Introduction

Heterocycles’ ring structures are composed by elements other than carbon, and are able to impact strongly the physicochemical properties of the substances (Martins et al. 2015). Also, the incorporation of heterocyclic like fragments is a commonly strategy used in medicinal chemistry to design prototypes with enhanced potency and selectivity, and bioisosteric replacements are often tried goaling to affect the lipophilicity, polarity, and aqueous solubility of the substances, as a way to obtain therapeutically improved medicines (Dua et al. 2011). In order to obtain these products, modern synthetic methodologies are available, allowing rapid access to a wide variety of functionalized heterocyclic compounds of critical importance to the medicinal chemist (Taylor et al. 2016; Khanna et al. 2018; El-Sayed et al. 2018; Przybylski et al. 2018; Brahmachari 2018).

Hydrazonyl compounds are also described with a wide range of pharmacological activities, such as antibacterial, antifungal, anticonvulsant, anti-inflammatory, antimalarial, antileishmanial, and antineoplastic, having also recognized activities in antimycobacterial field (Rollas and Küçükgüzel 2007; Verma et al. 2014; Rodrigues et al. 2014; De Souza et al. 2018; Cardoso et al. 2017; Amim et al. 2016; Candéa et al. 2009.

As a part of a study goaling to investigate the importance of the nitrogen to the biological activity presented by six membered heterocycle derivatives, this manuscript present the antimicobacterial activity of componds containing the nitrogen at position 2 and 4 of the heterocycle moiety (identified as 2-pyridinylhydrazone and 4-pyridinylhydrazone derivatives), and also nitrogen dissubstituted heterocycles, defined as pyrazinylhydrazone derivatives.

Results and discussion

The leishmanicidal activities of series 2 and 4 were previously reported by our research group (De Souza et al. 2018). Concerning to 4-pyridinyl derivatives, compounds 3b, 3d, 3e, 3g, and 3h are novel, and compounds 3c and 3f were reported previously, but their spectral data have not been described in the literature.

Synthesis and characterization

The synthesis of planned 2-pyridinylhydrazone, 2, 4-pyridinylhydrazone, 3, and 2-pyrazinylhydrazone, 4, derivatives were accomplished according to the reported procedure11 as shown in Scheme 1. It was employed, respectively, commercial 2-hydrazinylpyridine, 4-hydrazinylpyridine, or 2-hydrazinylpyrazine as starting material, and appropriated aromatic or heteroaromatic aldehydes in ethanol at room temperature for 5 min-78 h (Table 1). The compounds were obtained in 35–97% yields. It is important to mention that aromatic aldehydes containing hydroxyl, metoxyl and nitro substituents, and heteroaromatic aldehydes were selected due to their activity in different series previously study from our group being the most actives (Rodrigues et al. 2014; Coimbra et al. 2013; Nogueira et al. 2019; Cardoso et al. 2014).

Scheme 1
scheme 1

Synthesis of 2-pyridinylhydrazone derivatives 2a–i, 4-pyridinylhydrazone derivatives 3a–i and 2-pyrazinylhydrazone derivatives 4a-i

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Table 1 2-pyridinylhydrazone 2a–i, 4-pyridinylhydrazone 3a–i and 2-pyrazinylhydrazone 4a–i derivatives
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All these new compounds were identified by detailed spectral data, including 1H NMR, 13C NMR, high-resolution mass spectrometry and infrared spectrometry. In general, the 1H NMR spectrum showed the pyridine protons of compounds 2a-i at 6.72–8.17 and ranging from 7.19 to 13.20 for compounds 3a–i. In addition, the pyrazine protons of compounds 4a–i were observed at 7.94–8.70 ppm and the N=CH protons of all series are presented ranging from 7.27 to 8.37 ppm. The N=CH functional group is also observed in infrared spectra, exhibiting an IR band at 1557–1645 cm−1.

Anti-mycobacterial activity

The antimycobacterial activity of compounds 2, 3, and 4 are shown in Table 2. This work is a continuation of previous studies of our group, when it was observed the highlighted activities presented by 2-hydroxylphenyl and heteroaryl substituted hydrazones. The most active compound of all series, the 4-pyridinylhydrazone, 5-nitrothien-2-yl 3h, present a remarkable activity, being more active than the first line tuberculostatic drug ethambutol.

Table 2 The in vitro activity of compounds 2, 3, and 4 against Mycobacterium tuberculosis H37Rv strain (ATCC 27294, susceptible to ethambutol)
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The 2-hydroxylphenyl derivatives, a-f compounds, are much more active than heteroarylic compounds in 2 and 4 series. In previous studies a role considered for the salicyldehyde hydrazones that of a chelator, and this capacity seems to be optimized when there is a nitrogen at position 2 of the main ring. Salicyldehyde derivatives are known to be able to act as tridentate O,N,N-ligands, and thereby form strong complexes with metals. It is considered that the removal of a metal vital for cell reproduction on complexation by the drug can take a major role in these drug interactions.

For compounds 3 the opposite is observed. Heteroaryl derivatives 3g-h showed an improved activity if compared with hidroxylated compounds 3a–f. The activity of the 5-nitrothienyl and 5-nitrofuranyl derivatives has been attributed to the redox potentials of such compounds (Mahmud et al. 1998; Rakesh et al. 2014; Squella et al. 2005).

The lipophilicities of 2, 3, and 4, expressed as logP values, were determined using the Data Warrior program and are listed in Table 1. Pharmacocinetic studies indicate that a good balance between permeability and aqueous solubility for a drug is indicated by a logP value between 0 and 3. The value found for the most active compound of this study, 3h, falls within this range, being 2.80.

Other compounds with remarkable activities are the 2-hydroxylphenyl derivatives 2b, 2e, 4d, and 4f, being only the pyrazinyl derivatives 4d and 4f, with logP values of 2.4991 and 1.9232, included in the ideal range. The obtained values suggest that these substances will be more soluble in water than 3h.

The toxicity of the compounds 2, 3, and 4 was also determinated using the Data Warrior program, see Table 2. The most promising compound of this study, compound 3h, as well as 2e, 4d, and 4f, were found to be nonmutagenic or tumorigenic.

Based on the results obtained for compound 3h, we can conclude that this compound has a remarkable profile and could be considered as a reasonable starting point to find new compounds with improved antitubercular activity.

Conclusion

In this study, we reported the antimycobacterial activity of 27 pyrimidinyl and pyrazinyl derivatives, which have been evaluated against M. tuberculosis ATTC 27294 using the MTT assay. Compounds 3h, 2e, 4d, and 4f displayed the best tuberculostatic profiles in all experiments. The most active compound, a 2-hydroxyl-5-nitrophenyl, 4-pyridinylhydrazone derivative 3h, showed good biodisponibility and nonmutagenic or tumorigenic profiles in preliminaries in silico studies, and exceptional in vitro activity, being compared with the reference drug ethambutol. These results suggest that this compound could be a useful starting point for further studies for new tuberculostatic drugs and confirm the potential of 4-pyridinylhydrazone derivatives as lead compounds in antituberculosis drug discovery.

Material and methods

Experimental

Melting points were determined using an MQAPF-302 (MicroQuímica Ltd, Santa Catarina, Brazil) apparatus. Infrared spectra were recorded on a Thermo Nicolet Nexus 6700 spectrometer as potassium bromide pellets and frequencies are expressed in cm−1. NMR spectra were were analysed using a Bruker Avance 400 operating at 400.00 MHz (1H) and 100.0 MHz (13C) in DMSO-d6. Chemical shifts are reported in ppm (δ) relative to tetramethylsilane and J-coupling in Hertz (Hz). Proton and carbon spectra were typically obtained at room temperature. High-resolution mass spectra were acquired on a Bruker compact-TOF. The progress of the reactions was monitored by thin-layer chromatography (TLC) on 2.0 cm × 6.0 cm aluminum sheets (silica gel 60, HF-254, Merck) with a thickness of 0.25 mm, using ultraviolet light irradiation. For column chromatography, Merck silica gel (70–230 or 230–400 mesh) was used.

General procedure for the synthesis of 2-pyridinylhydrazone derivatives 2a-i (GP1)

To a stirred solution of 2-hydrazinylpyridine (1.0 mmol) in ethanol (10 mL) was added the appropriate benzaldehyde (0.9 mmol), and the reaction mixture was stirred for 5 min – 78 h at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was purified by washing with cold water (3 × 10 mL), affording the 2-pyridinylhydrazone derivatives 2a–i as solid in 37–81% yields.

General procedure for the synthesis of 4-pyridinylhydrazone derivatives 3a–i (GP2)

To a stirred solution of 4-hydrazinylpyridine (1.0 mmol) in ethanol (10 mL) was added the appropriate benzaldehyde (1.0 mmol), and the reaction mixture was stirred for 20 min –24 h at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was purified by washing with cold diethyl ether (3 × 10 mL), leading to the 4-pyridinylhydrazone derivatives 3a–i as solid in 37–97% yields.

General procedure for the synthesis of 2-pyrazinylhydrazone derivatives 4a–i (GP3)

To a stirred solution of 2-hydrazinylpyrazine (1.0 mmol) in ethanol (10 mL) was added the appropriate benzaldehyde (1.05 mmol), and the reaction mixture was stirred for 2–48 h at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was purified by washing with cold water (3 × 10 mL), cold ether (3 × 10 mL), or by column chromatography purification using hexane/ethyl acetate (50 → 100%) as eluent, thus affording the 2-pyrazinylhydrazone derivatives 4a–i in 35–87% yields.

The series 2 and 4 compounds were previously synthesized by our research group, and the antileishmanial activities published (De Souza et al. 2018). The compounds of series 3 are described below.

(E)-2-((2-(pyridin-4-yl)hydrazono)methyl)phenol (3a)

GP2 was followed using 4-hydrazinylpyridine (109 mg, 1.0 mmol), ethanol (10 mL), and was added 2-hydroxybenzaldehyde (122 mg, 1.0 mmol). The desired compound was obtained as amorphous colorless solid (172 mg, 81%); m.p. 261–263 °C; IR (KBr) ν 3085, 1633 cm−1; 1H NMR (400 MHz, DMSO-d6): δ = 13.92 (s, 1H, NH), 12.76 (s, 1H, H-2’ or H-4’), 10.30 (s, 1H, OH), 8.64 (s, 1H, H-2’ or H-4’), 8.32 (s, 1H, H-1’ or H-5’), 8.31 (s, 1H, H-1’ or H-5’), 7.84 (dd, J = 7.7, 1.6 Hz, 1H, H-5), 7.51 (s, 1H, N=CH), 7.28 (ddd, J = 8.3, 7.7, 1.6 Hz, 1H, H-3), 6.98 (dd, 1H, J = 8.3, 0.8 Hz, H-2), 6.89 (dt, J = 7.7, 0.8 Hz, 1H, H-4) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 156.7 (C-1), 154.5 (C-2’), 144.4 (C-4’), 140.8 (CH=NNH), 139.8 (C-3), 131.8 (C-5), 126.0 (C-6’), 119.6 (C-4), 119.3 (C-6), 116.2 (C-2), 107.8 (C-5’), 106.0 (C-1’) ppm; HRESIMS m/z: 214.0983 C12H12N3O (calcd. 214.0975).

(E)-3-((2-(pyridin-4-yl)hydrazono)methyl)benzene-1,2-diol (3b):

GP2 was followed using 4-hydrazinylpyridine (109 mg, 1.0 mmol), ethanol (10 mL), and was added 2,3-dihydroxybenzaldehyde (138 mg, 1.0 mmol). The desired compound was obtained as amorphous white solid (220 mg, 96%); m.p. 257–259 °C; IR (KBr) ν 2930, 1634 cm−1; 1H NMR (400 MHz, DMSO-d6): δ = 12.69 (s, 1H, H-2’ or H-4’), 9.72 (s, 1H, OH), 9.32 (s, 1H, OH), 8.62 (s, 1H, H-2’ or H-4’), 8.32 (s, 1H, H-1’ or H-5’), 8.30 (s, 1H, H-1’ or H-5’), 7.28 (dd, J = 7.9, 1.5 Hz, 1H, H-5), 7.27 (s, 1H, N=CH), 6.89 (dd, J = 7.9, 1.5 Hz, 1H, H-3), 6.71 (t, J = 7.9 Hz, 1H, H-4) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 154.3 (C-1), 145.7 (C-2), 145.5 (C-2’), 144.8 (C-4’), 140.6 (C=NNH), 120.3 (C-6’), 120.3 (C-5), 119.2 (C-4), 119.2 (C-6), 117.1 (C-3), 116.3 (C-5’), 106.9 (C-1’) ppm; HRESIMS m/z: 230.0932 C12H12N3O2 (calcd. 230.0925).

(E)-4-((2-(pyridin-4-yl)hydrazono)methyl)benzene-1,3-diol (3c):

GP2 was followed using 4-hydrazinylpyridine (109 mg, 1.0 mmol), ethanol (10 mL), and was added 2,4-dihydroxybenzaldehyde (138 mg, 1.0 mmol). The desired compound was obtained as amorphous pale yellow solid (222 mg, 97%); m.p. 200–203 °C; IR (KBr) ν 3164, 1624 cm−1; 1H NMR (400 MHz, DMSO-d6): δ = 13.71 (s, 1H, NH), 12.51 (s, 1H, H-2’ or H-4’), 10.21 (s, 1H, OH), 10.03 (s, 1H, OH), 8.50 (s, 1H, H-2’ or H-4’), 8.25 (s, 2H, H-1’ and H-5’), 7.63 (d, J = 8.6 Hz, 1H, H-5), 7.41 (s, 1H, N=CH), 6.42 (d, J = 2.2 Hz, 1H, H-2), 6.35 (dd, J = 8.6 Hz, 2.2, 1H, H-4) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 161.2 (C-1); 158.4 (C-3); 154.0 (C-2’); 145.3 (C-4’); 140.7 (C=NNH); 139.3 (C-5); 127.7 (C-6’); 111.1 (C-6); 108.1 (C-5’); 107.5 (C-1’); 105.5 (C-4); 102.3 (C-2) ppm HRESIMS m/z: 230.0935 C12H12N3O2 (calcd. 230.0925).

(E)-2-((2-(pyridin-4-yl)hydrazono)methyl)benzene-1,4-diol (3d):

GP2 was followed using 4-hydrazinylpyridine (109 mg, 1.0 mmol), ethanol (10 mL), and was added 2,5-dihydroxybenzaldehyde (138 mg, 1.0 mmol). The desired compound was obtained as amorphous white solid (215 mg, 94%); m.p. 243–245 °C; IR (KBr) ν 3236, 1635 cm−1; 1H NMR (400 MHz, DMSO-d6): δ = 13.92 (s, 1H, NH), 12.69 (s, 1H, H-2’ or H-4’), 9.59 (s, 1H, OH), 9.02 (s, 1H, OH), 8.56 (s, 1H, H-2’ or H-4’), 8.32 (s, 2H, H-1’ and H-5’), 7.44 (s, 1H, N=CH), 7.22 (d, J = 2.8 Hz, 1H, H-5), 6.79 (d, J = 8.7 Hz, 1H, H-2), 6.74 (dd, J = 8.7, 2.8 Hz, 1H, H-3) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 154.4 (C-1); 149.9 (C-2’); 149.8 (C-4’); 144.6 (C-4); 140.8 (C=NNH); 139.7 (C-6’); 119.8 (C-6); 119.6 (C-3); 117.1 (C-2); 110.7 (C-5); 107.9 (C-5’); 105.8 (C-1’) ppm; HRESIMS m/z: 230.0927 C12H12N3O2 (calcd. 230.0925).

(E)-5-methyl-2-((2-(pyridin-4-yl)hydrazono)methyl)phenol (3e):

GP2 was followed using 4-hydrazinylpyridine (109 mg, 1.0 mmol), ethanol (10 mL), and was added 2-dihydroxy-4-methylbenzaldehyde (136 mg, 1.0 mmol). The desired compound was obtained as amorphous white solid (216 mg, 95%); m.p. 290–292 °C; IR (KBr) ν 3191, 1635 cm−1; 1H NMR (400 MHz, DMSO-d6): δ = 13.98 (s, 1H, NH), 12.81 (s, 1H, H-2’ or H-4’), 10.24 (s, 1H, OH), 8.61 (s, 1H, H-2’ or H-4’), 8.30 (s, 2H, H1’ and H-5’), 7.71 (d, J = 8.0 Hz, 1H, H-5), 7.48 (s, 1H, N=CH), 6.81 (s, 1H, H-2), 6.71 (d, J = 8.0 Hz, 1H, H-4), 2.27 (s, 3H, CH3) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 156.8 (C-1); 154.4 (C-3); 144.8 (C-2’); 142.1 (C-4’); 140.8 (C=NNH); 139.7 (C-5); 126.1 (C-6’); 120.5 (C-4); 117.1 (C-6); 116.6 (C-2); 107.9 (C-5’); 105.9 (C-1’); 21.2 (CH3) ppm; HRESIMS m/z: 228.1138 C13H14N3O (calcd. 228.1137).

(E)-4-nitro-2-((2-(pyridin-4-yl)hydrazono)methyl)phenol (3f):

GP2 was followed using 4-hydrazinylpyridine (109 mg, 1.0 mmol), ethanol (10 mL), and was added 2-hydroxy-5-nitrobenzaldehyde (167 mg, 1.0 mmol). The desired compound was obtained as amorphous white solid (245 mg, 95%); m.p. 294–296 °C; IR (KBr) ν 1635, 1519, 1336 cm−1; 1H NMR (400 MHz, DMSO-d6): δ = 12.93 (s, 1H, H-2’ or H-4’), 8.65 (d, J = 2.9 Hz, 1H, H-5), 8.61 (s, 1H, H-2’ or H-4’), 8.38 (s, 1H, H-1’ or H-5’), 8.36 (s, 1H, H-1’ or H-5’), 8.18 (dd, J = 9.1, 2.9 Hz, 1H, H-3), 7.58 (s, 1H, N=CH), 7.21 (d, J = 9.1 Hz, 1H, H-2) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 162.1 (C-1); 154.7 (C-4); 141.6 (C=NNH); 139.9 (C-2’); 139.9 (C-4’); 126.8 (C-5); 121.3 (C-3); 120.5 (C-6’); 120.5 (C-6); 116.8 (C-2); 108.1 (C-5’); 106.5 (C-1’) ppm; HRESIMS m/z: 259.0822 C12H11N4O3 (calcd. 259.0826).

(E)-4-(2-((5-nitrofuran-2-yl)methylene)hydrazinyl)pyridine (3g):

GP2 was followed using 4-hydrazinylpyridine (109 mg, 1.0 mmol), ethanol (10 mL), and was added 5-nitrofuran-2-carbaldehyde (141 mg, 1.0 mmol). The desired compound was obtained as amorphous yellow solid (162 mg, 70%); m.p. 280–282 °C; IR (KBr) ν 1643, 1534, 1346 cm−1; 1H NMR (400 MHz, DMSO-d6): δ = 14.20 (s, 1H, NH), 13.20 (s, 1H, H-2’ or H-4’), 8.45 (s, 1H, H-1’ or H-5’), 8.43 (s, 1H, H-1’ or H-5’), 8.30 (s, 1H, H-2’ or H-4’), 7.83 (d, J = 4.0 Hz, 1H, H-3), 7.41 (s, 1H, N=CH), 7.38 (d, J = 4.0 Hz, 1H, H-4) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 154.8 (C-2); 152.0 (C-5); 151.0 (C-2’); 151.0 (C-4’); 141.2 (C=NNH); 134.9 (C-6’); 116.1 (C-3); 116.1 (C-4); 114.7 (C-5’); 108.2 (C-1’) ppm; HRESIMS m/z: 233.0677 C10H9N4O3 (calcd. 233.0670).

(E)-4-(2-((5-nitrothiophen-2-yl)methylene)hydrazinyl)pyridine (3h):

GP2 was followed using 4-hydrazinylpyridine (109 mg, 1.0 mmol), ethanol (10 mL), and was added 5-nitrothiophene-2-carbaldehyde (157 mg, 1.0 mmol). The desired compound was obtained as amorphous white solid (201 mg, 81%); m.p. 288–290 °C; IR (KBr) ν 1639, 1522, 1329 cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 14.11 (s, 1H, NH), 13.11 (s, 1H, H-2’ or H-4’), 8.52 (s, 1H, H-2’ or H-4’), 8.42 (s, 1H, H-1’ or H-5’), 8.41 (s, 1H, H-1’ or H-5’), 8.16 (d, J = 4.3 Hz, 1H, H-3), 7.65 (d, J = 4.3 Hz, 1H, H-4), 7.41 (s, 1H, N=CH) ppm; 13C-NMR (125 MHz, DMSO-d6): δ = 154.6 (C-5); 151.1 (C-2); 145.7 (C-2’); 145.7 (C-4’); 141.2 (C=NNH); 140.4 (C-6’); 130.6 (C-3); 130.6 (C-4); 130.1 (C-5’); 130.1 (C-1’) ppm; HRESIMS m/z: 249.0450 C10H9N4O2S (calcd. 249.0441).

(E)-2-((2-(pyridin-4-yl)hydrazono)methyl)pyridine (3i):

GP2 was followed using 4-hydrazinylpyridine (109 mg, 1.0 mmol), ethanol (10 mL), and was added 2-pyridinecarboxaldehyde (107 mg, 1.0 mmol). The desired compound was obtained as amorphous yellow solid (74 mg, 37%); m.p. 250–252 °C; IR (KBr) ν 1645cm−1; 1H NMR (400 MHz, DMSO-d6): δ = 8.60 (m, 1H, H-5), 8.33 (s, 1H, H-2’ or H-4’), 8.32 (s, 1H, H-2’ or H-4’), 8.14 (s, 1H, N=CH), 8.03 (d, J = 4.8 Hz, 1H, H-2), 7.86 (t, J = 7.6 Hz, 1H, H-4), 7.38 (ddd, J = 7.6, 4.8, 1.1 Hz, 1H, H-3), 7.20 (s, 1H, H-1’ or H-5’), 7.19 (s, 1H, H-1’ or H-5’) ppm; 13C-NMR (125 MHz, DMSO-d6): δ = 153.3 (C-6); 152.1 (C-2); 149.4 (C-2’); 146.4 (C-4’); 143.2 (C=NNH); 136.7 (C-4); 123.8 (C-3); 123.8 (C-6’); 119.7 (C-5); 119.7 (C-5’); 107.4 (C-1’) ppm; HRESIMS m/z: 199.0982 C11H11N4+ (calcd. 199.0979).

Anti-mycobacterial activity

The antimycobacterial activities of compounds 2, 3, and 4 were assessed against M. tuberculosis ATCC 27294 using Mosman’s MTT (3-(4,5-dimethylthylthiazol-2yl)-2,5-dimethyl tetrazolium bromide; Merck) microcultured tetrazolium assay (Mosmann 1983). Briefly, the cells were plated in flat-bottom 96 well plates (2.5 × 106 cells/well/100 μL) cultured for 24 h in a controlled atmosphere (CO2 5% at 37 °C), and nonadherent cells were washed by gentle flushing with RPMI 1640 supplemented with fetal bovine serum (10%, Sigma) and gentamicin (25 μg/mL). Adherent cells were infected or not with BCG (2.5 × 106 UFC/well/100 μL) cultured in the presence of medium alone, Tween 20 (3%) (live and dead controls, respectively), or different concentrations of compounds (1.0, 10.0, and 100 μg/mL) in a triplicate assay. After 48 h, stock MTT solution (5 mg/mL of saline; 20 mL/well) was added to the culture and 4 h later, the plate was centrifuged for 2 min at 2800 rpm, supernatant was discharged, and dimethyl sulfoxide (DMSO) (100 μL/well) was added to formazan crystals solubilization, and the absorbance was ready at 540 nm in a plate reader (Biorad – 450).