Cytotoxicity and Leishmanicidal Activity of Isoniazid-Derived Hydrazones and 2-Pyrazineformamide Thiosemicarbazones

Departamento de Química Geral e Inorgânica, Instituto de Química, Universidade do Estado do Rio de Janeiro (UERJ), 20550-900 Rio de Janeiro-RJ, Brazil Laboratório de Imunofarmacologia Parasitária, Departamento de Microbiologia Imunologia e Parasitologia, Universidade do Estado do Rio de Janeiro (UERJ), 20551-030 Rio de Janeiro-RJ, Brazil Instituto de Tecnologia em Fármacos (Farmanguinhos), Fundação Oswaldo Cruz (Fiocruz), 21041-250 Rio de Janeiro-RJ, Brazil Laboratório de Oncologia Experimental, Universidade Federal do Ceará (UFC), 60430-270 Fortaleza-CE, Brazil Instituto de Química, Universidade Federal Fluminense (UFF), 24020-141 Niterói-RJ, Brazil Instituto Militar de Engenharia (IME), 22290-270 Rio de Janeiro-RJ, Brazil


Introduction
Cancer is one of the leading causes of death worldwide, with about 14 million new cases and 8.2 million cancer related deaths in 2012.Africa, Asia and Central and South America together correspond to 70% of the world's deaths. 1 Although chemotherapy has been an important form of cancer treatment, it presents some challenges such as need to improve the selectivity of drugs and consequent reduction of undesirable side effects and the high incidence of cell resistance, 2 which demonstrate the urgency for the development of new anticancer agents.
Leishmaniases are another set of diseases that require attention.They are caused by protozoa of the genus Leishmania, and present a spectrum of clinical forms, including cutaneous, mucosal and disseminated and diffuse cutaneous leishmaniasis. 3 Leishmania braziliensis is one of the most important species that cause American tegumentary leishmaniasis and the major agent causing the mucosal forms in Brazil. 4Although regarded as neglected disease, leishmaniasis occurs in 98 countries, with an annual incidence of 1 to 1.5 million people worldwide. 1,5he treatment of leishmaniasis is restricted to a few drugs, quite costly and increasingly challenged by the development of parasite resistance. 6In this scenario, the search for drugs that are less toxic, more effective, and less costly is critical for the treatment of leishmaniasis in endemic countries.
INH and PZA (Figure 1), along with ethambutol and rifampin, are some of the most important drugs used in the treatment of tuberculosis. 18Mendez et al. 19 revealed that PZA displays in vitro antileishmanial activity on both promastigotes and amastigotes forms of Leishmania major with half maximal inhibitory concentration (IC 50 ) of 16.1 and 8.2 µM, respectively, after 48 h treatment.Moreover, PZA dramatically decreased lesion development and the parasite burden in C57BL/6 mice infected with the parasite. 19Thus, we have been interested in evaluating the effects of structural modification of PZA, as well as INH, by including a hydrazone or thiosemicarbazone scaffold on its antileishmanial action.

Physical measurements
All common chemicals were purchased from Aldrich and used without further purification.Melting points were determined on a Uni-Melt capillary melting point apparatus (Arthur Hoover Thomas Company).Infrared (IR) spectra were recorded on a Perkin Elmer Fourier transform infrared (FTIR) Spectrum GX spectrometer using KBr plates (4000-400 cm -1 ).Electronic spectra were recorded on an Agilent Technologies 8453 spectrophotometer at room temperature, using spectrophotometric grade solvents (dimethylformamide (DMF) or dimethylsulfoxide (DMSO)).Nuclear magnetic resonance (NMR) spectra were obtained on a Varian multinuclear UNITY-300 (600 MHz), for compounds 1-3 and 5-9, or on a Bruker (400 MHz) spectrometer, for compound 4, using DMSO-d 6 as solvent and tetramethylsilane (TMS) as internal reference.The 1 H resonances were assigned based on chemical shifts and multiplicities of 1 H NMR and 2D NMR correlation spectroscopy (COSY).The carbon type (C, CH x ) was determined by using attached proton test (APT) experiments.Assignments of the protonated carbons were made by 2D heteronuclear single quantum coherence (HSQC).Information on the configurational arrangement of compound 4 was obtained by using nuclear Overhauser effect spectroscopy (NOESY) experiment.High resolution mass spectra of the novel compounds in methanol solution were obtained in the negative ion mode on a Bruker micrOTOF-Q II electrospray ionization-tandem quadrupole-time-of-flight (ESI-Qq-TOF) (for compounds 8 and 9) or in the positive ion mode on a Bruker TOF Compact Electrospray (for compound 4) instrument.
The novel compounds 2-pyridineformamideisonicotinoyl hydrazone (HPAmIH, 4), N(4)-ethyl-2-pyrazineformamidethiosemicarbazone (HPzAm4E, 8) and N(4)-phenyl-2pyrazineformamide-thiosemicarbazone (HPzAm4Ph, 9) were prepared following the literature procedure for the reduction of 2-cyanopyridine and 2-cyanopyrazine. 24Thus, sodium (0.0493 g, 2.1 mmol) was solubilized in 25 mL of dry methanol.2-Cyanopyridine (1.249 g, 12.0 mmol, for production of compound 4) or 2-cyanopyrazine (1.261 g, 12.0 mmol, for production of compounds 8 and 9) was then added, and the mixture was stirred for 0.5 h.After that, for the production of 4, 8 and 9, equimolar amount of isoniazid (1.645 g), N(4)-ethyl-3-thiosemicarbazide (1.430 g) or N(4)-phenyl-3-thiosemicarbazide (2.006 g), respectively, was added in small portions over a period of 0.5 h.The mixture was refluxed for 4 h and stirred for 24 h.The resulting solids were then filtered off, washed with methanol followed by diethyl ether, and dried in vacuo.(8) were obtained by slow evaporation of 1 and 8 in an ethanolic solution, in the presence and absence of nitrate, respectively.Data were collected at room temperature on a Bruker D8 VENTURE equipped with Mo Kα high-brilliance microfocus source (IµS) radiation (λ = 0.71073 Å) and a PHOTON 100 CMOS detector.The instrument was controlled with the APEX2 software package. 25Data were processed using the integrated plug-in in the controlling software package (SAINT), 25 and corrected for absorption by the multiscan semi-empirical method implemented in SADABS. 25Using Olex2, 26 the structure was solved with the SHELXS-97 27 structure solution program using direct methods and refined with the SHELXL-2013 27 refinement package using least squares minimization.Positional and anisotropic atomic displacement parameters were refined for all non-hydrogen atoms.Hydrogen atoms were placed geometrically and the positional parameters were refined using a riding model.

Leishmanicidal activity
Antileishmanial activity studies were performed against intracellular amastigote forms of Leishmania (Viannia) braziliensis (MCAN/BR/98/R619).For this, resident macrophages were obtained from peritoneal cells of Swiss Webster mice after a peritoneal injection of 5 mL of Roswell Park Memorial Institute (RPMI) medium.For adherence, the peritoneal cells (2 × 10 6 per mL) were plated onto glass coverslips placed within the wells of a 24-well culture plate (0.5 mL well -1 ) and incubated at 37 o C in 5% CO 2 for 1 h.After removing the non-adherent cells, the monolayers of macrophages were infected with promastigotes of L. braziliensis (ratio of 5 promastigotes for each macrophage) for 4 h at 37 o C and 5% CO 2 .The infected macrophages were washed and incubated with different concentrations of the tested compounds in RPMI medium supplemented with 10% of fetal bovine serum for 48 h at 37 o C and 5% CO 2 .Each compound was previously dissolved in DMSO (stock solution).The final concentration of DMSO in the RPMI culture medium was kept below 0.3% (v/v).Controls were incubated with medium alone or with 0.3% of DMSO.The monolayers were then stained with Giemsa stain, and at least 100 infected macrophages per sample were counted under optical microscopy.After that, the infection index (% infected macrophages × number of amastigotes / total number of macrophages) was calculated.IC 50 was determined by logarithmic regression analysis using GraphPad Prism 5.All assays were performed in at least three replicates and the results are shown as mean of three independent experiments.
To evaluate the toxicity of INH and PZA derivative molecules on mammalian cells, monolayers of murine macrophages (4 × 10 6 in 200 µL) were incubated with the compounds (1.5-400 µmol L -1 ) dissolved in RPMI medium supplemented with 10% of fetal bovine serum for 48 h at 37 o C and 5% CO 2 .Controls were treated with medium or 1% of DMSO (the major final concentration of the diluent).Viability of macrophages was then assessed by measuring the mitochondrial dependent reduction of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan.For this purpose, MTT (10 µL at 10 mg mL -1 ) was added and monolayers of macrophages were incubated at 37 o C and 5% CO 2 for 3 h.The medium was removed and formazan crystals were dissolved in 180 µL of DMSO.The absorbance was read at 570 nm using a microplate spectrophotometer.The 50% of cytotoxic concentration (CC 50 ) was determined by logarithmic regression analysis using GraphPad Prism 5.
Tumor cell culture and cytotoxic activity SF-295, HCT-116, OVCAR-8 and HL-60 cancer cell lines, from the National Cancer Institute, were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol L -1 glutamine, 100 µg mL -1 penicillin, and 100 µg mL -1 streptomycin at 37 o C and 5% CO 2 .Each compound was previously dissolved in DMSO (stock solution).The final concentration of DMSO in the RPMI culture medium was kept below 0.1% (v/v).PZA, INH and 1-9 (5 µg mL -1 ) were incubated with SF-295, HCT-116 and OVCAR-8 cell lines, for 72 h.The cell viability was determined by reduction of MTT to formazan. 28ompounds that inhibited the proliferation in more than 50% were selected for determination of IC 50 against SF-295, HCT-116, OVCAR-8 and HL-60 tumor cell lines.To this end, 5-0.009 µg mL -1 range for compound concentration was used.All experiments were performed in at least three replicates per compound and results shown are the average of three independent experiments.

Results and Discussion
Characterization of 4, 8 and 9 Compound 4, as well as 5, melts at a temperature above 240 ºC.The similar hydrazones 1-3, in turn, present Vol.27, No. 4, 2016   melting point lower than 200 ºC.The higher melting point values for 4 and 5 are probably due to the presence of NH 2 group, which may be involved in intermolecular hydrogen bonds.Vibrations in the infrared spectrum of 4 attributed to n a (NH 2 ), n s (NH 2 ) and n(N-H) were observed at 3392-3133 cm -1 .Absorptions at 1659 and 1621 cm -1 were attributed to n(C=O) and d(NH 2 ), respectively.These absorptions are shifted in relation to those present in the infrared spectrum of INH.One absorption at 1546 cm -1 attributed to n(C=N) was observed in the spectrum of 4, but is absent in the IR spectrum of INH, suggesting that the condensation reaction took place, giving rise to an imine group in the molecular structure of 4. The C-H out-of-plane bending (γ-CH) or ring bending (β-ring) absorptions from pyridine rings were present at 842, 804, 750 and 679 cm -1 .
Vibrations attributed to n a (NH 2 ), n s (NH 2 ) and n(N-H) were observed at 3275-3414 cm -1 in the IR spectra of 8 and 9. Absorptions at 1655, 1533 and 778 cm -1 in the spectrum of 8 were attributed to d(NH 2 ), n(C=N) and n(C=S), respectively.These bands were observed at 1658, 1545 and 748 cm -1 , respectively, in the spectrum of 9.The in-plane deformation mode of the pyrazine ring was present at 566 and 594 cm -1 in the spectra of 8 and 9, respectively.
We observed duplicated signals in the 1 H and 13 C NMR spectra of 4, suggesting a mixture of two isomers, with the major one accounting for 90%.An analysis of the 1 H NMR spectrum of 4 shows that the maximum difference in d between the signals of the isomers is observed for C3H proton of the ortho-pyridine group (∆(dH(3)) = 0.4 ppm).For other protons of both pyridine rings, the difference in chemical shifts between the two isomers is significantly smaller and in general does not exceed 0.1 ppm.For the amines N3H and N5H 2 , we observed a difference in d of 0.22 and 0.20 ppm, respectively.In order to obtain more information about the identity of the isomers, NOESY analysis was performed (Supplementary Information).For the major isomer, we observed spatial interaction between N5H 2 and H3, as well as between N3H and N5H 2 .These interactions were not observed for the minor isomer.Thus, according to the analysis, the major isomer adopts the E configuration in solution, whereas the minor one corresponds to the Z configurational isomer.The proposed arrangements justify the most significant differences observed between chemical shifts of C3H and amine groups in both isomers.NMR spectra of similar hydrazones with the same behavior in solution had previously been observed and duplicated signals had been attributed to the presence of E and Z configurational isomers in solution 22,29 (Figure S16).
Only one signal for each proton was observed in the 1 H NMR spectra of 8 and 9.The N3H chemical shifts at 10.09 and 10.52 ppm for 8 and 9, respectively, indicate the molecules adopt the E configuration.Chemical shifts higher than 175 ppm observed for the signals of C8 in both compounds are typical of C=S, which suggest the thiosemicarbazones are in the thione form in solution. 16Ray diffraction analysis X-Ray crystal data of compounds [H 2 PCIH]NO 3 •H 2 O (1a) and HPzAm4E ( 8) are summarized in Table S1.Selected intra-molecular bond distances and angles for 1a and 8, as well as for HBPIH (3), 21 HPzAm4DH•H 2 O 23 and HPzAm4M•2H 2 O 23 are given in Tables S2 and S3.Selected dihedral angles, hydrogen bond distances (Å) and angles ( o ) for 1a and 8 are shown in Tables S4 and S5, respectively.The Oak Ridge thermal ellipsoid plot (ORTEP) representation of asymmetric unit, showing ellipsoids displayed at 50% probability level, is shown in Figure 2.

Antileishmanial activity
To evaluate the antileishmanial effect of compounds we used intracellular amastigote of L. braziliensis, since this parasite form is relevant for infected host treatment.All the compounds, including PZA and INH, reduced the infection index in relation to untreated control (p < 0.05), with IC 50 in the 33.36-10.70µM range (Table 1).The isoniazidderived hydrazones 1-5 were as potent as INH, while the thiosemicarbazone derivatives were in general more active than PZA.HPzAm4M (7) (IC 50 = 10.70 ± 2.84 µM), for example, was about 3-fold more active than the original molecule PZA (IC 50 = 33.36 ± 7.60 µM).
Microscopic observation of stained infected macrophages (Figure 5) illustrates the antiparasitic activity of tested compounds without apparent toxicity to host cell.The treatment with 25 µmol L-1 of HPzAm4M ( 7) induces a major reduction in the number of amastigotes inside macrophages (Figure 5b) when compared to untreated control (Figure 5a).Under these experimental conditions, the antileishmanial effect of compounds was independent of nitric oxide production by infected macrophages (data not shown).
Meglumine antimonate (glucantime), a drug of choice for the treatment of leishmaniasis, was used as control.Its structure and composition is not completely understood and, in aqueous solution, it exists as a complex mixture in equilibrium. 30Thus, the IC 50 value for glucantime was expressed as µg mL -1 units.We found that all compounds are more potent against L. braziliensis than the antileishmaninal drug.
Toxicity studies of the tested compounds on macrophage allow us to evaluate its selectivity index (SI).INH, PZA and their derivatives were selectively active against intracellular amastigote of L. braziliensis.The compounds with better SI values are HAPIH (2, SI = 30.00)and HPzAm4DH (6, SI = 20.83).These results suggest the studied hydrazones and thiosemicarbazones have potential application as antileishmanial agents.

Activity against human tumor cell lines
The cytotoxic effects of the studied compounds at the 5 µg mL -1 concentration are listed in Table 2.In general, the thiosemicarbazones exhibit low cytotoxicity against all cell lines, except HPzAm4E (8), which presents moderate action against HCT-116 and OVCAR-8 cells.HAPIH (2) and HBPIH (3) are the most potent hydrazones, whose activity is superior to INH.
Compounds 2 and 3 were selected for further determining IC 50 of SF-295, HCT-116, OVCAR-8 and HL-60 cells (Table 3).The hydrazones present similar activity against all cell lines and are only approximately 1.6    up to 10-fold less effective than the control (doxorubicin) in inhibiting the cell growth.

Conclusions
PZA, INH and their derivatives exhibit leishmanicidal effects on Leishmania braziliensis.All compounds were more potent than glucantime and selectively active against intracellular amastigote of L. braziliensis in relation to macrophages.
Pyrazinamide-derived thiosemicarbazones have proved to have poor activity against SF-295, OVCAR-8 and HCT-116 cell lines.On the other hand, the isoniazid-derived hydrazones showed activity against the tumor cell lines at the tested concentration.The hydrazones HBPIH (3) and HAPIH (2) are the most efficient cytotoxic compounds, whose activities against the three cancer cell lines were higher than isoniazid.These compounds have also proved to be only approximately 1.6 up to ten times less effective than the control (doxorubicin) to inhibit the SF-295, OVCAR-8, HCT-116 and HL-60 tumor cell lines growth.Thus, the studies indicate the structural modification of isoniazid was an interesting strategy to improve its cytotoxicity against the tested cell lines.

Figure 5 .
Figure 5. Aspect of infected macrophages: monolayers of murine macrophages were infected with promastigote form of L. braziliensis (ratio 5:1) during 4 h.After washing three times the infected monolayers were incubated with HPzAm4M (7) for 48 h.The monolayers were stained with Giemsa.The photographs show infected macrophages that (a) received no drug treatment (control) or (b) were treated with 25 µM of HPzAm4M (7).