact on the mortality of tested cells, however, its concentration was the highest among all tested compounds (37 µM). All applied concentrations of that derivative resulted in 7-fold higher cytotoxicity as compared to the LD 10 value.
Significant concentration-dependent changes in shape, size, and density of HaCaT cells were observed under a light microscope after 24 h treatment with lowest concentrations of all investigated compounds (Figure 3). At given concentrations, derivatives inhibited viable cell proliferation by about 30-40% (compounds 13 and 25) or 65% (11) (Figure 4). This suggests a direct effect on the HaCaT viability, which was confirmed by the MTT assay. Derivatives 13 and 25 have demonstrated an increased proliferation rate.
The tested substances modified cell viability as well as mortality and growth rate of HaCaT cultures. The presented results demonstrate that derivative 11, one of the strongest antistaphylococcal and antibiofilm agents, at its lowest concentration, possessed the lowest toxic effect on HaCaT cells.
Our studies suggest that all tested substances in their lowest concentrations were less toxic in normal immortalized cell lines in comparison to cancer cell lines, however, further studies are needed to describe the underlining mechanism for future application. describe the underlining mechanism for future application.

Chemistry
All reagents and solvents were purchased from Alfa Aesar, Sigma Aldrich or POCh (Polskie Odczynniki Chemiczne, Gliwice, Poland). The infrared (IR) spectra were obtained on Perkin Elmer Spectrum 1000 spectrometer. The nuclear magnetic resonance (NMR) spectra were recorded on Varian VNMRS 300 Oxford NMR spectrometer, using tetramethylsilane (TMS) as the internal reference. Mass spectral electrospray ionization (ESI) measurements were carried out on Waters ZQ Micro-mass instruments with quadruple mass analyzer, at a declustering potential of 40-60 V. Flash chromatography was performed on Merck silica gel 60 (200-400 mesh) using chloroform eluent.

Chemistry
All reagents and solvents were purchased from Alfa Aesar, Sigma Aldrich or POCh (Polskie Odczynniki Chemiczne, Gliwice, Poland). The infrared (IR) spectra were obtained on Perkin Elmer Spectrum 1000 spectrometer. The nuclear magnetic resonance (NMR) spectra were recorded on Varian VNMRS 300 Oxford NMR spectrometer, using tetramethylsilane (TMS) as the internal reference. Mass spectral electrospray ionization (ESI) measurements were carried out on Waters ZQ Micro-mass instruments with quadruple mass analyzer, at a declustering potential of 40-60 V.

Chemistry
All reagents and solvents were purchased from Alfa Aesar, Sigma Aldrich or POCh (Polskie Odczynniki Chemiczne, Gliwice, Poland). The infrared (IR) spectra were obtained on Perkin Elmer Spectrum 1000 spectrometer. The nuclear magnetic resonance (NMR) spectra were recorded on Varian VNMRS 300 Oxford NMR spectrometer, using tetramethylsilane (TMS) as the internal reference. Mass spectral electrospray ionization (ESI) measurements were carried out on Waters ZQ Micro-mass instruments with quadruple mass analyzer, at a declustering potential of 40-60 V. Flash chromatography was performed on Merck silica gel 60 (200-400 mesh) using chloroform eluent.

X-ray Crystalography
The X-ray diffraction intensities were measured for 20 and 24 on a SuperNova diffractometer with monochromatized CuKα radiation (λ = 1.54184 Å). Data sets were collected using the ω scan technique. The programs CrysAlisPro [46] were used for data collection, cell refinement and data reduction; empirical absorption correction was applied using spherical harmonics implemented in SCALE3 ABSPACK scaling algorithm. The structure was solved by direct methods using SHELXS-97 and refined by the full-matrix least-squares on F 2 using the SHELXL-97 [47]. Non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were positioned geometrically and allowed to ride on their parent atoms, with U iso (H) = 1.2 U eq (C).

In Vitro Evaluation of Antimicrobial Activity
The antimicrobial activity of the compounds was tested on Gram-positive bacteria ( Antibacterial activity was examined by the disc-diffusion method under standard conditions using Mueller-Hinton II agar medium (Becton Dickinson) according to CLSI (previously NCCLS) guidelines [39]. Antifungal activities were assessed using Mueller-Hinton agar + 2% glucose and 0.5 µg/mL Methylene Blue Dye Medium [40]. Sterile filter paper discs (9 mm diameter, Whatman No 3 chromatography paper) were dripped with tested compound solutions (in DMSO) to load 400 µg of a given compound per disc. Dry discs were placed on the surface of appropriate agar medium. The results (diameter of the growth inhibition zone) were read after 18 h of incubation at 35 • C. Minimal Inhibitory Concentration (MIC) was tested by the twofold serial microdilution method (in 96-well microtiter plates) using Mueller-Hinton Broth medium (Beckton Dickinson) for bacteria or RPMI-1640 medium for Candida species according to CLSI guidelines [41,42]. The stock solution of tested agent was prepared in DMSO and diluted in sterile water. Concentrations of tested agents ranged from 0.125 to 512 µg/mL. The final inoculum of all studied microorganisms was 10 5 CFU/ mL −1 (colony forming units per mL). Minimal inhibitory concentrations (the lowest concentration of a tested agent that prevents visible growth of a microorganism) were read after 18 h (bacteria) or 24 h (yeasts) of incubation at 35 • C.

Cytotoxicity and Anti-HIV Assays
Cell line supporting the multiplication of Human Immunodeficiency Virus type-1 (HIV-1) was the CD4+ human T-cells, derived from human hematological tumors, containing an integrated HTLV-1 genome (MT-4). Cells were purchased from American Type Culture Collection (ATCC). Human Immunodeficiency Virus type-1 (HIV-1) IIIB laboratory strain was obtained from the supernatant of the persistently infected H9/IIIB cells (NIH 1983).
The activity of compounds against HIV-1 was based on inhibition of virus-induced cytopathogenicity in MT-4 cell acutely infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 µL of RPMI containing 1 × 10 4 MT-4 cells were added to each well of flat-bottom microtiter trays, containing 50 µL of RPMI without or with serial dilutions of test compounds. Then, 20 µL of a HIV-1 suspension containing 100 CCID 50 were added. After a 4-day incubation at 37 • C, cell viability was determined by the MTT method.
Cell lines derived from human solid tumors were: Skin melanoma (SK-MEL-28); prostate carcinoma (DU-145). Normal tissues foreskin fibroblasts (CRL-7065) were also used. Cells were seeded at 1 × 10 5 cells/mL in 96 well plates in specific media supplemented with 10% FCS and antibiotics, as above. Cell cultures were then incubated at 37 • C in a humidified, 5% CO 2 atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability was determined after 96 h at 37 • C by the MTT method.

Cell Culture: Conditions and Treatments
Human immortal keratinocyte cell line from adult human skin (HaCaT) was purchased from American Type Culture Collection (Rockville, MD, USA), and cultured in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with antibiotics (penicillin and streptomycin), 10% heat-inactivated FBS-fetal bovine serum (Gibco Life Technologies, Carlsbad, CA, USA) at 37 • C and 5% CO 2 atmosphere. Cells were passaged using trypsin-EDTA (Gibco Life Technologies, Carlsbad, CA, USA) and cultured in 24-well plates (2.5 × 10 4 cells per well). Experiments were conducted in DMEM with 2% FBS.

Cell Viability Assessment (Mitochondrial Function Assessment)
The cell viability assay was assessed by the determination of MTT salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich Chemie, Buchs, Germany) conversion by mitochondrial dehydrogenase. The cells were incubated for 24 h in 24-well plates with tested compounds (11,13 and 25) at given concentrations, and subsequently for another 2 h with 0.5 mg/mL of MTT solution, which was converted in living cells under the effect of mitochondrial dehydrogenase into insoluble formazan. Then the converted dye was solubilized with the use of 0.04 M HCl in absolute isopropanol. Absorbance of solubilized formazan was measured spectrophotometrically at 570 nm (using Epoch microplate reader, BioTek Inc., Winooski, VT, USA, equipped with Gen5 software (BioTech Instruments, Inc., Biokom, Janki, Polska).
Cell viability was presented as a percent of MTT in the treated cells versus the control (cells incubated in serum-free DMEM without extracts). The relative MTT level (%) was calculated as Release of lactate dehydrogenase (LDH) from the cytosol to culture medium is a marker of cell death. The assay was performed after 24 h incubation of HaCaT cells in 24-well plates with investigated concentrations of each compound. The activity of LDH released from the cytosol of damaged cells to the supernatant was measured using the protocol of the cytotoxicity detection kit LDH test described by the manufacturer (Roche Diagnostics, Berlin, Germany). Absorbance was measured at 490 nm using a microplate reader (using Epoch microplate reader, BioTek Inc., Winooski, VT, USA) equipped with Gen5 software (BioTech Instruments, Inc., Biokom, Janki, Polska) Compounds mediated cytotoxicity expressed as the LDH release (%) was determined by the following equation: [(A test sample-A low control)/(A high control-A low control)] × 100% (A-absorbance); where "low control" were cells in DMEM with 2% FBS without tested compounds, and "high control" were cells incubated in DMEM with 2% FBS with 10% Triton X-100 (100% LDH release).

Proliferation Rate in Cultures of HaCaT Cells
After 48 h incubation with the lowest concentration of the tested compound the increased number of viable cells were assessed by direct counting of cells using trypan blue with a Countess Automated Cell Counter (Invitrogen, Carlsbad, CA, USA). The results are presented as a % of viable and dead cells versus control.

Conclusions
To sum up, this work has revealed the synthesis of new thiourea derivatives and their in vitro antibacterial activity against clinically relevant pathogens. The title compounds showed potent to moderate antimicrobial activity. Representative aryl-thioureas 20, 22, 23 and bicyclic-thiourea derivative 24 were even more active towards selected clinical staphylococci than the reference drug, Ciprofloxacin. To complete the biological profile of the presented thiourea series, their cytotoxic activities in MT-4 cells, as well as human hematological and solid tumors cell lines were assessed. Several compounds (1, 4, 6, 9-11, 13, 14, 17, 19) turned out cytotoxic in the low micromolar range (CC 50 < 10 µM). Performed studies proved that synthesized derivatives weakly to moderately influenced the viability and growth of the human immortal keratinocyte cell line (HaCaT).