Efficient Synthesis and Antimicrobial Activities of Long Alkyl Chain Trifluoromethyl- 1H-pyrazol-1-(thio)carboxamides and Trifluoromethyl-1H-pyrazol-1-yl-thiazoles

The synthesis of 3-alkyl-5-trifluoromethyl-1Hpyrazole-1-carboxamides, 3-alkyl5-trifluoromethyl-1H-pyrazole-1-thiocarboxamides, and 2-(3-alkyl-5-trifluoromethyl-1H-pyrazol1-yl)-thiazoles derivatives are reported. [3 + 2] cyclocondensations for a series of long alkyl chain 1,1,1-trifluoro-4-methoxyalk-3-en-2-ones and semicarbazide or thiosemicarbazide were carried out in ethanol, an eco-friendly medium. The series of trifluoromethyl-1H-pyrazol-1-ylthiocarboxamide following a [3 + 2] cyclocondensation with 2-bromoacetophenone were converted into two series of 2-(3-alkyl-5-trifluoromethyl-1H-pyrazol-1-yl)-thiazoles. Good yields (69-96%) of the isolated products were obtained. The structures of the new long alkyl chain 1H-pyrazoles and 2-(1H-pyrazol-1-yl)thiazoles were characterized using H, C, and F nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization tandem mass spectrometric (ESI MS/MS) data. Moreover, some of the products were evaluated for their antimicrobial activity against Gram-negative Escherichia coli American Type Culture Collection (ATCC) 35218, Salmonella enteritidis ATCC 13076, and Pseudomonas aeruginosa ATCC 15692; and Gram-positive Staphylococcus aureus ATCC 6538, methicillin resistant Staphylococcus aureus (MRSA clinical isolate), Streptococcus sp. (clinical isolate), and Candida albicans ATCC 14053 and Candida krusei ATCC 6258 fungi. All the tested 1H-pyrazoles exhibited antibacterial and antifungal activities at the tested concentrations. The compounds from series 4 were found to be powerful against MRSA.


Introduction
Bacterial and fungal diseases affect millions of human worldwide every day. Several million doses of antibiotics are prescribed annually for these infections, contributing to the current problem of increased antibiotic resistance. The global charge to society of this is a very high annual financial cost to treat antibiotic-resistant infections in addition to the lives lost due to inefficient therapies. 1,2 Thiazole compounds have been described as antimicrobial agents. 3,4 This heterocycle is a prevalent scaffold in a number of naturally occurring and synthetic compounds with attractive pharmacological activities such as anti-inflammatory, analgesic, anticonvulsant, antitumor, antiviral, antifungal, and antibacterial, including antitubercular. 5,6 Niridazole and levamisole, thiazole derivatives, are used as antihelmintic drugs; 7 ritonavir is an antiretroviral drug, from the protease inhibitor class, used to treat HIV (human immunodeficiency virus) infections and AIDS (acquired immunodeficiency syndrome). 8 Therefore, a great deal of interest has been focused on the design and biological activity of thiazole derivatives. On the other hand, CF 3 -containing 1H-pyrazoles have emerged as a group of compounds possessing a broad spectrum of useful biological properties such as herbicides, fungicides, and analgesic activities, and because of this, their synthesis has also received intense attention. 9,10 The 4-alkoxy-1,1,1-trihalo-3-alken-2-ones have been proven to be key intermediates in the heterocycle synthesis protocols. 11,12 They represent a range of precursors containing a 1,3-dielectrophilic system with differentiated reactivity carbons applied in the development of heterocycle
for the 2a-2g series; however, the signals due to NH 2 for 3a-3g appear as two broad singlets at 6.22 and 7.14 ppm. The signal for H4 from the aromatic 1H-pyrazol ring of product 5 was at d 6.6-6.7 ppm, while that for H5 of the 4-phenylthiazole ring of products 4 and 5 had a signal at d 6.90-7.25 ppm. In the 13 C NMR spectra are similar for the 4,5-dihydro-1H-pyrazole series, the quartet signals due to carbons coupling to fluorine atoms, the C5 at 90-92 ppm with 2 J CF 34 Hz, and CF 3 group were observed at about 123 ppm with J CF 284-286 Hz. The quartet signal related to C5-CF 3 from the aromatic 1H-pyrazole ring was at about d 132 ppm with J CF 42 Hz. Besides the signal set related to the straight saturated chain substituent, including the terminal methyl, the overlapping methylenes and, α and β methylene to pyrazole ring at consistent chemical shifts (see Supplementary Information).

Biological studies
All the novel trifluoromethyl-1H-pyrazol-1-(thio) carboxamides (2, 3) and trifluoromethyl-1H-pyrazol-1-yl-thiazoles (4, 5) were evaluated in vitro for antibacterial activity against a representative human pathogenic Gram-positive bacteria, such as S. aureus American Type Culture Collection (ATCC) 6538, clinically isolated methicillin-resistant S. aureus (MRSA), and clinically isolated Streptococcus sp.; and Gram-negative bacteria, such as S. enteritidis ATCC 13076, E. coli ATCC 35218, and P. aeruginosa ATCC 15692. The antifungal profile of the compounds was evaluated against C. albicans ATCC 14053 and C. krusei ATCC 6258. An agar-diffusion method was utilized for the initial rating of antimicrobial activities. Chloramphenicol and ketoconazole were used as standard antibacterial and antifungal agents. The antimicrobial activities of 1H-pyrazoles (2)(3)(4)(5) are summarized in Tables 2  and 3. The measurements were recorded for each new compound as the average diameter of the inhibition zones of bacterial or fungal growth around the disks in mm, for the triplicate experiment. The minimum inhibitory concentration (MIC) in μg mL −1 was measured for all compounds for a representative microorganism, which showed large growth inhibition zones, using the twofold  serial dilution method. The inhibition zone diameter values shown in Table 2 refer to the tested concentration of 2500 μg mL −1 and the MICs (μg mL −1 ) for selected microorganisms are shown in Table 3. From the results, it has been observed that all the tested compounds possess moderate to poor antimicrobial activity against selected bacteria strains. On the basis of the inhibition zone test against S. aureus, compounds 2d (R = n-C 9 H 19 ), 4d (R = n-C 9 H 19 ) and those in series 5 were found to have inhibitory activity when compared with the standard drug chloramphenicol. The selective activity on Gram-positive bacteria of the studied compounds may indicate good permeability by the outer layer of the membrane of these monoderms bacteria, a thick peptidoglycan layer crossed by teichoic and lipoteichoic acids. But they do not cross the more complex outer membrane of Gram-negative bacteria with the same ease. 18 In vitro MIC and minimal bactericidal concentrations (MBC) determined by means of a standard twofold dilution method are reported in Table 3. In this study, both Gram-positive cocci and Gram-negative bacteria showed poor susceptibilities to the tested trifluoromethyl-1H-pyrazol-1-(thio)carboxamides (2, 3) and trifluoromethyl-1H-pyrazol-1-yl-thiazoles (4, 5) when compared to standard chloramphenicol. The results showed that the unbranched C6-C13 alkyl chain in trifluoromethyl-1H-pyrazol-1-(thio)carboxamides (2, 3) and trifluoromethyl-1H-pyrazol-1-yl-thiazoles (4, 5) was not very relevant to antimicrobial activity. The investigation of the antibacterial screening data revealed that the tested compounds exhibit poor to moderate bacterial inhibitions. Compounds 4a-4g showed good inhibition against S. aureus with an MIC 150 μg mL −1 ; however, they were not efficient against E. coli. The 1H-pyrazoles (2, 3), and pyrazolyl-thiazole (5) showed poor inhibition against tested bacteria. The newly prepared compounds (2-5) were screened for their antifungal activity against C. albicans and C. krusei; the inhibition zone tests had some relevance for both, however, when the minimal fungicidal concentration (MFC) for the C. albicans was determined, the values obtained for the series (2-5) show very low activity in relation to the standard ketoconazole.

Experimental
The starting 1,1,1-trifluoro-4-methoxy-3-alken-2-ones (3) were synthesized by the acylation of the respective fatty alkyl methyl ketones with trifluoroacetic anhydride in pyridine and dichloromethane or chloroform, which resulted in good yields of 90%. 1 H, 13  Chemical shifts (d) for 1 H are given in parts per million (ppm) from tetramethylsilane (TMS), for 19 F (d) Cl 3 CF was the standard, setting the signal at 0.0 ppm, and coupling constants (J) are given in Hz. Melting points were determined using open capillaries on an Electrothermal Mel-Temp 3.0 apparatus and are uncorrected. Electrospray ionization (ESI) high-resolution mass spectra (HRMS) were determined using an Agilent 6460 Triple Quadrupole connected to a 1200 series LC and equipped with a solvent degasser, binary pump, column oven, auto-sampler, and an ESI source. The Agilent QQQ 6460 tandem mass spectrometer was operated in the positive jet stream ESI mode. Nitrogen was used as a nebulizer, turbo (heater) gas, curtain gas, and collision-activated dissociation gas. The capillary voltage was set to +3500 V, and the nozzle voltage was set to +500 V. The ion source gas temperature was 300 °C with a flow rate of 5 L min −1 . The jet stream sheath gas temperature was 250 °C with a flow rate of 11 L min −1 . All samples were infused into the ESI source at a flow rate of 5 μL min −1 . Data was acquired in positive MS total ion scan mode (mass scan range m/z 50-650) and in positive MS/MS product ion scan mode. The mass spectra recorded were evaluated using the Qualitative Analysis software from Agilent Technologies.
The antimicrobial susceptibility assay was performed using the disc diffusion method described by Bauer et al. 19 in Mueller-Hinton agar. The suspensions containing different strains were seeded on a plate containing Mueller-Hinton agar. 10 μL of the compounds at a concentration of 100 mg mL −1 was added to the discs which in turn were deposited on a plate. The plate, with the discs, was incubated for 24 h at 37 °C, then the inhibition zones were measured with a calliper ruler. For the determination of the MIC and MBC values of the substances against the test microorganisms, the broth dilution method was used according to the Clinical Laboratory Standards Institute (CLSI). 20 Stock solutions of the substances were prepared in 20% dimethyl sulfoxide (DMSO) and a twofold dilution series from 50.0-0.39 mg mL −1 was prepared using sterile distilled water. 100 μL from each of the dilutions was added to 96-well microtiter plates. Bacterial and yeast cultures were standardized to 10 8 colony forming unit (CFU) mL −1 using a 0.5 McFarland standard solution. The fungal spore suspensions were prepared using sterile 0.1% Tween 80 and adjusted to about 10 6 spores mL −1 . 100 μL of each microorganism suspension was then added into the wells and incubated at 37 °C for 24 h in aerobic conditions. The culture medium alone and medium containing bacteria without testing compounds were considered as controls. Triplicate wells were applied for each concentration of the individual test material. Chloramphenicol and ketoconazole were used as standard antibacterial and antifungal drugs, respectively. MIC and MBC/MFC values were detected using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromine] method. 21 A concentration of 5 mg mL −1 MTT was prepared in phosphate-buffered saline (PBS pH 7.2) of Mueller-Hinton broth. It was used in suspensions containing Candida albicans, Staphylococcus aureus, and Escherichia coli. After dilution, 10 μL of the bacterial suspension was added in all wells; the plate was then incubated for 24 h at 37 °C. After incubation, 100 μL of 2,3,5-triphenyltetrazolium chloride was used to indicate microbial growth.  (CF 3 , q, J CF 287 Hz), 156.5 (C3), 156.6 (CO); 19  General procedure for the synthesis of 3-alkyl-5-hydroxy-5-trifluoromethyl-4,5-dihydro-1H-pyrazol-1-thiocarboxamides (3a-3g) A solution of the thiosemicarbazide (1.2 mmol) and a 1,1,1-trifluoro-4-methoxyalk-3-en-2-one (1 mmol) in EtOH (6 mL) was kept under stirring at 78 °C until the starting dielectrophile was fully consumed, monitored using TLC, for 16 h. After cooling the solution, the precipitated product (3a-3g) was filtered off, washed with cooled EtOH (2 × 10 mL), and dried in air to give the following: NH 2 ), 7.11 (broad s, 1H, NH 2 ), 7.96 (s, 1H, OH); 13  A solution of a 3-alkyl-5-trifluoromethyl-5-hydroxy-4,5-dihydro-1H-pyrazol-1-thiocarboxamide (3a-3g) (1.0 mmol) and 2-bromoacetophenone (1 mmol, 0.2 g) in EtOH (6 mL) was kept under stirring at 30 °C until the starting 2-bromoacetophenone was fully consumed, monitored using TLC, 8 h for 3a-3e, 3g and 24 h for 3f. Then EtOH was evaporated under reduced pressure, and the crude product (4a-4g) was solubilized in chloroform (6 mL), washed with water (2 × 6 mL), and dried over MgSO 4 to give the following: