Cytotoxic , Trypanocidal Activities and Physicochemical Parameters of nor-β-Lapachone-based 1 , 2 , 3-Triazoles

A atividade citotóxica de cinco derivados 1,2,3-triazólicos da nor-β-lapachona e do precursor azidonaftoquinona, foi avaliada contra seis linhagens de células neoplásicas: SF-295 (sistema nervoso central), HCT-8 (cólon), MDAMB-435 (melanoma), HL-60 (leucemia), PC-3 (próstata) e B-16 (melanoma murino). Valores de IC 50 entre 0,43 e 9,48 μM foram obtidos. A 3-(4-(1-hidróxicicloexil)-1H-1,2,3-triazol-1-il)-2,2-dimetilnaftol[1,2-b]furan-4,5-dione apresentou-se altamente citotóxica contra MDAMB-435, com IC 50 menor do que o encontrado para doxorubicina (controle positivo). Na tentativa de obter correlação entre parâmetros físico-químicos (potencial de redução e clog P) e atividade citotóxica, estudos eletroquímicos foram realizados em tampão acetato, pH 4,5, em eletrodo de carbono vítreo, e valores de log P calculados (clog P). Apesar da ausência de interação estrutural conjugativa entre o sistema quinônico e o anel triazólico, observou-se a influência do anel heterocíclico no comportamento voltamétrico, com deslocamento anódico dos potenciais de redução. Não se verificou correlação entre os parâmetros eletroquímicos (Ep Ic ) e a atividade citotóxica. Por outro lado, a comparação de Ep Ic com atividades tripanocidas, já descritas, reafirmou a tendência de maior atividade biológica para quinonas mais eletrofílicas (> Ep Ic ). Apesar da ausência de correlação direta, foi possível verificar que clog P influencia a atividade citotóxica: quanto menor a lipofilia (< clog P), menor a citotoxicidade (> IC 50 ).

The cytotoxicities of five nor-β-lapachone-based 1,2,3-triazoles and the precursor azidonaphthoquinone were assayed against six neoplasic cancer cell lines: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (melanoma), HL-60 (leukaemia), PC-3 (prostate) and B-16 (murine melanoma).IC 50 values ranging from 0.43 to 9.48 μM were obtained.3-(4-(1-hydroxycyclohexyl)-1H-1,2,3-triazol-1yl)-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione proved highly cytotoxic to MDAMB-435, with IC 50 even lower than the one from doxorubicin (positive control).In an attempt to correlate physicochemical parameters (reduction potentials and calculated log P) with cytotoxic activity, electrochemical studies were conducted in acetate buffer, pH 4.5, using a vitreous carbon electrode and calculated log P values were obtained.Despite the absence of a structural conjugative interaction between the two systems (quinone and triazole), the heterocyclic group was found to influence the voltammetric behaviour, as indicated by anodic shifts in the reduction potentials.No correlation was found between Ep Ic and cytotoxicity.In contrast, a comparison of Ep Ic with previously reported trypanocidal activities reconfirmed the trend for higher activity coupled with better quinone electrophilicity (> Ep Ic ).A leading term in the correlation of cytoxicity, despite the absence of a linear correlation, was the calculated log P: the lower the lipophilicity, the lower the cytoxicity (> IC 50 ).

Introduction
Cancer is the second leading cause of death worldwide after cardiovascular diseases.][4][5][6] Quinones play a major role as bioreductive drugs. In this regard, quinones are known to play a pivotal role in energy metabolism and in many other key biochemical processes, mainly in chemotherapy, which uses redox cycling drugs. 9,11he electrochemical properties of quinone compounds are obviously very important for their bioreductive activation to either the semiquinone or hydroquinone. 9,11here are several examples of correlations between electrochemical potentials and biological activities in this class of compounds.For example, a definite correlation has been found between redox potentials and the inhibitory effects of naphthoquinones on Epstein-Barr virus early antigen activation [12][13][14] and their cytotoxicity. 15,16In other cases, this correlation has not been evinced. 17,18here are also several examples of quinone activity outside the field of cancer, 19 e.g., in Chagas' disease, where a correlation has been found between trypanocidal activity and redox potential. 9,11,20hagas' disease is a long-term debilitating disease caused by the flagellate protozoan Trypanosoma cruzi, which is mainly transmitted by triatomine insects or by blood transfusion.However, several routes of transmission that do not involve insect vectors have also been described, such as transmission via blood products or transplantation of infected organs, and vertical transmission.It is one of the most serious endemic parasitic diseases of Latin America, with a social and economic impact far outweighing the combined effects of other parasitic diseases. 21At present, the number of people infected with Chagas' disease worldwide is estimated to be about 10-12 million.The process of urbanization in Latin America and migratory population movements from endemic countries have led to the disease being diagnosed in non-endemic areas. 22 special feature of T. cruzi is its unique sensitivity to the action of intracellular generators of H 2 O 2 .T. cruzi possesses an original redox defence system based upon trypanothione and trypanothione reductase, a NADPH-dependent flavoprotein, which regenerates trypanothione from its oxidised form (disulphide form).It lacks catalase and glutatione peroxidase and is therefore considerably more sensitive to H 2 O 2 -induced oxidative stress than its biological hosts.To date, Chagas' disease has defied all attempts to develop an efficient chemotherapy. 21Despite the recognition of the importance of redox cyclers as potent trypanocidal agents, few reports have shown a possible correlation between redox potentials of quinones and trypanocidal activities. 206][27][28][29][30] An important naphthoquinone obtained from lapachol (1b) is β-lapachone (2b) 9 (Figure 1), which is currently under multiple Phase II clinical trials. 31or-β-lapachone (2a) also has anticancer activity against SF-295 (central nervous system), MDAMB-435 (melanoma), and HL-60 (leukaemia) within the range of 0.3-1.75μM. 32Our group recently demonstrated that the introduction of arylamine groups (Figurea 1, 3) in the dihydrofuran ring of 2a enhanced the anticancer activity of this substance against six cancer cell lines with the IC 50 below 1.5 μM. 32,2,3-Triazoles are an important class of compounds possessing a variety of biological activities, 33 e.g., as antiplatelet agents, 34 as dopamine D2 receptor ligands related to schizophrenia, 35 and as anticonvulsant, 36 anti-inflammatory, 37 anti-allergic, [38][39][40][41] antiviral [42][43][44][45] and antimicrobial agents.[46][47][48][49][50][51] Figure 1. Soe natural and semi-synthetic naphthoquinones endowed with anticancer activity.
Since triazoles and naphthoquinones are important compounds with independent pharmacological activity, it would be relevant to produce some compounds by molecular hybridization using these moieties.The use of this approach in the design of new drugs has proved useful in modifying the selectivity profile and in reducing undesirable side effects. 52Additionally, this approach has proved successful for obtaining trypanocidal compounds (Scheme 1). 53All synthesized naphthoquinoidal 1,2,3-triazoles proved more active than the original quinones against the infective bloodstream trypomastigote form of T. cruzi, with IC 50 /day values in the range of 17 to 359 μM, the apolar phenyl substituted triazole 8 being the most active compound (Table 1). 53ased on an investigation of additional activities for this new class of compounds, this communication reports on the cytotoxic activities against six neoplasic cancer cell lines: SF-295 (central nervous system), HCT-8 (colon), MDAMB-435 (melanoma), HL-60 (leukaemia), PC-3 (prostate) and B-16 (murine melanoma) and against one normal cell, the murine fibroblast L-929, of azidoquinone 5 and of the naphthoquinones containing a 1,2,3-triazole ring in the C-3 position of the dihydrofuran ring of 2a, named 6-10.An analysis was also made of their haemolytic activity, assayed with erythrocyte suspensions, in order to contribute to the clarification of the mechanism of biological action.
Due to the importance of electrochemical studies of quinones in biological mechanisms of action, 9,11 voltammetric parameters (Ep Ic ) are also presented, aiming to correlate biological and physicochemical parameters.The experiments were conducted in a protic medium (to resemble a hydrophilic environment).Additionally, based on the same rationale, 9,11 previously reported data on trypanocidal activity 53 are compared with measured reduction potentials.
Biological activity towards a live host involves a complex outcome that is not usually dominated by only one parameter.Generally, various physicochemical properties impact the pharmacokinetic and metabolic fate of a biologically active compound; hence, a thorough understanding of such characteristics is crucial in studies of modes of action. 54In drug metabolism and pharmacokinetics, one of the most important parameters is lipophilicity, adequately described by experimentally obtained log P, 54 or by a calculated one (clog P) 46 ; therefore, in the present case, we also report clog P for all the compounds.

Chemistry
The compounds were synthesized by the Huisgen reaction 55 using copper catalysis.As shown in Scheme 2, the syntheses of 6-10 were carried out from the azidoquinone 5 by 1,3-dipolar cycloaddition. 56The 3-bromo-nor-βlapachone (4), easily obtained from nor-lapachol (1a), 57 is the precursor of 5, obtained so far by substitution with sodium azide.The synthesis and characterization of triazolic compounds (6-10) have already been reported, 53 except for compound 10, whose spectroscopic data are included in the experimental section.

Biology
Compounds 5-10 were tested in vitro against six cancer cell lines and a normal murine fibroblast L-929, in comparison with the quinones 2a, 2b and doxorubicin, the positive control, through an MTT assay. 58Concentrations that induce 50% inhibition of cell growth (IC 50 ) in μM are reported in Table 1.Based on their activity, the compounds were classified against at least two cancer cell lines as highly active (IC 50 < 2 μM), moderately active (2 μM < IC 50 < 10 μM), or inactive (IC 50 > 10 μM).
The great majority of the compounds are strongly or moderately cytotoxic against all cancer cell lines with IC 50 in the range of 0.43 to 9.48 μM, although they are less cytotoxic than standards 2a, 2b and doxorubicin, with few exceptions (see compound 9, Table 1).The most sensitive cancer cell lines are MDAMB-435 and B-16, as already evidenced for 2a, 2b and arylaminonaphthoquinones, 32 unlike doxorubicin, which is more active against HL-60, HCT-8 and B-16.Compound 5 (IC 50 = 1.19 μM) was selectively active against MDAMB-435.Among the triazolic naphthoquinones, compound 9 showed the highest activity against cancer cells (IC 50 ranging from 0.43 up to 1.83 μM) with the strongest activity against MDAMB-435.Additionally, it showed high selectivity compared with other cancer cells (2-4 × more selective) as well as towards normal cells (L-929, IC 50 = 2.85 μM) (Table 1).The cytoxicity of compound 9 for melanoma cells is similar to that of the original quinones (2a and 2b) and higher than doxorubicin.Towards HL-60, 9 is more active (IC 50 = 1.17 μM) than the original quinones 2a and 2b.Compound 8 showed higher cytotoxic activity than compounds 6 and 7.The presence of aromatic substituent (for 8) and non-aromatic (9 and 10) rings on the triazole moiety seems relevant for activity, possibly indicative of lipophilic effects.The absence of haemolytic activity (EC 50 > 508.35 μM) suggests that the cytotoxicity of the compounds was not related to membrane damage of mouse erythrocytes.

Electrochemistry and clog P
The importance of the data on electron transfer involved in biological activities of quinones prompted us to conduct electrochemical studies of the majority of quinones, using cyclic voltammetry, in a protic (acetate buffer, pH 4.5) medium.A relevant fact is that this paper offers the very first description of the electrochemistry of compounds 2a and 5-10.
In this study, we used the potential of the first reduction wave (Ep Ic ), which gives a quantitative measure of ease of reduction of an oxidant or electron acceptor, for purposes of comparison.Figures 2 A-D  well-behaved quinones.The CVs show a reversible, diffusional and bielectronic pair of reduction waves [(Ep Ic -Ep 1/2 ) experimental = 37-44 mV, (Ep Ic -Ep 1/2 ) theoretical ~ 30 mV], leading to the formation of the respective hydroquinones, with participation of two protons. 9,11n this case, Ep Ic values range from -0.155 V (compound 9) up to -0.249 V (compound 7) vs. Ag/AgCl,Cl - (0.1 mol L -1 ), all the values representing easily reducible compounds.The ease of reduction in the series, expressed by Ep Ic (Table 1), is: Despite absence of a structural conjugative interaction between the two systems (quinone and triazole) due to the presence of a spacer (C sp3 ), the heterocyclic group influences the voltammetric behaviour, showing evidence of anodic shifts in the reduction potentials.This fact has been observed in several instances, e.g., the reduction of 2-methyl-5-nitroimidazole substituted at the N 1 -ethyl side chain with halogens. 59In the present case, it is possible that the electrogenerated hydroquinone is stabilized by hydrogen bonding with the heterocyclic nitrogen, which would favour reduction.
Although there is no direct correlation between these values and cytotoxicity, all the reduction potentials are in the region that allows the enzymatic reduction of quinones and further reaction with oxygen, explaining the presence of cytotoxicity for the great majority of assayed quinones. 9,11Banasri and coworkers 18 also investigated the redox behaviour of aminoquinones and compared them with their cytotoxicity activities, but found no evidence of a correlation.Evidently the electrochemical parameters of quinonoids, albeit essential for cellular event analysis, do not always show a quantitative correlation with their cytotoxic properties.In fact, many other factors such as lipophilicity, membrane permeability, site-specific binding, etc., need critical consideration in order to rationalize the relationship of structural modulation and cytotoxicity. 54og P values were calculated (Table 1) for all derivatives by semi-empirical methods.The values for the less cytotoxic compounds 5-7 were the lowest in the series.The more active compounds 8-10 showed the highest clog P values, even compared with references, 2a and 2b.Partitioning can be related to either absorption or permeation.It is reasonable to suppose that those compounds can be better absorbed and permeated in the cell of the organism.As in the present case, the lipophilicity of catechols has been shown to enhance cytotoxicity. 60The lipophilicity of quinones is similar to that of the parent catechols.This means that the transport of both catechols and quinones into and out of the cell is similar, so there is no significant differentiation between reduced and oxidized forms with respect to transport through the cellular membrane. 61As mentioned in the introduction, it is worthy comparing electrochemical results and trypanocidal activities.The IC 50 order shown in Table 1 With regard to this activity, despite the impossibility of establishing a direct correlation due to the small number of compounds, it was observed that the more electrophilic compounds are better trypanocidal agents.

Conclusions
The nor-β-lapachone-based 1,2,3-triazoles were generally cytotoxic against the cancer cell lines tested, with IC 50 below 5 μM.The results demonstrated that these triazoles are a promising addition to the arsenal of anticancer drugs.An enhancement of the activity between the triazole and 1,2-naphthoquinone moieties was demonstrated in the case of compound 9.The synthetic compounds 8-10 appear to be very interesting as new prototypes that could be exploited for the preparation of analogues, opening prospects for the development of new anticancer drugs, despite the fact that the two standard naphthoquinones, nor-β-lapachone and β-lapachone and doxorubicin were, generally, more potent.As for the calculated clog P values, the data (Table 1) allowed for the discrimination between highly and moderately cytotoxic compounds.Higher lipophilicity values (clog P) were associated with better cytotoxicity.Despite the importance of electrochemical data, in this case, no correlation was found between Ep Ic and cytotoxicity.In contrast, the comparison of Ep Ic with previously reported trypanocidal activities reconfirmed the trend for higher activity coupled with better quinone electrophilicity (> Ep Ic ).

Cell membrane disruption
The tests were performed in 96-well plates using a 2% mouse erythrocyte suspension in 0.85% NaCl containing 10 mmol L -1 CaCl 2 , following the method described by Jimenez et al. 64 The compounds diluted as mentioned above were tested at concentrations ranging from 1.5 to 200 μg mL -1 .After 30 min of incubation at room temperature and centrifugation, the supernatant was removed and the released haemoglobin was measured spectrophotometrically at 540 nm.DMSO was used as a negative control and Triton X-100 (1%) was used as a positive control.After incubation at room temperature for 30 min and centrifugation, the supernatant was removed and the released haemoglobin was measured spectrophotometrically at 540 nm.ED 50 is the calculated dose that effectively induced lysis in 50% of the Triton X-100.

Electrochemistry
The electrochemical experiments of cyclic voltammetry (CV) were performed using an Autolab (Echo-Chemie, Utrecht, Netherlands) PGSTAT 20.The working electrode was a BAS (Bioanalytical Systems, West.Lafayette, IN, USA) GC electrode of 3 mm diameter, the counter electrode was a platinum coil, and the reference electrode was Ag|AgCl,Cl − (0.1 mol L -1 ), and all were contained in a one-compartment electrochemical cell with 10 mL capacity.In the CV experiments, the scan rate varied from 0.020 to 2 V s -1 , while the reported parameters were determined at a rate of 0.100 V s -1 .It was necessary to degas the cell with a nitrogen flow for reduction studies.All the experiments were conducted at room temperature (25 ± 1ºC).
Aqueous acetate buffer solutions (0.2 mol L -1 ; pH 4.5) were used in all the experiments and were prepared from analytical grade reagents and purified water (conductivity < 0.1 μS cm -1 ) obtained from a Millipore (Bedford, MA, USA) Milli-Q system.A Marconi (Piracicaba, Brazil) pHmeter with a combined glass electrode (model MAPA200; series 0113992) was used to measure the buffer pH.

Lipophilicity calculation (clog P)
Theoretical parameters of lipophilicity (clog P) were calculated (Table 1) for all the derivatives using the semiempirical methods: AM1, using the Villar method included in the Spartan package, and OSIRIS Property Explorer (Thomas Sander, Actelion Pharmaceuticals Ltd) freely available on the web site http://www.organic-chemistry.org/prog/peo/.These programs were used to calculate the theoretical log P based on the experimentally determined partition coefficients between n-octanol and water of commercially available drugs.

-10, 2a, 2b and
doxorubicin (Doxo), with the respective confidence intervals.Ep Ic values in V vs. Ag/AgCl,Cl − , aqueous (acetate buffer pH 4.5), at ν = 0.100 V s -1 .Calculated log P (clog P) 53Data are presented as IC 50 values and 95% confidence intervals obtained by nonlinear regression for all cell lines from three independent experiments.Doxorubicin was used as positive control.Data for 2a, 2b and doxorubicin have already been published 32 for all cell lines, except for PC-3 and B-16; b Activity of compounds against trypomastigote forms of T. cruzi [IC 50 /day μmol L -1 )]53; nd: not determined.