Synthesis, antileishmanial activity and QSAR studies of 2-chloro- N -arylacetamides

We describe herein the synthesis and evaluation of the antileishmanial activity against promastigote forms of Leishmania amazonensis and cytotoxicity to murine macrophages of a series of 2-chloro-N-arylacetamide derivatives. All compounds were active, except one (compound 3). Compound 5 presented the most promising results, showing good antileishmanial activity (CI50=5.39±0.67 µM) and moderate selectivity (SI=6.36), indicating that further development of this class is worthwhile. Preliminary QSAR studies, although not predictive, furnished some insights on the importance of electronic character of aryl substituent to biological activity, as well as an indirect influence of hydrophobicity on activity.


INTRODUCTION
Leishmaniasis is a neglected tropical disease caused by several species of Leishmania protozoa and transmitted to humans by the bite of the infected phlebotomine sandfly.This important public health problem affects about 12 million people worldwide, with an annual incidence of approximately 1.3 million new cases.Leishmaniasis is endemic in 98 countries, spread through Latin America, Africa, Asia and southern Europe (WHO, 2015).
There is no effective vaccine in preventing this disease and the available chemotherapy consists of pentavalent antimony compounds, paromomycin, pentamidine, amphotericin B and miltefosine, which are not satisfactory drugs for the treatment due to high toxicity and costs, as well as their parenteral administration for long periods and recent cases of resistance (Jain, Jain, 2013;McGwire, Satoskar, 2014).Therefore, the search for new cheap, potent and safe therapeutic alternatives is needed and has been pursued by several groups (Birhan, Bekhit, Hymete, 2014;Coimbra et al., 2013;Gómez-Pérez et al., 2014;Bonano et al., 2014;Gellis et al., 2012;Machado et al., 2012).
Trypanosomatids present a unique thiol-based metabolism which protects these parasites against oxidative stress, ensuring their survival in a hostile environment (Krauth-Siegel, Comini, 2008).They also possess cysteine proteases, thiol-containing enzymes that are essential to life cycle and pathogenicity of the parasites (Vermelho et al., 2010), so inhibition of that metabolic pathway or these enzymes might impair the viability of trypanosomatids.The biological activity of 2-chloro-N-arylacetamides against trypanosomatids has not been described so far.It is likely that these compounds alkylate such pivotal targets, causing serious damages to parasites.Thus, in this work, we report the synthesis of ten 2-chloro-N-arylacetamide derivatives in order to evaluate their in vitro antileishmanial activity and calculate their half maximal inhibitory concentrations (IC 50 ) against Leishmania amazonensis.The cytotoxicity of the synthesized compounds to murine macrophages was also evaluated and their half maximal inhibitory concentrations (CC 50 ) against these cells were calculated.The selectivity index (SI) was also calculated based on the ratio between CC 50 and IC 50 .We prepared compounds with different features regarding hydrophobic, steric and electronic characters, which would enable us to study qualitative and quantitatively, by QSAR (Quantitative Structure-Activity Relationship) analysis, the relationship between the structures of tested substances and their biological activity.

MATERIAL AND METHODS
General: All reactants and solvents were acquired from commercial suppliers and used without further purification.Melting points were determined on Microquímica MQAPF 301 apparatus.IR spectra were obtained on a Spectrum One, Perkin-Elmer ATR system. 1 H and 13 C NMR spectra were performed on a Bruker Avance DPX-200 spectrometer and the proton and carbon chemical shifts (δ) are given with respect to TMS.
Mice: Peritoneal macrophages were obtained from female BALB/c mice (8 weeks old), which were purchased from the Institute of Biological Sciences (ICB) from UFMG, and maintained under specific pathogen-free conditions.The experimental protocol (code 136/2012) was approved by the Animal Use Committee from UFMG (CETEA/UFMG) Antileishmanial activity: The inhibition of Leishmania growth was assessed in vitro by cultivating stationary-phase promastigotes of L. amazonensis (1 x 10 6 cells) in the presence of each individual compound (0 to 100 µM), in 96-well culture plates (Nunc, Nunclon  , Roskilde, Denmark), for 48 h at 24 o C. A previous titration curve was performed to determine the best time of inhibition of L. amazonensis growth by incubating the parasites with the evaluated products for different periods (data not shown).Cell viability was assessed by measuring the cleavage of 2 mg/mL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma).Absorbances were measured by using a multi-well scanning spectrophotometer (Molecular Devices, Spectra Max Plus, Canada), at a wavelength of 570 nm.Amphotericin B (AmpB, 1 µM) was used as a positive control.The concentration of the compounds needed to inhibit 50% of the Leishmania viability (IC 50 ) was determined by applying the sigmoidal regression of the concentration-response curves, using different concentrations of the compounds.Data shown are representative of three independent experiments, performed in triplicate, which presented similar results.

Cytotoxicity assay:
The inhibition of 50% of the macrophage viability (CC 50 ) was calculated by cultivating macrophages (5 x 10 5 cells) in the presence of each individual compound (0 to 200 µM) in 96-well plates for 48 h, at 37 o C. A previous titration curve was performed to determine the best time of inhibition of macrophages viability by incubating the parasites with the evaluated products for different periods (data not shown).Cell viability was assessed by the MTT assay, and AmpB (1 µM) was used as a control.The selectivity index (SI) was calculated by determining the ratio between CC 50 and IC 50 .Data shown are representative of three independent experiments, performed in triplicate, which presented similar results.
Statistical analysis: Data were analyzed using the GraphPad Prism software (version 5.0 for Windows).The difference among the groups was evaluated by the one-way ANOVA, followed by Bonferroni´s post-test for multiple comparisons.Differences were considered significant when P<0.05.QSAR: QSAR regression analyses were executed with BuildQSAR program (Oliveira, Gaudio, 2001) and Multiple Linear Regression (MLR) was employed.2D-QSAR study was performed using as biological parameter log (1/IC 50 ), derived from IC 50 values for active compounds.ClogP and the phenyl substituent constants σ R and MR R were evaluated as physicochemical descriptors.Briefly, ClogP is the calculated octanol-water partition coefficient, σ R is Hammet's electronic constant and the MR R is the contribution of substituent to molar refractivity, being dependent on volume and polarizability.ClogP was calculated by MarvinSketch 5.9.3 (ChemAxon, 2012) and the substituent constants σ R and MR R were obtained from Hansch and Leo (1979).

RESULTS AND DISCUSSION
Compounds 1-10 were prepared according to Imamura et al. (2005), by reacting the appropriate aniline with 2-chloroacetic anhydride in THF, at room temperature (Figure 1).
We evaluated the in vitro antileishmanial activity of compounds 1-10 against stationary-phase promastigote forms of L. amazonensis and the results are shown in Table I.Except for compound 3, all 2-chloro-N-arylacetamide derivatives showed significant antileishmanial activity.The most active compounds were 2, 5 and 8, with IC 50 values below 6 µM.Interestingly, these compounds possess in common an electron-withdrawing aryl substituent, which may contribute to improve their potential as alkylating agents, by increasing the electrophilic character of the carbon alpha to chloro atom by resonance effect.
The cytotoxicity of compounds was determined using macrophages derived from female BALB/c mice and the values of CC 50 are shown in Table I.The selectivity index (SI) for each compound (the ratio between the CC 50 and IC 50 values) was calculated and the data are also shown in Table I.According to Pires et al. (2013), a SI value above 10 is recommended to ensure the safety of a compound.The most selective 2-chloro-N-arylacetamide derivative was the compound 5, with a SI of 6.36.Since these compounds probably act as alkylating agents, this result indicates that the compound 5 could alkylate parasite nucleophiles with a moderate selectivity when compared with mammalian cell nucleophiles.
In order to study quantitatively the relationship between physicochemical properties of synthesized  compounds and their antileishmanial activity, a 2D-QSAR study was performed.The hydrophobic, electronic and steric parameters were evaluated using ClogP and the phenyl substituent constants σ R and MR R , respectively.IC 50 values of active compounds were converted into log (1/IC 50 ) and used as biological parameters in the QSAR study (Table II).
The data set consists of nine points.We evaluated different linear models with one or two independent variables to obey the condition n/p≥4, where n is the number of data points and p is the number of descriptors used in QSAR model (Verma, Hansch, 2010).The MLR module of BuildQSAR program was used (Oliveira, Gaudio, 2001).The obtained QSAR models with statistical parameters are shown in Table III.
To determine which QSAR model better describes our data, we evaluated several statistical parameters, such as the fraction of variance (r 2 ), the correlation coefficient (r) and the measure of statistical significance (p).Among the QSAR models, we found the best correlation between independent and dependent variables with model 1.In this model, the compound 4 was considered an outlier.Among the predicted QSAR equations, model 1 presented the highest r 2 value, indicating that this model can explain the activity variation in a higher percentage.Moreover, the low value of p indicates a higher statistical significance of the model regarding the others.The data of observed activity values and predicted activity values (calculated using model 1) is given in Table II.By the model 1, a good correlation between the antileishmanial activity of this class and Hammet's electronic constant was observed.This constant represents  the electronic effect of a particular substituent.Positive σ R values mean that the group has an electron withdrawing effect and negative values mean that the group has an electron donating effect.The parameter r 2 indicates that this model can explain 85.9% of activity variation.The p-value indicates a statistical significance of 99.91%.
Since we worked with a small data set and the external validation of QSAR models could not be done, the obtained model does not have a predictive value.However, the correlation found between the antileishmanial activity and Hammet's electronic constant (σ R ) can help us to get some insights about the mechanism of action of these compounds.The positive coefficient of σ R means a direct correlation between the dependent and independent variables, corroborating previous observations that structurally-related compounds bearing electron-withdrawing groups present greater activity than those with electron donating groups (Céspedes-Camacho et al., 2012;Neau et al., 1982).Although compound 4 (R = SO 2 NH 2 ) presents a substituent with high positive value of σ R , it does not follow this tendency, behaving like an outlier.This could be explained by its lower hydrophobicity, indicated by its low value of ClogP (0.35), which may be hindering its passage across the lipophylic membranes of parasite.In the same way, one could suggest a reason why the compound 3 was inactive.In physiological pH, the carboxylic group of compound 3 is predominantly ionized.Then, if we consider the ionized forms in calculating partition coefficient, we obtain a ClogP (or calculated distribution coefficient -ClogD) of -1.66.This suggests that hydrophobicity could also affect the activity when compounds present low values of ClogP, although the high σ R value.This observation is in accordance with Jablonkai (2003), which demonstrates that the herbicidal activity of chloracetamides is not simply dependent on chemical reactivity, but related to molecular properties, including lipophilicity.

CONCLUSION
In conclusion, we have described herein the synthesis and evaluation of the antileishmanial activity against promastigote forms of L. amazonensis and cytotoxicity to murine macrophages of ten 2-chloro-N-arylacetamide derivatives.All compounds, but one, present good antileishmanial activity, demonstrating for the first time the biological potential of this class against Leishmaniasis.Moreover, compound 5 (R=COCH 3 ) showed the most promising results, with good antileishmanial activity and moderate selectivity.By QSAR studies, the influence of electronic character of aryl substituent to biological activity was demonstrated.The indirect influence of hydrophobicity on this activity was also observed.In view of the promising antileishmanial activity of some compounds of this class, the synthesis of analogues possessing high SI is worthwhile.Work in this direction is underway and the results will be reported in due time.

TABLE III -
QSAR models with statistical parameters.
a n/p: number of data points/number of descriptors; b r: correlation coefficient; c r 2 : fraction of variance; d p: statistical significance.