A facile synthesis, and antimicrobial and anticancer activities of some pyridines, thioamides, thiazole, urea, quinazoline, β-naphthyl carbamate, and pyrano[2,3-d]thiazole derivatives

Background Chalcones have a place with the flavonoid family and show a few very important pharmacological activities. They can used as initial compounds for synthesis of several heterocyclic compounds. The compounds with the backbone of chalcones have been reported to possess various biological activities. Results Pyridine and thioamide derivatives were obtained from the reaction of 3-(furan-2-yl)-1-(p-tolyl)prop-2-en-1-one with the appropriate amount of malononitrile, benzoylacetonitrile, ethyl cyanoacetate and thiosemicarbazide in the presence of ammonium acetate. The reaction of 3,5-di(furan-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide with ethyl 2-chloro-3-oxobutanoate, 3-chloropentane-2,4-dione or ethyl chloroacetate produced thiazole derivatives. Pyrano[2,3-d]thiazole derivatives were obtained as well from thiazolone to arylidene malononitrile. The structures of the title compounds were clarified by elemental analyses, and FTIR, MS and NMR spectra. Some compounds were screened against various microorganisms (i.e., bacteria +ve, bacteria −ve and fungi). We observed that compounds (3a), (4a), (4d), (5), (7) and compound (8) exhibited high cytotoxicity against the MCF-7 cell line, with IC50 values of 23.6, 13.5, 15.1, 9.56, 14.2 and 23.5 μmol mL−1, respectively, while compound (9) was displayed the lowest values against MCF-7 cell lines. Conclusions Efficient synthetic routes for some prepared pyridines, pyrazoline, thioamide, thiazoles and pyrano[2,3-d]thiazole were created. Moreover, selected newly-synthesized products were evaluated for their antitumor activity against two carcinoma cell lines: breast MCF-7 and colon HCT-116 human cancer cell lines.


Cytotoxicity evaluations
The in vitro growth inhibitory activity of the synthesized compounds 3a, 4a, 4d-4f, 5, 7, 8, 9, 11a, and 11b was investigated against two carcinoma cell lines: breast MCF-7 and colon HCT-116 human cancer cell lines in comparison with the Imatinib anticancer standard drug (cisplatin) under the same conditions using the crystal violet viability assay. Data generated were used to plot a dose response curve where the concentration of test compounds required to kill 50% of the cell population (IC 50 ) was determined and is summarized in Table 1. The IC 50 values of the synthesized compounds 4a, 4d, 5, 7, and 8, (IC 50 = 9.65-23.6 μmol mL −1 ) were comparable to that of Imatinib. We observed that compounds 3a, 4a, 4d, 5, 7, and 8 exhibited high cytotoxicity against the MCF-7 cell line, with IC 50 values of 23.6, 13.5, 15.1, 9.56, 14.2 and 23.5 μmol/mL, respectively, while compound 9 was observed as having the lowest against the MCF-7 cell lines. Our results showed that compounds 4e, 4f, 11a and 11b had the lowest IC 50 values against HCT-116 cancer cells.

Antimicrobial activity
Nineteen of the newly synthesized target compounds were evaluated for their in vitro antibacterial activity against Streptococcus pneumonia and Bacillus subtilis (as examples of Gram-positive bacteria) and Pseudomonas aeruginosa and Escherichia coli (as examples of Gramnegative bacteria). They were also evaluated for their in vitro antifungal activity against a representative panel of fungal strains i.e., Aspergillus fumigatus and Candida albicans fungal strains. Ampicillin and Gentamicin are used as reference drugs for in vitro antibacterial activity while Amphotericin B is a reference drug for in vitro antifungal activity, respectively, at The Regional Center for Mycology and Biotechnology at Al-Azhar University (Nasr City, Cairo, Egypt). The results of testing for antimicrobial effects are summarized in Table 2.

General information
All melting points were measured with a Gallenkamp melting point apparatus (Weiss-Gallenkamp, London, Scheme 1 Synthesis of pyridine derivatives (2-4) and thioamide (5) UK). The infrared spectra were recorded using potassium bromide disks on pye Uni-cam SP 3300 and Shimadzu FT-IR 8101 PC infrared spectrophotometers (Pye Unicam Ltd. Cambridge, England, and Shimadzu, Tokyo, Japan, respectively). The NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer (Varian, Palo Alto, CA, USA). 1 H spectra were run at 300 MHz and 13 C spectra were run at 75.46 MHz in deuterated chloroform (CDCl 3 ) or dimethyl sulphoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer (Shimadzu) at 70 eV. Elemental analyses were carried out at the Microanalytical Center of Cairo University. The antimicrobial and antcancer screening was performed at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.

General methods for the synthesis of pyridines (2-4)a-f
Method A A mixture of the appropriate chalcones (1a-f) (10 mmol), and the appropriate amount of malononitrile, benzoylacetonitrile, or ethyl cyanoacetate (10 mmol) in glacial acetic acid containing ammonium acetate (0.77 g, 10 mmol) was refluxed for 3-4 h, and the acetic acid was evaporated under reduced pressure, left to cool, then poured. gradually with stirring onto crushed ice. The solid formed was filtered off, dried, and recrystallized from an appropriate solvent to obtain the corresponding pyridines (2-4)a-f, respectively.
Method B A mixture of the appropriate aldehydes (10 mmol), arylketone (10 mmol), and the appropriate amount of malononitrile, benzoylacetonitrile, or ethyl cyanoacetate (10 mmol) in n-butanol (20 mL) containing ammonium acetate (6.00 g, 77 mmol) was refluxed for 3-4 h, then the solvent evaporated under reduced pressure, left to cool, then poured gradually with stirring onto crushed ice. The solid formed was filtered off, dried, and recrystallized from an appropriate solvent to obtain products that were identical in all respects (mp, mixed mp, and IR spectra) with the corresponding pyridines (2-4)a-f, respectively. The products (2-4)a-f together with their physical constants are listed below.

Evaluation of the antitumor activity using viability Assay
Crystal violet stain (1%), composed of 0.5% (w/v) crystal violet and 50% methanol, was made up to volume with ddH 2 O and filtered through a Whitman No. 1 filter paper.

Cytotoxicity evaluation using viability assay
Human hepatocellular breast (MCF-7) and colon (HCT-116) carcinoma cells were obtained from the VACSERA Tissue Culture Unit. The cells were propagated in Dulbecco's modified Eagle's medium (DMEM), and supplemented with 10% heat-inactivated fetal bovine serum, 1% l-glutamine, HEPES buffer and 50 μmol mL −1 gentamycin. All cells were maintained at 37 °C in a humidified atmosphere with 5% CO 2 and were sub-cultured twice a week.

Evaluation of cytotoxicity activity
Cytotoxicity of all compounds was tested in MCF-7 and HCT-116 cells. All experiments and data concerning the cytotoxicity evaluation were performed at the Regional Center for Mycology and Biotechnology RCMB, Al-Azhar University, Cairo, Egypt. For the cytotoxicity assay, cells were seeded in a 96-well plate at a cell concentration of 1 × 10 4 cells per well in 100 μL of growth medium. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. Serial two-fold dilutions of the tested compounds were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette. The microtiter plates were incubated at 37 °C in a humidified incubator with 5% CO 2 for a period of 48 h. Three wells were used for each concentration of the test sample. Control cells were incubated without the test sample and with or without DMSO. The little percentage of DMSO present in the wells (maximal 0.1%) was found not to affect the experiment. After incubation of the cells for at 37 °C, various concentrations of the sample were added, and the incubation continued for 24 h before viable cell yield was determined using a colorimetric method. In brief, after the end of the incubation period, media were aspirated and the crystal violet solution (1%) was added to each well for at least 30 min. The stain was removed and the plates were rinsed using tap water until all excess stain was removed. Glacial acetic acid (30%) was then added to all wells and mixed thoroughly, before the absorbance of the plates was measured (after being gently shaken) on a Microplate reader (TECAN, Inc.), using a test wavelength of 490 nm. All results were corrected for background absorbance detected in wells without added stain. Treated samples were compared with the cell control in the absence of the tested compounds. All experiments were carried out in triplicate. The cell cytotoxic effect of each tested compound was calculated. Optical density was measured with a microplate reader (SunRise, TECAN, Inc., USA) to determine the number of viable cells, and the percentage of viability was calculated as the percentage of cell viability = [1 − (ODt/ ODc)] × 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relationship between the surviving cells and drug concentration was plotted to obtain the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each concentration using Graphpad Prism software (San Diego, CA. USA).

Antimicrobial activity assay
Chemical compounds under investigation were individually tested against a panel of Gram-positive and Gram-negative bacterial pathogens, and fungi. Antimicrobial tests were conducted using the agar well-diffusion method [36][37][38]. After the media had cooled and solidified, wells (6 mm in diameter) were made in the solidified agar, before microbial inoculum was uniformly spread using a sterile cotton swab on a sterile Petri dish containing nutrient agar (NA) medium, or Sabouraud dextrose agar (SDA) media for bacteria and fungi, respectively. An amount of 100 µL of the tested compound solution was prepared by dissolving 1 mg of the compound in 1 mL of dimethylsulfoxide (DMSO). The inoculated plates were then incubated for 24 h at 37 °C for bacteria and yeast, and 48 h at 28 °C for fungi. Negative controls were prepared using DMSO employed for dissolving the tested compound. Amphotericin B (1 mg/mL), Ampicillin (1 mg/mL), and Gentamicin (1 mg/mL) were used as standards for bacteria and fungi, respectively. After incubation, antimicrobial activity was evaluated by measuring the zone of inhibition against the tested microorganisms. Antimicrobial activity was expressed as inhibition diameter zones in millimeters (mm).

Conclusions
In summary, new and efficient synthetic routes of some prepared pyridines, pyrazoline, thiazoles, and pyrano[2,3-d]thiazole were achieved. The structure of the newly prepared compounds was established based on elemental analysis, spectral data, and alternative methods wherever possible. The synthesized compounds (3a, 4a, 4d-4f, 5, 7-9, 11a, and 11b) were investigated against two carcinoma cell lines: breast MCF-7 and colon HCT-116 human cancer cell lines. Our results showed that compounds 4e, 4f, 11a, and 11b had the lowest IC 50 values against HCT-116 cancer cells. In addition, the selected newly prepared compounds were evaluated for their antimicrobial activity against Gram-positive and Gram-negative bacteria as well as some fungal-plants.
The results proved that some prepared compounds showed an adequate inhibitory efficiency of growth of Gram-positive and Gram-negative bacteria.