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

Abstract

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.

Keywords:
pyrazole; pyridine; hydrazide; antimicrobial activity; DPPH

Introduction

Free radicals are related to a large number of diseases affecting humans; for example, cardiovascular problems, cancer, immune system decline, and brain dysfunctions like the neurodegenerative Alzheimer and Parkinson disease.^1-4 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.⁵

Consequently, 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.⁶

Methods 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.^7-10 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 strains.^11,12 The 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.¹³ Although 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.¹⁴

The 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.¹⁵ Therefore, 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 antipyretic²⁰ and selective enzyme inhibitory activities.²¹

Specifically, 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

A 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-(β-diketones).

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)-1,1,1-trifluoroalk-3-en-2-ones with carbohydrazides, at yields of 73-89%.³¹ In 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-1-yl)-5-(1H-pyrazol-1-yl-1-carbonyl)pyridines with yields of 62-97%.³² It 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.³³ Following 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.³⁴ In the same year, a synthetic route for the selective "deprotection" reaction (amide hydrolysis reaction) for 2-(1H-pyrazol-1-yl)-5-(1H-pyrazol-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.³⁵ In 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,^32-35 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-4-alkyl(aryl/heteroaryl-1,1,1-trifluoro(chloro)alk-3-en-2-ones 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.

Results and discussion

Synthesis and structure

Firstly, the 4-methoxy-4-(alkyl/aryl/heteroaryl)-1,1,1-trihaloalk-3-en-2-ones (4a, 5a),³⁶ (4b, 5b, 5c, 5d),³⁷ and (5e)³⁸ were obtained from the acylation reaction of 2-methoxypropenyl ether or acetals derived from acetophenones or 2-acetylfuran with trifluoroacetic anhydride or trichloroacetyl chloride, in accordance with the methodology developed in our laboratory. Subsequently, we reacted ketones 4 and 5 with substituted pyrazolyl-nicotinohydrazides (1-3)³⁵ in ethanol (green solvent), obtaining novel bis-pyrazolyl-pyridines (6-10) in a one-step reaction (Scheme 1, Table 1), which were then dehydrated and furnished, for example, the corresponding bis-pyrazolyl-pyridine systems 11e and 12c (Scheme 2, Table 1). The synthesis of all compounds was monitored by thin-layer chromatography (TLC).

Scheme 1
Synthetic route for the synthesis of bis-pyrazolyl-pyridines 6-10.

Table 1
Scope and yields for the isolated compounds 1-12

Scheme 2
Dehydration reactions for bis-pyrazolyl-pyridines 8c, 9e.

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 ¹H 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 (Et₃N) 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 ¹H NMR spectra. In the following step, compound 13 was easily dehydrated with SOCl₂, 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.³⁹ In 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.^40,41 Thus, 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 ¹H NMR analysis-with a much more satisfactory yield of 71% (Scheme 3).

Scheme 3
Synthesis of bis-pyrazolyl-pyridine 13 and its dehydration reaction to 14.

The structures of the new pyrazoline-pyridine derivatives 6-10 were deduced from ¹H, ¹³C, and ¹⁹F NMR spectra, and by comparison with the NMR data of other pyrazole derivatives previously synthesized in our laboratory.^32-35,41 The ¹H NMR analysis in dimethylsulfoxide, DMSO-d₆, revealed a chemical shift for the H-4 as a sharp singlet in the range of 7.04 ppm (R² = CH₃), 7.84 ppm (R² = Ph), and 7.59 ppm (R² = 2-furyl) at the pyrazole ring (cycle B). For the pyrazoline ring (cycle C), the methylene protons of H-4 showed a characteristic doublet on average at δ 3.83 ppm, and the other doublet at δ 3.53 ppm, respectively, with a germinal coupling constant on average at 19.0 Hz. The hydroxyl proton was shown at an average of δ 8.30 ppm. All compounds present the typical ¹³C 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 ³J 3 Hz. The C-5 exhibit signals at around 132.8 ppm with a characteristic quartet of ²J 40 Hz, because they are attached to the CF₃ group. The CF₃ shows a typical quartet at an average of 120.5 ppm with ¹J 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 ²J 34 Hz, because they are attached to the CF₃ group. The CF₃ shows a typical quartet at an average of 123.1 ppm, with ¹J 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 ¹⁹F NMR showed a typical singlet at an average of δ –56.6 ppm for the CF₃ pyrazole (cycle B) and δ –76.2 ppm for the CF₃ pyrazoline (cycle C). After dehydration, compound 11e presented the ¹⁹F singlet signal in δ –58.54 ppm of pyrazole (cycle C). The structures of 13-14, for which X = H, were then deduced from NMR spectra (¹H, ¹³C, and ¹⁹F) 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 δ 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 (CH₃) at 1.98 and 1.92 ppm. Vinylic hydrogens from the pyrazoline ring (cycle C) of compound 14 were seen at δ 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).

Figure 1
A perspective view of molecule 2-(3-methyl-5-trifluoromethyl- 1H-pyrazol-1-yl)-5-(3-methyl-5-hydroxy-5-trichloromethyl-4,5-dihydro- 1H-pyrazol-1-yl-1-carbonyl)pyridine (6a) with atoms labeled (CCDC 1040652). Displacement ellipsoids are drawn at the 50% probability level.

Figure 2
A perspective view of molecule 2-(5-trifluoromethyl-3-methyl- 1H-pyrazol-1-yl)-5-(5-trifluoromethyl-4,5-dihydro-5-hydroxy-3-methyl- 1H-pyrazol-1-yl-1-carbonyl)pyridine (7a) with atoms labeled (CCDC 1040653). Displacement ellipsoids are drawn at the 50% probability level.

Figure 3
A perspective view of molecule 2-(5-trifluoromethyl-3-phenyl- 1H-pyrazol-1-yl)-5-(3,5-dimethyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (14) with atoms labeled (CCDC 1040654). Displacement ellipsoids are drawn at the 50% probability level.

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 CX₃, R¹ and R² 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 CX₃ group but also of the R¹ and R² substituents. For example: a comparison between compounds 8d (CX₃ = CCl₃) and 9d (CX₃ = CF₃), both containing R¹ and R² = Ph as substituents, showed none activity. However, a comparison between compounds 8d and 6a (CX₃ = CCl₃, R¹ and R² = Me), showed activity only for 6a. Moreover, we found none biological activity for compounds where CX₃ = CF₃ or CCl₃ and R¹ ≠ R² (9c, 9f, 9g, 10h and 11e), but good results for CX₃ = 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 (R¹ = Me, R² = Ph; CX₃ = 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 (R¹ and R² = CH₃; CX₃ = CCl₃) 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 2). The other tested substances did not show antimicrobial activity for the micro-organisms and concentrations employed.

Table 2
Antifungal activity of compounds 6a and 14

Characteristic	Microorganisma	Sample 6a		Sample 14		Standardd
		MICb / μM	MLCc / μM	MICb / μM	MLCc / μM	MICb / μM
Yeast	Candida krusei	265	531	> 1215	−	52
	Candida glabrata	265	531	> 1215	−	26
	Candida dubliniensis	> 1062	−	303	303	0.81
	Candida parapsilosis	> 1062	−	303	607	6.5
	Cryptococcus neoformans	1062	1062	303	607	13
Dermatophyte	Microsporum canis	1062	1062	151	303	0.20
	Trichophyton mentagrophytes	265	265	76	76	0.013
	Trichophyton rubrum	265	265	76	151	0.10
Filamentous	Sporothrix schenckii	265	531	> 1215	−	0.54
	Rhizopus oryzae	531	1062	−	−	1.08

• ^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).

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 CX₃, R¹ and R² 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.

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

Compound	Inhibition / %
	10 μg mL-1	50 μg mL-1	100 μg mL-1	500 μg mL-1	1000 μg mL-1
1	6.54 ± 2.7	–1.52 ± 1.9	2.77 ± 2.3	12.5 ± 0.6	25.8 ± 1.1
2	5.55 ± 3.1	–1.46 ± 1.0	1.15 ± 3.6	17.16 ± 0.6	29.57 ± 1.8
6b	–2.68 ± 8.4	0.01 ± 5.4	3.21 ± 6.9	–20.87 ± 0.4	–43.96 ± 8.6
8c	–0.61 ± 4.8	21.38 ± 1.9	43.34 ± 2.4	77.25 ± 6.5	85.74 ± 2.9
8d	–9.17 ± 8.8	–4.97 ± 13.9	–9.77 ± 5.4	–8.94 ± 10.1	–41.67 ± 7.8
9c	–7.80 ± 9.6	6.37 ± 5.2	–9.5 ± 4.5	4.4 ± 7.6	20.67 ± 2.4
10h	–9.40 ± 6.9	–3.93 ± 12.4	–2.2 ± 6.5	16 ± 2.4	21.67 ± 3.1
11e	–2.73 ±9.4	–4.60 ± 10.7	–4.93 ± 4.2	–12.2 ± 17.6	–19.8 ± 10.35
12c	–9.21 ± 5.3	–2.93 ± 3.0	–4.02 ± 6.5	–23.84 ± 1.1	–42.05 ± 6.5
13	–10.89 ± 6.0	–11.21 ± 2.9	–11.04 ± 5.5	–5.77 ± 1.9	3.65 ± 2.4
14	–8.73 ± 8.8	14.2 ± 7.9	–6.57 ± 3.6	11.1 ± 12.8	17.07 ± 2.9

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 IC₅₀ = 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.

Compounds 6b and 13 can act either as pro-oxidants or as antioxidants, depending on the concentration. It can be seen that, for precursor compound 2 (R²= Ph), antioxidant activity was 29.57% at a concentration of 1 mg mL^-1. When evaluated, for compound 8c (R¹= CH₃; R²= Ph; CX₃ = CCl₃) free radical inhibition reached 85.74%; compound 8d (R¹ and R² = Ph; CX₃ = CCl₃) obtained prooxidant activity (–41.67%); in substance 9c (R¹ = CH₃; R²=Ph; CX₃ =CF₃) there was a decrease in the antioxidant activity (20.67%); and for compound 12c, a product of the dehydration reaction of 8c (R¹= CH₃; R²= Ph; CX₃ = CCl₃), the assay value was –42.05% (prooxidant). Derivative compounds of pentane-2,4-dione 13-14 (R¹=CH₃; R²= Ph; CX₃ = CH₃) presented activity results of 3.65% and 17.07%, respectively, at a concentration of 1 mg mL^-1 (Table 3). According to Herbert,⁴² some ingredients may serve both as antioxidants and pro-oxidants, depending on conditions, including the concentration of these chemicals and if transition metals or oxygen are present.

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.^43,44 This 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.⁸

Table 4
Total antioxidant capacity of the compounds by phosphomolybdenum method, presented as mean ± standard derivation

Compound	Total antioxidant capacity / %
	10 μg mL-1	50 μg mL-1	100 μg mL-1	500 μg mL-1	1000 μg mL-1
1	25.28 ± 0.3	97.54 ±1.9	137.62 ± 4.3	> 275	> 275
2	22.53 ± 0.9	87.92 ± 2.2	138.27 ± 2.6	> 275	> 275
8c	8.58 ± 1.3	42.63 ± 1.6	73.95 ± 2.2	158.44 ± 8.8	167.10 ± 10.1
8d	10.55 ± 0.5	25.54 ± 1.0	30.01 ± 0.4	37.24 ± 1.1	37.61 ± 1.5
9c	7.55 ± 0.6	13.88 ± 2.8	23.74 ± 2.1	72.87 ± 6.9	87.35 ± 6.7
9d	15.66 ± 1.2	24.02 ± 1.9	29.85 ± 1.7	38.08 ± 2.3	48.52 ± 3.1

Our results demonstrate that the chemical structures of compounds 1 and 2 changed only in the R² position (CH₃ and C₆H₅, 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 CH₃ in the R¹ for compound 8c promoted higher antioxidant activity than found for compound 9c with C6H5 in the R¹ position. Therefore, the antioxidant capacity of the compounds was found to decrease in the following order: 1 ca. 2 > 8c > 9c > 9d > 8d.

Conclusions

In summary, we have described a useful approach for building bis-pyrazoles with the insertion of different non-halogenated, trifluoromethyl, and trichloromethyl groups, in both pyrazole derivative rings, with good yields when the structures are determined by NMR (¹H, ¹³C, ¹⁹F) 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.

Experimental

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 ¹³C spectra were acquired on a Bruker DPX 400 (¹H at 400.13 MHz and ¹³C at 100.61 MHz) and a Bruker Avance III DPX 600 spectrometer (¹H at 600 MHz and ¹³C at 150 MHz), with 5 mm sample tubes, 298 K, digital resolution of ± 0.01 ppm, in CDCl₃ or DMSO-d₆, and using TMS as an internal reference.

The ¹⁹F 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 of ± 0.01 ppm, in CDCl₃ or DMSO-d₆, and using CFCl3 (δ 0.0 ppm) as the external reference. Mass spectra were registered in a HP 5973 MSD connected to a HP 6890 GC and interfaced by a Pentium PC. The CHN elemental analyses were performed on a Perkin-Elmer 2400 CHN elemental analyzer (University of São Paulo, SP, Brazil), and the high resolution mass spectrometry was performed using an Agilent-QTOF 6530 spectrometer (UFSM, RS, Brazil) and a Micro TOF Bruker Daltonic spectrometer (University of São Paulo, SP, Brazil). Diffraction measurements were done by graphite-monochromatized Mo Kα radiation, with λ 0.71073 Å, on a Bruker SMART CCD diffractometer.^45,46 The structures of 6a,7a and 14 were solved with direct methods using the SHELXS-97 program, and refined on F² by full-matrix least-squares using the SHELXL-97 package.³⁹

The absorption correction was performed by Gaussian methods.⁴⁷ Anisotropic displacement parameters for non-hydrogen atoms were applied. The hydrogen atoms were placed at calculated positions with 0.96 Å (methyl CH₃) 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.⁴⁷

General 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/H₂O, and dried under reduced pressure in a desiccator containing P₂O₅.

Figure 4
General chemical structure of compounds 1-3.

6-(5-Trifluoromethyl-3-methyl-1H-pyrazol-1-yl)nicotinohydrazide (1)³⁵

White solid; yield 73%; mp 156-157 ºC (70%, mp 154-156 ºC);³⁵¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 9.98 (s, 1H, NH), 8.87 (s, 1H, H-2), 8.39 (dd, 1H, J 2.0, 8.0, H-4), 7.92 (d, 1H, J 9.0, H-5), 4.58 (s, 2H, NH2), (Pyr-B): δ 7.04 (s, 1H, H-4), 2.34 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 163.3 (CO), 151.7 (C-6), 115.1 (C-5), 138.1 (C-4), 127.9 (C-3), 146.5 (C-2), (Pyr-B): δ 131.5 (q, J 40.0, C-5), 112.3 (q, J 3.0, C-4), 119.7 (q, J 268.0, CF₃), 150.2 (C-3), 12.9 (CH₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆) Pyrazole B: δ –56.61 (CF₃); HRMS (FTMS + pESI) m/z, calcd. for C₁₁H₁₀F₃N₅O [M]⁺: 286.0915; found: 286.0987.

6-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)nicotinohydrazide (2)³⁵

White solid; yield 62%; mp 217-219 ºC (62%, mp 218-219 ºC);³⁵¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 10.06 (s, 1H, NH), 8.93 (s, 1H, H-2), 8.47 (dd, 1H, J 2.0, 8.0, H-4), 8.11 (d, 1H, J 9.0, H-5), 4.60 (s, 2H, NH2), (Pyr-B): δ 8.02-8.04 (m, 2H, Ph), 7.82 (s, 1H, H-4), 7.44-7.54 (m, 3H, Ph); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 163.1 (CO), 151.8 (C-6), 115.5 (C-5), 138.2 (C-4), 128.3 (C-3), 146.5 (C-2), (Pyr-B): δ 132.5 (q, J 40.0, C-5), 109.8 (q, J 3.0, C-4), 151.7 (C-3), 130.5 (Ph), 129.1 (Ph), 128.8 (Ph), 125.7 (Ph), 119.6 (q, J 267.0, CF₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): Pyr-B: δ –56.58 (CF₃); HRMS (FTMS + pESI) m/z, calcd. for C₁₆H₁₂F₃N₅O [M]⁺: 348.1072; found: 348.1064.

6-(5-Trifluoromethyl-3-(fur-2-yl)-1H-pyrazol-1-yl)nicotinohydrazide (3)

Pale brown solid; yield 57%; mp 204-206 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.02 (d, 1H, J 8.0, H-5), 8.46 (dd, 1H, J 2.0, 8.0, H-4), 8.92 (d, 1H, J 2.0, H-2), 10.04 (s, 1H, NH), 4.61 (s, 2H, NH2), (Pyr-B): δ 7.84 (s, 1H, 2-furyl), 7.56 (s, 1H, H-4), 7.08 (d, 1H, J 3.0, 2-furyl), 6.67 (dd, 1H, J 3.0, 2-furyl);¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 163.1 (CO), 151.5 (C-6), 115.4 (C-5), 138.3 (C-4), 128.3 (C-3), 145.6 (C-2), (Pyr-B): δ 132.2 (q, J 41.0, C-5B), 109.4 (q, J 3.0, C-4B), 119.4 (q, J 269, CF₃), 146.5 (C-3), 144.2 (2-furyl), 143.8 (2-furyl), 111.7 (2-furyl), 109.2 (2-furyl); ¹⁹F NMR (564.68 MHz, DMSO-d₆): Pyr-B: δ –56.73 (CF₃); HRMS (FTMS + pESI) m/z, calcd. for C₁₆H₁₃Cl₃F₃N₅O₂ [M]⁺: 338.0866; found: 338.0832.

General procedure for the synthesis of 2-[3-alkyl(aryl/heteroaryl)-5-trifluoromethyl-1H-pyrazol-1-yl]-5-[3-alkyl(aryl/heteroaryl)-5-trihalomethyl-5-hydroxy-4,5-dihydro-1H-pyrazol-1-yl-1-carbonyl]-pyridines (6-10)

To a solution of absolute ethanol (10 mL) and 4-alkyl(aryl/heteroaryl)-4-methoxy-1,1,1-trihaloalk-3-en-2-ones (4,5)^36-38 (1 mmol) at room temperature and under magnetic stirring, pyrazolyl nicotinohydrazide (1-3)³⁵ (1 mmol) was added. For precursor 1, the reaction needed a small quantity of acetic acid (0.2 mL). The reaction stayed for 16 h at reflux temperature (78 ºC) under magnetic agitation. After the reaction time, solids 6-10 (Figure 5) were isolated either by cold filtration (if precipitation occurred when cooled) or the solvent was partly reduced in a rotary evaporator and only then isolated by cold filtration in a Büchner funnel, washed in ethanol and water (4:1), cooled and recrystallized from ethanol/acetone (2:1), washed in ethanol, and then dried in a low pressurized desiccator containing P₂O₅.

Figure 5
General chemical structure of compounds 6-10.

2-(5-Trifluoromethyl-3-methyl-1H-pyrazol-1-yl)-5-(5-trichloromethyl-4,5-dihydro-5-hydroxy-3-methyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (6a)

Yellow crystalline solid; yield 71%; mp 109-111 ºC; ¹H NMR (400.13 MHz, CDCl₃): (Py-A): δ 8.98 (d, 1H, J 2.0, H-6), 8.29 (dd, 1H, J 2.0, 8.0, H-4), 7.91 (d, 1H, J 8.0, H-3), (Pyr-B): δ 6.70 (s, 1H, H-4), 2.37 (s, 3H, CH₃), (Pyr-C): δ 3.58 (d, 1H, J 19.0, H-4), 3.32 (d, 1H, J 19.0, H-4), 2.04 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, CDCl₃): (Py-A): δ 152.7 (C-2), 114.1 (C-3), 140.2 (C-4), 128.0 (C-5), 150.0 (C-6), (Pyr-B): δ 150.3 (C-3), 112.2 (q, J 3.0, C-4), 133.2 (q, J 40.0, C-5), 119.8 (q, J 268.0, CF₃), 13.4 (CH₃), (Pyr-C): δ 169.8 (CO), 157.0 (C-3), 50.1 (C-4), 102.5 (C-5), 103.6 (CCl₃), 15.6 (CH₃); ¹⁹F NMR (564.68 MHz, CDCl₃): (Pyr-B): δ –56.60 (CF₃); anal. calcd. for C₁₆H₁₃Cl₃F₃N₅O₂ (470.66): C, 40.83, H, 2.78; N, 14.88; found: C, 40.93, H, 2.82, N, 15.09%.

2-(5-Trifluoromethyl-3-methyl-1H-pyrazol-1-yl)-5-(5-trichloromethyl-4,5-dihydro-5-hydroxy-3-phenyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (6b)

Pale orange solid; yield 80%; mp 127-128 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.82 (s, 1H, H-6), 8.40 (d, 1H, J 10.0, H-4), 8.00 (d, 1H, J 8.0, H-3), (Pyr-B): δ 7.07 (s, 1H, H-4), 2.37 (s, 3H, CH₃), (Pyr-C): δ 8.30 (s, OH), 7.71 (d, 2H, J 7.0, Ph), 7.5-7.44 (m, 3H, Ph), 4.11 (d, 1H, J 19.0, H-4), 3.88 (d, 1H, J 19.0, H-4); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 148.2 (C-6), 130.7 (C-5), 140.0 (C-4), 114.5 (C-3), 151.4 (C-2), (Pyr-B): δ 131.6 (q, J 40.0, C-5), 112.4 (q, J 3.0, C-4), 150.3 (C-3), 119.6 (q, J 268.0, CF₃), 12.8 (CH₃), (Pyr-C): δ 166.1 (CO), 102.4 (C-5), 46.8 (C-4), 154.0 (C-3), 129.9 (Ph), 129.5 (Ph), 128.6 (Ph), 126.4 (Ph), 103.2 (CCl₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –56.56 (CF₃); anal. calcd. for C21H15Cl3F3N5O (532.73): C 47.35, H, 2.84, N, 13.15; found: C, 47.71, H, 3.11, N, 13.42%.

2-(5-Trifluoromethyl-3-methyl-1H-pyrazol-1-yl)-5-(5-trifluoromethyl-4,5-dihydro-5-hydroxy-3-methyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (7a)

Yellow crystalline solid; yield 91%; mp 135-137 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.75 (d, 1H, J 2.0, H-6), 8.29 (dd, 1H, J 2.0, 8.0, H-4), 7.92 (d, 1H, J 8.0, H-3), (Pyr-B): δ 7.05 (s, 1H, H-4), 2.36 (s, 3H, CH₃), (Pyr-C): δ 7.97 (s, 1H, OH), 3.53 (d, 1H, H-4, J 19.0), 3.16 (d, 1H, J 19.0, H-4), 2.01 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 148.2 (C-6), 129.9 (C-5), 140.1 (C-4), 114.5 (C-3), 151.3 (C-2), (Pyr-B): δ 131.5 (q, C-5, J 40.0), 112.5 (q, C-4, J 3.0); 150.3 (C-3), 119.7 (q, CF₃, J 267.0), 12.9 (CH₃), (Pyr-C): δ 163.6 (CO), 91.3 (q, C-5, J 34.0), 123.2 (q, CF₃, J 286.0), 47.6 (C-4), 155.4 (C-3), 15.2 (CH₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –56.63 (CF₃), (Pyr-C): δ –76.28 (CF₃); anal. calcd. for C₁₆H₁₃F₆N₅O₂ (421.29): C, 45.61, H, 3.11, N, 16.62; found: C, 45.29, H, 3.11, N, 16.04%.

2-(5-Trifluoromethyl-3-methyl-1H-pyrazol-1-yl)-5-(5-trifluoromethyl-4,5-dihydro-5-hydroxy-3-phenyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (7b)

Yellow solid; yield 81%; mp 114-116 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.83 (d, 1H, J 2.0, H-6), 8.42 (dd, 1H, J 2.0, 8.0, H-4), 8.0 (d, 1H, J 8.0, H-3), (Pyr-B): δ 7.01 (s, 1H, H-4), 2.36 (s, 3H, CH₃), (Pyr-C): δ 8.33 (s, OH), 7.73 (d, 2H, J 8.0, Ph), 7.49-7.45 (m, 3H, Ph), 4.01 (d, 1H, J 19.0, H-4), 3.68 (d, 1H, J 19.0, H-4); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 148.4 (C-6), 130.9 (C-5), 140.4 (C-4), 114.6 (C-3), 151.4 (C-2), (Pyr-B): δ 131.6 (q, J 40.0, C-5), 112.6 (q, J 2, C-4), 119.7 (q, J 268.0, CF₃), 150.5 (C-3), 13.0 (CH₃), (Pyr-C): δ 163.8 (CO), 92.1 (q, J 34, C-5), 44.2 (C-4), 153.0 (C-3), 129.8 (Ph), 129.7 (Ph), 128.8 (Ph), 126.6 (Ph), 123.2 (q, J 285.0, CF₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –56.56 (CF₃), (Pyr-C): δ –76.03 (CF₃); anal. calcd. for C₂₁H₁₅F₆N₅O₂ (483.36): C, 52.18, H, 3.13, N, 14.49; found: C, 51.64, H, 3.12, N, 14.25%.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(5-trichloromethyl-4,5-dihydro-5-hydroxy-3-methyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (8c)

Yellow solid; yield 83%; mp 140-142 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.79 (d, 1H, J 2.0, H-6), 8.11 (d, 1H, H-3, J 8 Hz), 8.34 (dd, J 2.0, 8.0, H-4), (Pyr-B): δ 8.02 (d, 2H, Ph), 7.80 (s, 1H, H-4), 7.5-7.46 (m, 3H, Ph), (Pyr-C): δ 7.52 (s, OH), 3.66 (d, 1H, J 19.0, H-4), 3.43 (d, 1H, J 19.0, H-4), 2.01 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, CDCl₃): (Py-A): δ 150.1 (C-6); 131.3 (C-5); 140.3 (C-4); 114.4 (C-3); 152.9 (C-2), (Pyr-B): δ 134.2 (q, J 41.0, C-5), 109.6 (q, J 3.0, C-4), 119.9 (q, CF₃, J 268.0), 152.5 (C-3), 129.2 (Ph), 128.9 (Ph); 128.4 (Ph); (Pyr-C): δ 169.8 (CO); 102.6 (C-5), 50.3 (C-4), 157.0 (C-3); 126.0 (Ph),103.7 (CCl₃) 15.6 (CH₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –57.89 (CF₃); anal. calcd. for C₂₁H₁₅Cl₃F₃N₅O (532.73): C, 47.35, H, 2.84, N, 13.15; found: C, 47.33, H, 2.86, N, 13.01%.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(5trichloromethyl-4,5-dihydro-5-hydroxy-3-phenyl-1H-pyrazol1-yl-1-carbonyl)pyridine (8d)

Yellow solid; yield 79%; mp 157-159 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.86 (s, 1H, H-6), 8.46 (d, 1H, J 8.0, H-4), 8.18 (d, 1H, J 8.0, H-3), (Pyr-B): δ 7.85 (s, 1H, H-4), 8.05 (d, 2H, J 7.0, Ph), (Pyr-C): δ 8.33 (s, OH), 7.73 (d, 2H, J 7.0, Ph), 7.55-7.46 (m, 6H, Ph), 4.12 (d, 1H, J 19.0, H-4), 3.89 (d, 1H, J 19.0, H-4); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 148.3 (C-6), 130.7 (C-5), 140.2 (C-4), 114.8 (C-3); 151.9 (C-2), (Pyr-B): δ 132.5 (q, J 40.0, C-5), 109.9 (C-4), 151.4 (C-3), 119.5 (q, J 268.0, CF₃), 130.3 (Ph), 129.0 (Ph), 128.6 (Ph), 125.7 (Ph); (Pyr-C): δ 165.9 (CO), 102.4 (C-5), 46.8 (C-4), 154.0 (C-3), 130.4 (Ph), 129.5 (Ph), 128.6 (Ph), 126.4 (Ph), 103.2 (CCl₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –56.56 (CF₃); anal. calcd. for C₂₆H₁₇F₃Cl₃N₅O₂ (594.79): C, 52.50, H, 2.88, N, 11.77; found: C, 52.49, H, 2.98, N, 11.99%.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(5-trifluoromethyl-4,5-dihydro-5-hydroxy-3-methyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (9c)

Yellow solid; yield 87%; mp 153-155 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.80 (d, 1H, J 2.0, H-6), 8.36 (dd, 1H, J 2.0, 8.0, H-4), 8.12 (s, 1H, H-3), (Pyr-B): δ 8.04 (d, 2H, J 7.0, Ph), 7.84 (s, 1H, H-4), 7.54 7.46 (m, 3H, Ph), (Pyr-C): δ 8.10 (s, 1H, OH), 3.55 (d, 1H, J 19.0, H-4), 3.18 (d, 1H, J 19.0, H-4), 2.02 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 148.2 (C-6); 130.5 (C-5), 140.2 (C-4), 114.9 (C-3); 151.9 (C-2); (Pyr-B): δ 132.5 (q, J 40.0, C-5), 110.0 (q, J 2.0, C-4), 151.3 (C-3), 130.3 (Ph), 129.2 (Ph), 128.8 (Ph), 125.7 (Ph), 119.6 (q, J 268.0, CF₃), (Pyr-C): δ 163.5 (CO), 91.4 (q, J 34.0, C-5), 47.6 (C-4), 155.5 (C-3), 123.2 (q, J 285.0, CF₃), 15.2 (CH₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –56.60 (CF₃), (Pyr-C): δ –76.23 (CF₃); anal. calcd. for C₂₁H₁₅F₆N₅O₂ (483.36): C, 52.18, H, 3.13, N, 14.49; found: C, 52.09, H, 3.33, N, 14.41%.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(5-trifluoromethyl-4,5-dihydro-5-hydroxy-3-phenyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (9d)

Pale beige solid; yield 75%; mp 192-194 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.88 (dd, 1H, J 2.0, 8.0, H-6), 8.48 (dd, 1H, J 2.0, 8.0, H-4), 8.17 (d, 1H, J 8.0, H-3), (Pyr-B): δ 8.05 (d, 2H, Ph, J 8.0), 7.85 (s, 1H, H-4), (Pyr-C): δ 8.35 (s, OH), 7.74 (d, 2H, Ph, J 2.0), 7.55-7.47 (m, 6H, Ph), 4.02 (d, 1H, J 19.0, H-4), 3.68 (d, 1H, J 19.0, H-4); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 149.1 (C-6), 131.4 (C-5), 141.1 (C-4), 115.5 (C-3); 152.5 (C-2), (Pyr-B): δ 133.1 (q, J 40.0, C-5), 110.7 (q, J 2.0, C-4), 152.0 (C-3), 120.2 (q, CF₃, J 268.0), 130.3 (Ph), 129.8 (Ph), 129.3 (Ph), 127.2 (Ph), (Pyr-C): δ 164.3 (CO), 92.7 (q, C-5, J 34.0), 44.7 (C-4), 153.6 (C-3), 131.6 (Ph), 130.7 (Ph), 129.4 (Ph), 126.4 (Ph), 123.7 (q, CF₃, J 285.0); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –51.80 (CF₃), (Pyr-C): δ –71.24 (CF₃); anal. calcd. for C₂₆H₁₇F₆N₅O₂ (545.44): C, 57.25, H, 3.14, N, 12.84; found: C, 57.32, H, 3.11, N, 12.62%.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(5-trifluoromethyl-4,5-dihydro-5-hydroxy-3-(4-methylphenyl)-1H-pyrazol-1-yl-1-carbonyl)pyridine (9e)

Pale yellow solid; yield 67%; mp 206-207 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.87 (d, 1H, H-6, J 2.0), 8.48 (dd, 1H, H-4, J 2.0, 8.0), 8.16 (d, 1H, H-3, J 9.0), (Pyr-B): δ 8.06 (d, 2H, Ph, J 8.0), 7.86 (s, 1H, H-4), 7.55-7.47 (m, 3H, Ph), (Pyr-C): δ 8.35 (s, OH), 7.62 (d, 2H, Ph, J 8.0), 7.28 (d, 2H, Ph, J 8.0), 3.99 (d, 1H, H-4, J 19.0), 3.66 (d, 1H, H-4, J 19.0), 2.35 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 148.3 (C-6), 130.5 (C-5),140.2 (C-4), 114.8 (C-3), 151.8 (C-2), (Pyr-B): δ 132.6 (q, C-5, J 40.0), 109.8 (q, C-4, J 2.0), 151.3 (C-3), 119.5 (q, CF₃, J 268.0), 130.1 (Ph), 129.1 (Ph), 126.9 (Ph), 125.7 (Ph), (Pyr-C): δ 163.5 (CO), 91.9 (q, C-5, J 34.0), 44.0 (C-4), 152.8 (C-3), 140.7 (Ph), 128.9 (Ph), 128.6 (Ph), 126.4 (Ph), 123.0 (q, CF₃, J 285.0), 20.7 (CH₃); anal. calcd. for C₂₇H₁₉F₆N₅O₂ (559.46): C, 57.96, H, 3.42, N, 12.52; found: C, 57.76, H, 3.58, N, 12.86%.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(5-trifluoromethyl-4,5-dihydro-5-hydroxy-3-(4-methoxyphenyl)-1H-pyrazol-1-yl-1-carbonyl)pyridine (9f)

Orange solid; yield 80%; mp 200-201 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.87 (d, 1H, H-6, J 2.0), 8.48 (dd, 1H, H-4, J 2.0, 8.0), 8.16 (d, 1H, H-3, J 8.0), (Pyr-B): δ 8.05 (d, 2H, Ph, J 7.0), 7.85 (s, 1H, H-4), 7.55-7.47 (m, 3H, Ph), (Pyr-C): δ 8.28 (s, 1H, OH), 7.68 (d, 2H, Ph, J 8.0), 7.03 (d, 2H, Ph, J 8.0), 3.97 (d, 1H, H-4, J 19.0), 3.81 (s, 3H, OCH₃), 3.64 (d, 1H, H-4, J 19.0); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 148.6 (C-6), 130.6 (C-5), 140.6 (C-4), 114.9 (C-3), 152.0 (C-2), (Pyr-B): δ 132.6 (q, C-5, J 40.0), 110.2 (d, C-4, J 3.0), 151.4 (C-3), 119.7 (q, CF₃, J 268.0), 130.3 (Ph), 129.3 (Ph), 128.9 (Ph), 122.3 (Ph), (Pyr-C): δ 163.6 (CO), 92.0 (q, C-5, J 34.0), 44.3 (C-4), 152.8 (C-3), 161.4 (Ph), 128.5 (Ph), 125.9 (Ph), 114.3 (Ph), 123.2 (q, CF₃, J 285.0), 55.3(OCH₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –56.55 (CF₃), (Pyr-C): δ –75.99 (CF₃); anal. calcd. for C₂₇H₁₉F₆N₅O₃ (575.46): C, 56.35, H, 3.33, N, 12.17; found: C, 56.38, H, 3.29, N, 12.12%.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(3-(fur-2-yl)-5-trifluoromethyl-4,5-dihydro-5-hydroxy-1H-pyrazol-1-yl-1-carbonyl)pyridine (9g)

Pale brown solid; yield 69%; mp 146-148 ºC; ¹H NMR (600 MHz, DMSO-d₆): (Py-A): δ 8.86 (d, 1H, H-6, J 2.0), 8.42 (dd, 1H, H-4, J 2.0, 8.0), 8.16 (d, 1H, H-3, J 8.0), (Pyr-B): δ 8.05 (d, 2H, Ph, J 7.0), 7.87 (s, 1H, H-4), 7.53 (t, 2H, Ph, J 7.0), 7.48 (t, 1H, Ph, J 7.0), (Pyr-C): δ 8.49 (s, 1H, OH), 7.91 (s, 1H, 2-furyl), 7.13 (d, 1H, 2-furyl, J 3.0), 6.69 (dd, 1H, 2-furyl, J 2.0, 2.0), 3.44 (d, 1H, H-4, J 19.0), 3.33 (d, 1H, H-4, J 19.0); ¹³C {¹H} NMR (150 MHz, DMSO-d₆): (Py-A): δ 148.5 (C-6); 130.6 (C-5), 140.6 (C-4), 115.2 (C-3), 152.0 (C-2), (Pyr-B): δ 132.6 (q, C-5, J 40.0), 110.3 (s, C-4), 146.2 (C-3), 121.5 (q, CF₃, J 268.0), 130.2 (Ph); 129.4 (Ph), 129.0 (Ph), 125.9 (Ph), (Pyr-C): δ 163.8 (CO), 91.7 (q, C-5, J 34.0), 43.9 (C-4), 151.5 (C-3), 123.1 (q, CF₃, J 285.0), 144.8 (2-furyl), 144.3 (2-furyl), 115.6 (2-furyl); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –56.57 (CF₃), (Pyr-C): δ –76.05 (CF₃); anal. calcd. for C₂₄H₁₅F₆N₅O₃ (535,39): C, 53.84, H, 2.82, N, 13.08; found: C, 54.19, H, 3.23, N, 12.78%.

2-(3-(Fur-2-yl)-5-trifluoromethyl-1H-pyrazol-1-yl)-5-(5-trichloromethyl-4,5-dihydro-5-hydroxy-3-phenyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (10h)

Pale brown solid; yield 88%; mp 144-145 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.87 (d, H-6, J 2.0), 8.47 (dd, H-4, J 2.0, 8.0), 8.11 (d, H-3, J 8.0), (Pyr-B): δ 7.62 (s, 1H, H-4), 7.88 (s, 1H, 2-furyl), 7.14 (d, 1H, 2-furyl, J 3.0), 6.70 (dd, 1H, 2-furyl, J 3.0, 2.0), (Pyr-C): δ 7.74 (d, 2H, Ph, J 8.0), 7.51-7.46 (m, 3H, Ph), 4.13 (d, 1H, H-4, J 19.0), 3.91 (d, 1H, H-4, J 19.0); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 145.6 (C-6), 130.7 (C-5), 140.2 (C-4), 144.9 (C-3), 151.2 (C-2), (Pyr-B): δ 132.4 (q, J 40.0, C-5), 109.4 (d, J 3.0, C-4), 148.3 (C-3), 144.2 (2-furyl); 143.7 (2-furyl), 116.3 (2-furyl), 119.3 (q, J 268.0, CF₃), (Pyr-C): δ 165.9 (CO), 102.4 (C-5), 46.8 (C-4), 154.1 (C-3), 130.4 (Ph), 129.5 (Ph), 128.6 (Ph), 126.4 (Ph), 103.2 (CCl₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –56.71 (CF₃); anal. calcd. for C₂₄H₁₅F₃Cl₃N₅O₃ (583.76): C, 49.29, H, 2.59, N, 11.98; found: C, 49.46, H, 2.89, N, 11.56%.

General procedure for the synthesis of 2-(3-alkyl(aryl/heteroaryl)-5-trifluoromethyl-1H-pyrazol-1-yl)-5-(3-alkyl(aryl/heteroaryl)-5-trihalomethyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (11e, 12c, 14)⁴⁹

A solution of 8c, 9e, 13 (2.8 mmol) and pyridine (3 mL, 37.1 mmol) in 50 mL of benzene was cooled to 0 ºC, and thionyl chloride (1.22 mL, 16.8 mmol) diluted in benzene (25 mL) was added dropwise over a period of 10 min. The solution was stirred for an additional 30 min, during which time the temperature was allowed to rise to 20 ºC. The mixture was then heated under reflux (bath temperature 80 ºC) for 1 h and filtered to remove the pyridine hydrochloride at room temperature. The solution was washed twice with water and dried over sodium sulfate. Evaporation of the solvent left a solid (Figure 6) which was recrystallized from ethanol.

Figure 6
General chemical structure of compounds 11e, 12c and 14.

Compound 12c was purified by column chromatography in a mixture of solvents (hexane and ethyl acetate at 6:4 ratio).

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(5-trifluoromethyl-3-(4-methylphenyl)-1H-pyrazol-1-yl-1-carbonyl)pyridine (11e)

Beige solid; yield 70%; mp 153-155ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 9.13 (s, 1H, H-6), 8.26 (d, 1H, H-4, J 8.0), 8.73 (d, 1H, H-3, J 9.0), (Pyr-B): δ 7.87 (s, 1H, H-4), 8.06 (d, 2H, Ph, J 7.0), 7.55-7.49 (m, 3H, Ph), (Pyr-C): δ 8.00 (s, 1H, H-4), 7.84 (s, 2H, Ph), 7.31 (d, 2H, Ph, J 7.0), 2.35 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, CDCl₃): (Py-A): δ 151.6 (C-6), 131.0 (C-5), 140.3 (C-4), 114.3 (C-3), 153.6 (C-2), (Pyr-B): δ 134.3 (q, C-5, J 41.0), 110.1 (q, C-4, J 3.0), 152.7 (C-3), 119.8 (q, CF₃, J 268.0), 129.7 (Ph), 129.3 (Ph), 127.2 (Ph), 126.0 (Ph), (Pyr-C): δ 163.0 (CO), 136.3 (q, C-5, J 42.0), 111.4 (q, C-4, J 3.0), 153.9 (C-3), 141.9 (Ph), 129.3 (Ph), 128.2 (Ph), 126.2 (Ph), 119.3 (q, CF₃, J 285.0), 21.3 (CH₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –56.58 (CF₃), (Pyr-C): δ –58.54 (CF₃); anal. calcd. for C₂₇H₁₇F₆N₅O (541.45): C, 59.89, H, 3.16, N, 12.93; found: C, 60.29, H, 3.91, N, 12.34%.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(5-trichloromethyl-3-methyl-1H-pyrazol-1-yl-1-carbonyl) pyridine (12c)

Yellow solid; yield 54%; mp 160-162 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 9.00 (s, 1H, H-6), 8.55 (d, 1H, H-4, J 8.0), 8.18 (d, 1H, H-3, J 8.0), (Pyr-B): δ 7.82 (s, 1H, H-4), 8.03 (d, 2H, Ph, J 7.0), 7.52-7.47 (m, 3H, Ph), (Pyr-C): δ 7.22 (s, 1H, H-4), 2.29 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 149.1 (C-6), 126.1 (C-5), 140.4 (C-4), 115.6 (C-3), 152.9 (C-2), (Pyr-B): δ 132.8 (q, C-5, J 41.0 ), 110.3 (q, C-4, J 3.0), 119.8 (q, CF₃, J 268.0), 152.2 (C-3), (Pyr-C): δ 165.4 (CO), 142.2 (C-5), 141.3 (C-4), 162.5 (C-3), 130.5 (Ph), 129.3 (Ph), 128.9 (Ph), 125.9 (Ph), 106.8 (CCl₃) 10.9 (CH₃); ¹⁹F NMR (564.68 MHz, CDCl₃): (Pyr-B): δ –57.94 (CF₃); HRMS (FTMS + pESI): m/z, calcd. for C₂₁H₁₃F₃Cl₃N₅O [M]⁺: 514.0216; found: 514.0302.

General procedure for the synthesis of 2-(5-trifluoromethyl-3-methyl-1H-pyrazol-1-yl)-5-(3,5-dimethyl-5-hydroxy-4,5-dihydro-1H-pyrazol-1-yl-1-carbonyl)pyridine (13)

Acetylacetone (0.2 mL, 0.196 g, 2 mmol) and drops (0.3 mL) of triethylamine (Et₃N) were added under magnetic stirring to a solution of treated absolute ethanol (10 mL) and hydrazide 2 (0.347 g, 1 mmol). The reaction stayed under reflux for 16 h. After the reaction time, the solvent was evaporated in a rotatory evaporator, the solid 13 (Figure 7) was isolated directly by filtration, washed with cold ethanol to remove the excess of acetylacetone, and the residual solvent was removed under reduced pressure.

Figure 7
Chemical structure of compound 13.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(4,5-dihydro-5-hydroxy-3,5-dimethyl-1H-pyrazol-1-yl-1-carbonyl) pyridine (13)

Beige solid; yield 72%; mp 131-133 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 8.80 (s, 1H, H-6), 8.34 (d, 1H, H-4, J 8.0), 8.06 (d, 3H, H-3, J 8.0), (Pyr-B): δ 8.02 (d, 2H, Ph, J 7.0), 7.78 (s, 1H, H-4), 7.53-7.46 (m, 3H, Ph), (Pyr-C): δ 6.51 (s, 1H, OH), 3.02 (d, 1H, H-4, J 18.0), 2.91 (d, 1H, H-4, J 18.0), 1.98 (s, 3H, CH₃), 1.92 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 148.1 (C-6), 130.6 (C-5), 140.0 (C-4), 114.9 (C-3), 151.7 (C-2), (Pyr-B): δ 132.5 (q, C-5, J 40.0), 109.7 (q, C-4, J 3.0), 150.9 (C-3), 119.6 (q, J 268.0, CF₃), 131.3 (Ph), 129.1 (Ph), 128.8 (Ph), 125.7 (Ph), (Pyr-C): δ 162.9 (CO), 91.5 (C-5), 51.7 (C-4), 156.1 (C-3), 25.7 (CH₃), 15.7 (CH₃); anal. calcd. for C₂₁H₁₈F₃N₅O₂ (429.39): C, 58.74, H, 4.23, N, 16.31; found: C, 58.41, H, 4.59, N, 16.04%.

General procedure for the synthesis of 2-(5-trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(3,5-dimethyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (14)

Acetylacetone (0.2 mL, 0.200 g, 2 mmol) and drops (0.3 mL) of acetic acid (AcOH) were added under magnetic stirring to a solution of treated absolute ethanol (10 mL) and hydrazide 2 (0.347 g, 1 mmol). The reaction stayed under reflux for 16 h. After the reaction time, the solvent was evaporated in a rotatory evaporator, and the isolated solid 14 (Figure 8) was washed with cold ethanol and dried in a vacuum apparatus.

Figure 8
Chemical structure of compound 14.

2-(5-Trifluoromethyl-3-phenyl-1H-pyrazol-1-yl)-5-(3,5-dimethyl-1H-pyrazol-1-yl-1-carbonyl)pyridine (14)

Pale yellow solid; yield 71%; mp 128-130 ºC; ¹H NMR (400.13 MHz, DMSO-d₆): (Py-A): δ 9.00 (s, 1H, H-6), 8.55 (d, 1H, H-4, J 8.0), 8.16 (d, 1H, H-3, J 8.0), (Pyr-B): δ 8.03 (d, 2H, Ph, J 7.0), 7.82 (s, 1H, H-4), 7.53-7.48 (m, 3H, Ph), (Pyr-C): δ 6.33 (s, 1H, H-4), 2.60 (s, 3H, CH₃), 2.20 (s, 3H, CH₃); ¹³C {¹H} NMR (100.61 MHz, DMSO-d₆): (Py-A): δ 149.0 (C-6), 114.6 (C-5), 130.4 (C-4), 141.7 (C-3), 151.8 (C-2), (Pyr-B): δ 132.6 (q, C-5, J 40.0), 110.2 (q, J 3.0, C-4), 152.0 (C-3), 129.1 (Ph), 128.7 (Ph), 128.5 (Ph), 125.7 (Ph), 119.5 (q, CF₃, J 268.0), (Pyr-C): δ 164.9 (CO), 144.5 (C-5), 111.6 (C-4), 152.3 (C-3), 13.6 (CH₃), 13.3 (CH₃); ¹⁹F NMR (564.68 MHz, DMSO-d₆): (Pyr-B): δ –57.88 (CF₃); anal. calcd. for C₂₁H₁₆F₃N₅O (411.38): C, 61.31, H, 3.92, N, 17.02; found: C, 61.18, H, 4.12, N, 16.95%.

Antimicrobial testing

Among the synthesized compounds, those representatives within the series (6a, 8d, 9c, 9d, 9f, 9g, 10h, 11e and 14) were screened in vitro for their antimicrobial activities. They were individually evaluated using either Gram-positive bacteria: Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 1228, Staphylococcus saprophyticus, Enterococcus faecalis ATCC 51299, Listeria monocytogenes, and Bacillus cereus ATCC 14579; or Gram-negative bacteria: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Shigella sonnei, and Salmonella enterica (typhi). These compounds were also evaluated for yeasts: Candida albicans ATCC 14057, Candida dubliniensis, Candida glabrata ATCC 2301, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22018, and Cryptococcus neoformans; and for filamentous fungi: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Fusarium solani ATCC 36031, Rhizopus oryzae, Microsporum canis, Trichophyton mentagrophytes ATCC 9533, Trichophyton rubrum, and Sporothrix schenckii. The MIC and MLC of each fraction of the tested microorganisms were determined by the broth microdilution methods in Muller-Hinton broth (bacteria) and in RPMI 1640 broth (fungi), following protocols M07-A9 (2012), M27-A3 (2008), and M38-A2 (2008) as approved by CLSI. For bacteria, the tested concentrations were 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, and 1.95 µg mL^-1. As for the fungi, the concentrations were 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.975 µg mL^-1. The MIC end point was taken as the lowest concentration of the compound or fraction inhibiting the total growth of microorganisms, and it was detected by lack of visual turbidity. To obtain the MLC, 10 µL was subcultured from each well that showed complete inhibition, from the last positive well onto Muller-Hinton agar (bacteria) or Sabouraud Dextrose agar plates (fungi). Plates were incubated at 35 ºC for 24 h (bacteria) or at 30 ºC for 48 h (fungi). The MLC was defined as the lowest concentration of the compound or fraction that showed either no growth or less than three colonies to obtain approximately 99-99.5% killing activity.⁵⁰

Antioxidant activity

Radical scavenging activity using DPPH method

The DPPH assay was conducted at the NEUROTOX Research Laboratory, in accordance with the lab's adapted protocol^7,51 in which 10 mg of each compound analyzed (1, 2, 6b, 8c, 8d, 9c, 10h, 11e, 12c, 13 and 14) as a free radical scavenger was individually solubilized in ethanol/acetone 1:1 (1 mL), in which stock solution A [ ]i = 10000 µg mL^-1 was used to prepare stock solution B [ ]i = 1000 µg mL^-1 (100 µL of stock solution A plus 900 µL of solubilizing mean). To each of the 96 wells in a microplate, 20 µL of ascorbic acid was added at concentrations of 1, 5, 10 and 100 µL mL^-1, as well as 80 µL of ethanol/acetone (1:1), for a final volume of 100 µL for each well. Subsequently, 20 and 10 µL of stock solution A and 20, 10, and 2 µL of stock solution B was added separately to other wells for each analyzed compound, and then 80, 90 and 98 µL of ethanol/acetone (1:1), thus totaling a volume of 100 µL in each well. Then, 100 µL of ethanol of DPPH solution 0.3 mmol L^-1 was added to each well, for a final volume of 200 µL. The samples' final concentrations in the wells were: [ ]f=1000, 500, 100, 50 and 10 µL mL^-1. Proper blanks were assayed simultaneously and samples were tested in duplicate in three plates. After 30 min of incubation at room temperature away from light, the 96 well culture plates were analyzed using a TP-THERMOPLATE^® at 518 nm wavelength. The percentage of antioxidant activity (%, AA) is the amount of DDPH consumed by antioxidants. Radical scavenging activity was calculated as equation 1.⁵²

Data was processed using the GraphPad Prism software and the results are presented as mean ± standard derivation (S.E.M.) of each concentration tested.

Determination of the TAC by the phosphomolybdenum method

The TAC of the compounds was evaluated by the phosphomolybdenum assay, as previously described by Prieto et al.,⁸ with a few modifications. Each compound was tested at 10, 50, 100, 500 and 1000 µg mL^-1 in 10% DMSO. Seven different concentrations of butylhydroxytoluene (BHT), 10, 50, 100, 500 and 1000 µg mL^-1 were used as positive controls. An aliquot of sample solution was combined with reagent solution (600 mmol L^-1 sulfuric acid, 28 mmol L^-1 sodium phosphate, and 4 mmol L^-1 ammonium molybdate). The tubes were capped and incubated in a water bath at 95 ºC for 90 min. After the samples had cooled to room temperature, the absorbance was measured at 695 nm against a blank (reagent and 0.05 mL of water). The TAC was expressed in relation to the BHT 50 µg mL^-1 absorbance (control 100%) and calculated by the equation 2.

• FAPERGS/CAPES has sponsored the publication of this article.
• Supplementary Information
  Crystallographic data reported in this paper for compounds 6a, 7a, and 14 have been deposited at the Cambridge Crystallographic Data Center (CCDC), No. 1040652, 1040653 and 1040654, respectively. Copies of the data can be obtained, free of charge, on application to CCDC 12 Union Road, Cambridge CB2 1EZ, UK (Fax [phone] or e-mail [e-mail]).
  Supplementary information (experimental details and analytical data for all new compounds, as well as the copies of ¹H and ¹³C NMR spectra) is available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgements

The authors thank the CAPES and the CNPq for fellowships and financial support (Proc. No. 303.013/2011-7 and 471.269/2013-1-Universal).

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Data availability

https://minio.scielo.br/documentstore/1678-4790/mCXtMssq97hFVtFtwDPkXdp/89f2474fb78f6ee98f86883b03901b2b42e944a6.pdf

Publication Dates

• Publication in this collection
  Nov 2015

History

• Received
  08 June 2015
• Accepted
  08 Sept 2015