Synthesis, Structure Elucidation, Antioxidant and Antimicrobial Activity of Novel 2-(5-Trifluoromethyl-1H-pyrazol-1-yl)-5-(5-trihalomethyl-1H-pyrazol-1-yl-1- carbonyl)pyridines

This paper describes an efficient approach for the synthesis of a novel series of sixteen 2-(5-trifluoromethyl-1H-pyrazol-1-yl)-5-(5-trihalomethyl-1H-pyrazol-1-yl-1-carbonyl) pyridines, for the first time with non-identical substituents in both pyrazole rings, through the cyclocondensation reaction of 4-methoxy-4-alkyl(aryl/heteroaryl-1,1,1-trihaloalk-3-en-2-ones [CX3C(O)CH=CR1OCH3, in which R1 = CH3, C6H5, 4-CH3C6H4, 4-OCH3C6H4, 2-furyl and X = F, Cl] or acetylacetone with some 6-[3-alkyl(aryl)-5-trifluoromethyl-1H-pyrazol-1-yl]nicotinohydrazides. Optimized yields of 67-91% were obtained when the reactions were performed in ethanol (green solvent) at reflux for 16 h. Subsequent antioxidant and antimicrobial evaluation revealed promising 1,1-diphenyl-2-picrylhydrazyl (DPPH) inhibition percentage and exhibited fungiostatic and fungicidal activities against yeasts, dermatophytes and filamentous, especially for the pyridine systems, when the both pyrazole rings attached to a pyridine ring contain CX3 groups (X = H, F, Cl) of different kinds. It is also observed the trichloromethyl substituted compounds presented higher antioxidant activity in comparison to their fluorinated analogous.


Introduction
2][3][4] The balance between the formation and removal of different types of radicals in the body must be regulated so that there is physiological cell maintenance.For this reason, the use of antioxidant agents in medicine has been a great field to explore, and more than this, these agents have been shown to have many industrial uses; for example, as food preservatives, cosmetic products, and gasoline additives. 5onsequently, there is great interest in the discovery of new antioxidant agents for preventing the presumably harmful effect that free radicals have on the human body.Antioxidant agents are capable of stabilizing or deactivating free radicals before they attack the biological targets in the cell system. 6ethods for the analysis of antioxidant activity have become relevant, because they help in the search for bioactive substances.Due to various types of radicals and different sites of action, several methods (with their peculiarities) are available and have been used to assess: the antioxidant potential of extracts, isolated or synthesized substances, peroxyl capture methods, metal reduction ability, hydroxyl radical capture, organic radical capture, etc. [7][8][9][10] It is important to select and employ a stable and rapid method to assess antioxidant activity.
On the other hand, antimicrobial and antibiotic substances have undergone major advance in the last five decades, with unlimited progress in drug therapy.As the advent of antibiotics and chemotherapeutics allowed the control and cure of infectious diseases, there was a marked change in the natural history of these diseases.However, the indiscriminate use of antibiotics in the 1970s resulted in the acceleration of the emergence of resistant bacterial Vol. 26, No. 11, 2015   strains. 11,12The search for new antimicrobial agents capable of inhibiting bacteria and fungi growth in a safer effective way that causes fewer side effects, has been the study object for many researchers.Infectious diseases caused by bacteria and fungi affect millions of people around the globe. 13Although invasive fungal infections can affect healthy people, most of these diseases occur in the context of a compromised host.
Despite the latest technology for antifungal therapy, mortality rates for invasive infections with the three most common species of human fungal pathogens remain at: 20-40% for Candida albicans; 50-90% for Aspergillus fumigatus; and 20-70% for Cryptococcus neoformans.The variety of antifungal agents is limited, particularly when compared to the number of agents available for bacterial infections.In fact, it took around 30 years for the development of the newest class of antifungal drugs. 14he great interest in novel drugs for the treatment of infectious diseases is still a challenge, due to a combination of factors, such as emerging infectious diseases and the increasing number of multidrug-resistant pathogens. 15Therefore, the pyrazole nucleus represents a very attractive scaffold for applications in the agrochemical and pharmaceutical industries, due to it being capable of exhibiting a wide range of bioactivities; for example, antimicrobial, 16 anticancer, 17 anticonvulsant, 18 antitubercular, 19 antipyretic 20 and selective enzyme inhibitory activities. 21pecifically, bis-pyrazoles have been reported to be effective components in capturing active oxygen and free radicals in vivo, 22,23 and other derivatives have been synthesized and used as promising agents with antifungal, 24 herbicidal, 25 and central nervous system activities. 26 brief review of the literature shows only a few reports on the synthesis of bis-pyrazole systems up until now.Soliman et al. 27 published the synthesis of bis-3,5-dimethylpyrazoles bridged by carbonyl groups with considerable antidiabetic activity, from the substitution reactions between ethyl chloroformate and 3,5-dimethylpyrazole. Two decades later, Hayter et al. 28 described the synthesis of bis-3-alkyl(aryl)-pyrazoles bridged by a benzene ring, from the cyclocondensation reaction of two equivalents of hydrazine and the appropriated bis-(βdiketones).
Kanagarajan et al. 29 reported improvements in the synthesis of carbocycle-bridged 5,5'-(1,4-phenylene)bis-(3-aryl-1H-pyrazole) derivatives through the reaction of bis-chalcones and hydrazine, which was performed under ultrasonic irradiation.In the field of luminescent materials, Bao et al. 30 reported a novel ligand containing bis-pyrazolone pyridine and with this they synthesized complexes with Eu III and Tb III .However, these approaches only explore simple hydrazine derivatives, most of them with carbocyclic bridges and leading to a limited scope and identical substituents in both pyrazole rings.
Our research group reported a one-pot regioselective synthesis of a series of bis-(3-aryl-5-trifluoromethyl-5-hydroxy-4,5-dihydro-1H-pyrazol-1-yl) methanones from the reactions of 4-alkoxy-4-aryl(heteroaryl)-1,1,1trifluoroalk-3-en-2-ones with carbohydrazides, at yields of 73-89%. 31In the same year, our research group started to explore the reactivity of 6-hydrazinonicotinic hydrazide in cyclocondensation reactions with trifluoromethyl vinyl ketones, at a 1:2 molar ratio, respectively.The first work resulted in the synthesis of a series of 2-(1H-pyrazol-1yl)-5-(1H-pyrazol-1-yl-1-carbonyl)pyridines with yields of 62-97%. 32It is also known that the 6-hydrazinonicotinic hydrazide has two distinct nucleophilic centers in its structure: a hydrazine and a hydrazide function, which would give this building block differentiated reactivity with electrophiles.However, in a recent study performed by us with 6-hydrazinonicotinic hydrazide, it was observed that no chemoselectivity is achieved when cyclocondensation reactions are promoted with the trifluoromethyl vinyl ketones at a 1:1 molar ratio, resulting in a mixture of products and partial recovery of the hydrazide. 33ollowing our continuous studies of the reactivity of 6-hydrazinonicotinic hydrazide, in 2014, we reported a convenient access by a green procedure in ethanol as solvent to a series of (E)-6-[2-ferrocenylalkylidenehydrazino] nicotinic hydrazides from the quimioselective reactions of 6-hydrazinonicotinc hydrazide with some acylferrocenes.Subsequently, cyclocondensation reactions of ferrocenylalkylidene hydrazones with 4-alkoxy-1,1,1-trifluoroalk-3-en-2-ones to obtain (E)-pyrazolylpyridinohydrazones, were also reported. 34In the same year, a synthetic route for the selective "deprotection" reaction (amide hydrolysis reaction) for 2-(1H-pyrazol-1-yl)-5-(1Hpyrazol-1-yl-1-carbonyl)pyridines, which furnished the corresponding methyl trifluoromethylpyrazolyl nicotinates, preserving only the pyrazole ring bonded to C-2 pyridine, and which originated from the hydrazine moiety, was reported by us. 35In a subsequent reaction with hydrazine hydrate, the ester function at the C-5 pyridine was restored to the initial hydrazide moiety, which led to the possibility of new cyclocondensation reactions and the construction of various heterocycles from this new nucleophilic precursor.
In view of this consistent literature review, and based on the results of our previous work, [32][33][34][35] the aim of this work is to present the results of an efficient methodology in order to construct bis-trihalomethylated pyrazolylpyridine systems containing two non-identical substituted pyrazole rings, from the [3 + 2] cyclocondensation reactions of pyrazol-1-yl-nicotinohydrazides with 4-alkoxy-4alkyl(aryl/heteroaryl-1,1,1-trifluoro(chloro)alk-3-en-2ones or acetylacetone, and then elucidating their chemical structures by nuclear magnetic resonance (NMR) and X-ray diffraction, as well as evaluating their antimicrobial and antioxidant activity in vitro.
Taking into consideration the results of the previous reactions involving halogenated ketones 4 and 5, we performed the synthesis of 13 with the hydrazide 2 and acetylacetone.The initial methodology tested employed ethanol as the solvent, at reflux temperature for 16 h.However, a mixture of pyrazoline-pyridine derivative 13 and the pyrazole-pyridine product 14 was obtained, being clearly observed by TLC and 1 H NMR analysis.Since it is well known that basic conditions favor the obtainment of pyrazolines, and that acidic catalysis can improve the formation of pyrazole products by dehydration reaction, we first tested a basic catalysis with triethylamine (Et 3 N) and ethanol reflux for 16 h.After the reaction time, the formation of pyrazoline (cycle C), confirmed as a single product 13, was characterized and proven by 1 H NMR spectra.In the following step, compound 13 was easily dehydrated with SOCl 2 , leading to the corresponding pyridine 14 (Scheme 3) which contains all three aromatic rings (cycles A, B and C) and according to the procedure reported in the literature for similar compounds. 39In previous works, researchers have demonstrated efficient and mild methodologies for intramolecular dehydration reaction for  non-halogenated 5-hydroxy-4,5-dihydro-pyrazoles, under acidic conditions. 40,41Thus, the cyclocondensation reaction was then performed by mild acidic conditions (ethanol, acetic acid mixture), under reflux for 16 h in ethanol.As a result, the pyridine 14 was isolated directly from 2, in a one-step, one-pot reaction, as a single product-identified by 1 H NMR analysis-with a much more satisfactory yield of 71% (Scheme 3).
4][35]41 The 1 H NMR analysis in dimethylsulfoxide, DMSO-d 6 , revealed a chemical shift for the H-4 as a sharp singlet in the range of 7.04 ppm (R 2 = CH 3 ), 7.84 ppm (R 2 = Ph), and 7.59 ppm (R 2 = 2-furyl) at the pyrazole ring (cycle B).For    C).The structures of 13-14, for which X = H, were then deduced from NMR spectra ( 1 H, 13 C, and 19 F) and also by comparison with the NMR data of other formerly synthesized pyrazoles 6-10 (in which X = Cl, F).Compound 13 showed: the hydroxyl at d 6.51 ppm; the doublet (H-4) with J 18.0 Hz at 3.02 and 2.91 ppm; and singlets each one of three hydrogen methyls (CH 3 ) at 1.98 and 1.92 ppm.Vinylic hydrogens from the pyrazoline ring (cycle C) of compound 14 were seen at d 6.33 ppm as a singlet (H-4) and at 2.60 and 2.20 ppm for the two methyls.Additionally, to confirm the structures, we performed X-ray diffraction measurements for a monocrystal of structures 6a (Figure 1), 7a (Figure 2) and 14 (Figure 3).

Biological activity
In the present work we evaluate the in vitro antimicrobial activity of nine representative compounds (6a, 8d, 9c, 9d, 9f, 9g, 10h, 11e and 14) against a panel of microorganisms including bacteria and fungi (yeasts, filaments and dermatophytes).These selected compounds present structural scaffolds that allowed to verify the influence of the CX 3 , R 1 and R 2 substituents on this   biological study.The assays were performed by broth microdilution techniques, in accordance with Clinical Laboratory Standard Institute (CLSI) guidelines, using Muller-Hinton broth (bacteria) and RPMI 1640 broth (fungi).The minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) were determined.Consequently, for these tests it was found that none of the tested compounds showed inhibition to Gram-positive and Gram-negative bacteria at concentrations ranging from 1000 to 1.95 µg mL -1 .As for the fungi, the concentrations used ranged from 500 to 0.98 µg mL -1 .We found that the fungiostatic and fungicidal activities depends not only of the CX 3 group but also of the R 1 and R 2 substituents.For example: a comparison between compounds 8d (CX 3 = CCl 3 ) and 9d (CX 3 = CF 3 ), both containing R 1 and R 2 = Ph as substituents, showed none activity.However, a comparison between compounds 8d and 6a (CX 3 = CCl 3 , R 1 and R 2 = Me), showed activity only for 6a.Moreover, we found none biological activity for compounds where CX 3 = CF 3 or CCl 3 and R 1 ≠ R 2 (9c, 9f, 9g, 10h and 11e), but good results for CX 3 = Me (14).Thus, we found that compounds 6a and 14 were active against the various fungi tested and their results are expressed in µM for a better comparison (Table 2).The best inhibition results were obtained with compound 14 (R 1 = Me, R  2).The other tested substances did not show antimicrobial activity for the micro-organisms and concentrations employed.

Antioxidant activity
In this work, two different chemical methods were used for the in vitro evaluation of antioxidant activity for compounds: the free radical capture method 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the method used for measuring the total antioxidant capacity based on the  reduction of Mo VI to Mo V by antioxidants, and subsequent formation of phosphate/Mo V complex.Both methods are photometric, using wavelengths of 518 nm (DPPH) and 695 nm (phosphomolybdenum).
Free radical scavenging activity by DPPH One of the most widely employed methods is DPPH because it is simple, efficient and inexpensive.It consists of assessing the performance of the compound as a radical (oxidant) or as a hydrogen donor (antioxidant).As the method works equally well with methanol or ethanol, the latter one was chosen because of its lower toxicity.The pyrazole pyridine targets 1, 2, 6b, 8c, 8d, 9c, 10h, 11e, 12c, 13 and 14 were compared with ascorbic acid as the reference compound (Table 3).These selected compounds allowed to verify the influence of the CX 3 , R 1 and R 2 substituents on the antioxidant property.The DPPH antioxidant assay measures the hydrogen-donating capacity of the molecules in the sample.When the stable free-radical DPPH is reduced by the sample, its color changes from violet/purple to pale yellow.This absorbance decline is measured and the scavenging or inhibitory capacity can be determined.
The results indicate the potential of compounds as antioxidants or pro-oxidants.In the calibration curve (ascorbic acid) the inhibition values obtained were: 10.60, 19.40, 51.20 and 93.50% for concentrations of 1, 5, 10 and 100 µL mL -1 , respectively, and with a value of IC 50 = 11.88 ± 0.82 µg mL -1 .Compounds 1, 2, 8c, 9c, 10h, and 14 were active as DPPH free radicals, trapping at concentrations > 100 µg mL -1 ; however, when compared to ascorbic acid, compound 8c presented relevant data at various concentrations.Compounds 8d, 11e and 12c were not able to inhibit DPPH at the concentrations tested, but were shown to be molecules with pro-oxidant activity.

Antioxidant capacity by phosphomolybdenum method
Our results demonstrated that all compounds presented a total antioxidant capacity (TAC, %) that is concentration dependent (Table 4).A standard butylhydroxytoluene (BHT) curve was used with positive control, and for estimating the TAC we used the absorbance of BHT (50 µg mL -1 ) as positive control (100%).This way, it was possible to affirm that compounds 1 and 2 (50 µg mL -1 ) presented a similar effect to BHT (50 µg mL -1 ).Additionally, compounds 8c and 9c showed similar antioxidant capacity to BHT (50 µg mL -1 ) at concentrations of 100 and 500 µg mL -1 , respectively.On the contrary, compounds 8d and 9d presented antioxidant activity lower than that found for the BHT (50 µg mL -1 ).The phosphomolybdenum method was used to characterize the antioxidant properties in natural and chemical compounds. 43,44This method comprehends if the compounds have the ability to promote the reduction of the Mo VI to Mo V due to an electron donation.The initial test solution has a yellow color, becoming green as the molybdenum phosphate solution decreases.This method has the advantage of assessing the antioxidant activity of both lipophilic components as hydrophilic. 8ur results demonstrate that the chemical structures of compounds 1 and 2 changed only in the R 2 position (CH 3 and C 6 H 5 , respectively) and thus probably did not interfere in the ability to donate an electron to the Mo VI , because these compounds showed a similar antioxidant effect, which was also higher when compared with the other compounds.Additionally, it was possible to observe that the compounds with X = Cl in the structure presented higher antioxidant activity than with X = F.Moreover, the presence of the CH 3 in the R 1 for compound 8c promoted higher antioxidant activity than found for compound 9c with C 6 H 5 in the R 1 position.Therefore, the antioxidant capacity of the compounds was found to decrease in the following order:

Conclusions
In summary, we have described a useful approach for building bis-pyrazoles with the insertion of different nonhalogenated, trifluoromethyl, and trichloromethyl groups, in both pyrazole derivative rings, with good yields when the structures are determined by NMR ( 1 H, 13 C, 19 F) and X-ray diffraction.When evaluated, compound 8c presented the highest antioxidant capacity, similar to BHT with 158.44% and 167.10% at a concentration of 100 and 500 µg mL -1 , respectively, and DPPH free radical inhibition reached 85.74%.However, when compared to ascorbic acid, 8c presented relevant data at various concentrations.Given all these considerations, it may contribute as a new antioxidant for preventing or reducing the development of pathologies associated with oxidative stress.
The preliminary in vitro antimicrobial screening results of novel 2-(5-trifluoromethyl-1H-pyrazol-1-yl)-5-(5-trihalomethyl-1H-pyrazol-1-yl-1-carbonyl)pyridines reported here revealed that the compounds are not good candidates for antibiotics, due to there being no inhibition of the bacteria tested.However, it is known that the azole heterocycle has good antifungal activity, and we envision compounds 6a and 14 as being potential agents against dermatophytes, because they showed promising results.
Furthermore, additional studies regarding the action mechanism are necessary for a complete understanding of the antifungal activity of these compounds and the development of new agents based on structures containing pyrazole derivatives, in the hope of generating new bioactive molecules that could be useful as potent agents.We also performed X-ray diffraction measurements for a monocrystal of representative compounds.

General
Unless otherwise indicated, all common reagents and solvents were used as obtained from commercial suppliers, without further purification.The melting points were determined using coverslips on a Microquímica MQAPF-302 apparatus and are uncorrected. 1H and 13 C spectra were acquired on a Bruker DPX 400 ( 1 H at 400.13 MHz and 13 C at 100.61 MHz) and a Bruker Avance III DPX 600 spectrometer ( 1 H at 600 MHz and 13 C at 150 MHz), with 5 mm sample tubes, 298 K, digital resolution of ± 0.01 ppm, in CDCl 3 or DMSO-d 6 , and using TMS as an internal reference.
The 19 F spectra were acquired on the same Bruker Avance III DPX 600, at 564.68 MHz, with 5 mm sample tubes, 0.3 mol L -1 solutions at 298 K, digital resolution  45,46 The structures of 6a, 7a and 14 were solved with direct methods using the SHELXS-97 program, and refined on F 2 by full-matrix least-squares using the SHELXL-97 package. 39he absorption correction was performed by Gaussian methods. 47Anisotropic displacement parameters for nonhydrogen atoms were applied.The hydrogen atoms were placed at calculated positions with 0.96 Å (methyl CH 3 ) and 0.93 Å (aromatic CH) using a riding model.The hydrogen isotropic thermal parameters were kept equal to U iso (H) = χU eq (carrier C atom), with χ 1.5 for methyl groups and χ 1.2 for all others.The valence angles C−C−H and H-C−H of the methyl groups were set to 109.5º, and the H atoms were allowed to rotate around the C−C bond.The molecular graph was prepared using ORTEP-3 for Windows. 47neral procedure for the synthesis of 6-[3-alkyl(aryl/ heteroaryl)-5-trifluoromethyl-1H-pyrazol-1-yl]nicotinohydrazides (1-3)   Methyl nicotinate hydrochlorides 35,48 (1 mmol) were added to a stirred solution of ethanol (5 mL) and hydrazine hydrate 24% (0.5 mL).After stirring the reaction mixture at reflux temperature for 20 h, the solids 1-3 (Figure 4) were isolated by filtration, washed with cold ethanol/H 2 O, and dried under reduced pressure in a desiccator containing P 2 O 5 .
the pyrazoline ring (cycle C), the methylene protons of H-4 showed a characteristic doublet on average at d 3.83 ppm, and the other doublet at d 3.53 ppm, respectively, with a germinal coupling constant on average at 19.0 Hz.The hydroxyl proton was shown at an average of d 8.30 ppm.All compounds present the typical13 C NMR chemical shifts of pyrazole rings (cycle B) at an average of 149.3 ppm (C-3), and the C-4 exhibit signals at 111.0 ppm with a characteristic quartet of 3 J 3 Hz.The C-5 exhibit signals at around 132.8 ppm with a characteristic quartet of 2 J 40 Hz, because they are attached to the CF 3 group.The CF 3 shows a typical quartet at an average of 120.5 ppm with 1 J 268 Hz.As for the pyrazoline rings (cycle C), values were at an average of 154.5 ppm (C-3) and 47.1 ppm (C-4).The compounds 7 and 9 (X = F) at C-5 exhibit signals around 92.0 ppm and a characteristic quartet of 2 J 34 Hz, because they are attached to the CF 3 group.The CF 3 shows a typical quartet at an average of 123.1 ppm, with 1 J 285 Hz.For compounds 6, 8, and 10, in which X = Cl, the C-5 exhibit signals around 102.5 ppm.The carbonyl carbon displayed a signal in the range of 166.6 ppm.The 19 F NMR showed a typical singlet at an average of d −56.6 ppm for the CF 3 pyrazole (cycle B) and d −76.2 ppm for the CF 3 pyrazoline (cycle C).After dehydration, compound 11e presented the 19 F singlet signal in d −58.54 ppm of pyrazole (cycle

Table 1 .
Scope and yields for the isolated compounds

1-12
2 = Ph; CX 3 = Me) for the yeasts C. dubliniensis, C. parapsilosis, and C. neoformans in 303 µM; and for the dermatophytes M. canis in 151 µM, and T. mentagrophytes and T. rubrum in 76 µM (31.25 µg mL -1 ), resulting in fungiostatic activity for 14.The following concentrations were lethal: 607 µM for C. parapsilosis and C. neoformans; 303 µM for C. dubliniensis and M. canis; 151 µM for T. rubrum; and 76 µM for T. mentagrophytes, but they also granted fungicidal activity for compound 14.However, compound 6a (R 1 and R 2 = CH 3 ; CX 3 = CCl 3 ) resulted in fungicidal activity for four micro-organisms, for which compound 14 obtained no results.Compound 6a inhibited the growth at a concentration of 265 µM for C. krusei, C. glabrata, and S. schenckii; and at a concentration of 531 µM for R. oryzae.It also presented fungicidal activity at a concentration of 531 µM for C. krusei, C. glabrata, and S. schenckii; and at 1062 µM for R. oryzae (Table

Table 2 .
Antifungal activity of compounds 6a and 14 a ATCC (American Type Culture Collection); b minimal inhibitory concentration (MIC); c minimal lethal concentration (MLC); d Fluconazole for yeasts and Terbinafine for dermatophytes (M.canis, T. mentagrophytes, T. rubrum) and Amphotericin B for filamentous fungi (S. schenckii and R. oryzae).

Table 3 .
Antioxidant activity of some synthesized compounds, by DPPH method