Synthesis and in vitro Evaluation of Novel Galactosyl-triazolo-benzenesulfonamides Against Trypanosoma cruzi

Os únicos fármacos aprovados para o tratamento da doença de Chagas, nifurtimox e benznidazol, apresentam efeitos colaterais e eficácia limitada na fase crônica da doença, destacando a necessidade de novos fármacos. Entre os diferentes alvos moleculares de fármacos identificados no parasita, uma trans-sialidase de Trypanosoma cruzi (TcTS) tem sido considerada essencial para o reconhecimento e invasão nas células do hospedeiro. Desta forma, o trabalho descreve a síntese eficiente e a avaliação biológica (inibição de TcTS e atividade antitripanossoma) de alguns triazol-arilsulfonamidas contendo galactose pela reação de 1,3-dipolar de cicloadição azida-alcino Cu(I) (CuAAC) em micro-ondas, usando como precursores os derivados de azido benzenossulfonamidas e alcino derivado de galactose. A maioria dos compostos testados contra TcTS mostrou inibição moderada a fraca (40%-15%), com exceção de um dos compostos (81%). Quanto ao ensaio de atividade antitripanossoma, alguns compostos [(IC50 70,9 μM) e (IC50 44,0 μM)] apresentaram atividades mais significativas, embora não tenham sido tão ativos como benznidazol (IC50 1,4 μM). Adicionalmente, a avaliação da citotoxicidade mostrou que todos os compostos não foram citotóxicos. Neste trabalho preliminar, alguns compostos foram considerados protótipos para posterior otimização.


Introduction
The last decade witnessed enormous advances in our understanding of Trypanosoma cruzi (T.cruzi) genome, the etiological agent of Chagas disease, which comprises 22,570 protein-coding genes, including the recent superfamily encoding mucins of the parasite surface, crucial to help the parasite to evade the immune system.This sequencing showed that over 50% of the genome is composed of repetitive sequences, including genes for Vol. 25, No. 10, 2014   surface superfamily of molecules, such as trans-sialidase, proteases and surface gp63 proteins associated with mucin (MASP's). 1][10][11] Furthermore, T. cruzi trans-sialidase (TcTS) affects parasite entry in cardiac cells, leading to the development of host cell inflammatory response and infection process, besides being extremely important in modulating the immune response against infection in the chronic phase of Chagas disease. 124][15][16][17] Only after the sialylation process, T. cruzi can invade and infect macrophages host cells.In general, it is established that parasite and host cell interactions are mediated by several molecules comprising calcium, glycoprotein Gp83, PKC, cruzipain, mucins and Gp85/ trans-sialidase primarily by the action of Gp82 component which triggers Ca 2+ mobilization from cell stores, and contribute for the invasion step. 12,18,19Thus, besides the relative relevance of TcTS for the parasite entry, sialylated mucins greatly contribute for parasite to escape from host immunological defense mechanisms hence acquisition of sialic acids leads mucins to interact with the inhibitory sialic acid-binding protein Siglec-E (sialic acid binding Ig like lectin-E), which disables immune cells activation. 20,213][24] Therefore, the role played by trans-sialidase in the pathogenesis of Chagas disease makes it a valuable and selective target to be explored in the search for new therapeutics.][27] Currently, the treatment of the Chagas disease is based on two drugs, nifurtimox (1) and benznidazole (2), which are effective during the acute phase of the disease albeit several drawbacks related to side effects and resistance development are described. 6,28,29Despite the significant reduction of parasite load and changes in the immune response during the course of benznidazole-combined therapy, for instance with allopurinol, 30 clomipramine, 31 amiodarone, 32 and posaconazole, 5 this promising discovery strategy based on the exploitation of new clinical activity observed for an old drug hitherto did not reach the market. 33ccording to the World Health Organization (WHO), Program for Tropical Disease Research (TDR) and BIO Ventures for Global Health, the major issues concerning the research and drug development in neglected disease, in which Chagas disease is a case in point, encompass implementation of new public policies and alternative strategies to translate basic research into new drug candidate. 34n this scenario, the major aim in designing antitrypanosomal drugs is to find a drug which will be effective in the chronic stage of the disease and remain active despite resistant variants, since an estimated 10,000 deaths still occur annually and 7 to 8 million people are infected worldwide. 35Among the numerous potent antitrypanosomal agents, sulfonamide derivatives have shown high in vitro activities against T. cruzi and transsialidase (Figure 1).Thus, based on molecular hybridization strategy, 36 a series of N-quinolin-8-yl-arylsulfonamides, 37 arylsulfonyl-2-methyl-1,2,3,4-tetrahydroquinolines 38 and N-(biphenyl-4-yl-sulfonyl)-nicotinamides 39 were described as potential antitrypanosomal agents, as respectively illustrated by compounds 3 (IC 50  31.75μM, Selectivity Index-SI 2.1), 4 (IC 50 11.44 μM, SI 21.7) and 5 (lytic concentration-LC 50 50.61μM, SI not available).Regarding trans-sialidase, chalcone-derived sulfonamides, such as 6, proved to be very active, with IC 50 ranging from 0.6 to 7.3 μM, which did not inhibit the human sialidase Neu2 at concentrations up to 200 μM. 40Alternatively, from a library of 1819 molecules, a potent N-thiadiazol-arylsulfonamide 7 transsialidase inhibitor (IC 50 280 μM) was successfully identified by virtual screening and docking simulations, being the arylsulfonamide core highlighted as sialic acid mimics that is able to interact in the TcTS donor sub-site. 27n the course of our work on novel hybrid molecules against T. cruzi, comprising β-galactosyl unit to interact in the TcTS acceptor sub-site, 41,42 and the importance of the above mentioned arylsulfonamides, we envisaged that combining both active core linked by a triazole bridge in a single molecule to give galactosyl-triazolobenzenesulfonamides might provide TcTS inhibitors and antitrypanosomal lead derivatives.Examples of syntheses of glycosylmimetics based on TcTS substrates, assembled by a triazole linker, have been previously described by our group. 43,44In this sense, we have decided to assemble these two important motifs (galactose and sulfonamide) for TcTS inhibition and antitrypanosomal activities via 1,2,3-triazole ring.Additionally, variations on heterocyclelinked sulphonamide core by the bioisosteric replacement of isoxazole, pyrimidine and pyridine rings or an acyl group may represent a convenient strategy to construct chemical diversity and regulate acidity.
Hence, we describe the synthesis of a series of watersoluble galactose-containing triazol-arylsulfonamides via microwave-assisted Cu(I) 1,3-dipolar azidealkyne cycloaddition (CuAAC) as efficient, practical and selective click chemistry strategy to provide 1,4-disubstituted 1,2,3-triazole hybrid molecules using azide benzenesulfonamides and galactose-derived alkyne as precursors.To pursue our goal, these compounds were evaluated as TcTS inhibitors and also as antitrypanosomal agents against T. cruzi (trypomastigote form).In addition, cytotoxicity was tested against a mammalian cell line.
With the azide benzenesulfonamides intermediates 30-36 in hands, the coupling of their azide functions with the O-propynyl galactoside 15 was carried out using the highly efficient CuAAC coupling in a sealed tube under microwave-assisted conditions, which shortened the required reaction time for up 1 h, instead of 16 h under conventional heating. 51In general, the reactions were conducted in DMF at 70-80 °C (150 W) for 10-60 min in the presence of copper sulfate and sodium ascorbate for in situ generation of Cu(I) catalyst.Under these conditions, a small chemical diversity of protected galactosyl-triazolobenzenesulfonamides 37-43 were obtained with complete regioselectivity since the 1,4-disubstituted triazoles were the unique isomer observed by 1 H NMR, with a single triazole hydrogen approximately at 8.0 ppm.Despite the previously reported synthesis of non-substituted derivative 37 in higher yield, 52 our novel series containing N-acetamide and -heterocycles groups was conveniently prepared in 50-63% yield.
Finally, removal of the sugar acetate protecting groups with catalytic NaOMe in MeOH, followed by treatment with DOWEX ® 50WX8 resin, afforded the target products 8-14 in nearly quantitative yield.

Inhibition of T. cruzi trans-sialidase (TcTS)
The in vitro inhibitory properties of compounds 8-14 against a recombinant T. cruzi trans-sialidase were assessed by a sensitive and practical continuous fluorimetric method that measures the residual hydrolase activity of TcTS on the donor substrate 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MuNANA) by cleaving the glycosidic bond that releases the fluorophore, methylumbelliferone (MU). 53Thus, an initial screening was performed using 1.0 mM concentration of products 8-14 in the presence of MuNANA (0.1 mM).For comparison purposes, the activities of 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (DANA) and pyridoxal phosphate were concomitantly measured in the same concentrations of the target compounds due to their respective weak and moderate activities on TcTS. 54ccording to Figure 2, compounds 8-14 showed a variable influence on TcTS enzyme inhibition, being compound 14 the most active of the series with an inhibition percentage of 81%, higher than pyridoxal phosphate (73%).On the other hand, the inhibition profile of compounds 9-13 was just moderate (40%), and compound 8 was weak (15%).Based on the greatest efficacy of 14 against TcTS, bearing a pyridine heterocycle at the benzenesulfonamide core, we pursued the enzymatic activity at lower concentrations (1.0, 0.75, 0.5 and 0.25 mM), however, the inhibition dropped significantly, giving an IC 50 of 0.61 mM.In fact, the inhibition of this enzyme presents a great challenge, since there are few examples of strong TcTS inhibitors that act at nM range. 27espite the lack of evidences that correlate TcTS and in vitro trypomastigote survival, we also investigated the potential antitrypanosomal properties of compounds 8-14 as TcTS-independent experiments.

Antitrypanosomal and cytotoxicity evaluations
In vitro growth-inhibitory properties of all compounds (8-14) against T. cruzi trypomastigotes bloodstream-form were achieved by a colorimetric reaction using chlorophenol red-β-D-galactoside (CPRG) as substrate for modified β-galactosidase Tulahuen strain of T. cruzi. 2,55From these experiments, the IC 50 for all derivatives were calculated considering the concentrations of compounds able to promote 50% parasite lysis, so-called antitrypanosomal activity, using benznidazole (Bz) as reference.From the IC 50 data (Table 1 and Figure 3), it was evident that benzenesulfonamide substituents affect the activity in a reasonable extension, although they were not as active as benznidazole (IC 50 1.4 μM).
It is noteworthy that pyridine derivative 14 that positively inhibit TcTS, failed to inhibit parasite growth as anticipated, suggesting that the antitrypanosomal activities observed for products 10 and 13 may occur by a different mechanism of action.On the other hand, the moderate TcTS activity showed by 14 could be insufficient to cause parasite death.
In addition, compounds 8-14 were examined for their ability to affect mammalian cells, such as cultured mouse spleen cells (Figure 4). 56As a general observation, all galactosyl-triazolo-benzenesulfonamides were not cytotoxic to cells in concentrations similar to those used in the evaluation of the antitrypanosomal activities (500 to 3.9 μM).Given the lack of cytotoxicity, the corresponding LD 50 values and, consequently, the selectivity index could not be calculated at the tested conditions.

Conclusions
In summary, our studies demonstrated that commercial available benzenesulfonamides can be converted to the corresponding azide derivatives using different solvents under microwave-assisted reaction.The coupling of the azide benzenesulfonamides, comprising the free sulfonamide function or substituted by acetamide or different heterocycles, with galactose-derived alkyne was successfully achieved via CuAAC reaction for the formation of a small set of regioselective 1,4-disubstituted galactosyl-triazolo-benzenesulfonamides in moderate to good yields.
Bioassays with products 8-14 revealed TcTS inhibition only for the galactosyl-triazolo-benzenesulfonamide 14, containing a sulfonamide-linked pyridine ring.Despite the weak activity in terms of IC 50 , its inhibition was superior to pyridoxal phosphate used as reference.On the other hand, the evaluation of products 8-14 against T. cruzi trypomastigote showed products 10 and 13, bearing respectively the 2,4-dimethoxypyrimidine and 5-methylisoxazole groups, as the most actives of the series, while the remaining products were two to four fold less active than 13.Bearing in mind the role played by TcTS in parasite cell surface glycosylation and the difficulties to associate TcTS inhibition with parasite viability in vitro assays, we found, indeed, a lack of correlation between the results obtained in enzyme and T. cruzi trypomastigote in vitro assays, suggesting that antitrypanosomal activities found for products 10 and 13 is not related to TcTS inhibition and may involve alternative mechanisms.Thus, based on the low cytotoxicity displayed by the series, compounds 10 and 13 can be considered as lead scaffolds for further optimization.

General
All chemicals were purchased as reagent grade and used without further purification.Solvents were dried according to standard methods. 57MuNANA (2'-(4-methylumbelliferyl)-α-D-N-acetyl-neuraminic acid sodium salt), used as a donor substrate for silylation reactions, was acquired from Toronto Research Chemicals Inc..The trans-sialidase used in this study was a Histagged 70 kDa recombinant material truncated to remove C-terminal repeats but retaining the catalytic N-terminal domain of the enzyme. 58Reactions were monitored by thin layer chromatography (TLC) on 0.25 nm precoated silica gel plates (Whatman, AL SIL G/UV, aluminium backing) with the indicated eluents.Compounds were visualized under UV light (254 nm) and/or dipping in ethanol-sulfuric acid (95:5, v/v), followed by heating the plate for a few minutes.Column chromatography was performed on Silica Gel 60 (Fluorochem, 35-70 mesh) or on a high performance flash chromatography (Biotage Horizon) system using 12 or 25 mm flash cartridges with the indicated eluents.The microwave-assisted reactions were performed in sealed tubes on a CEM Discover ® Microwave System.HPLC purifications were performed on a Shimadzu HPLC system using a Shim-PaK CLC-ODS (M) semi-preparative reverse phase column (250_10.0mm).Nuclear magnetic resonance spectra were recorded on Bruker Advance DRX 300 (300 MHz), DPX 400 (400 MHz) or DPX 500 (500 MHz) spectrometers.Chemical shifts (d) are given in parts per million downfield from tetramethylsilane.Assignments were made with the aid of HMQC and COSY experiments.Accurate mass electrospray ionization mass spectra (ESI-HRMS) were obtained using positive ionization mode on a Bruker Daltonics UltrOTOF-Q-ESI-TOF mass spectrometer.

Synthesis of azide benzenesulfonamides 30-36
General procedure Sodium azide (78 mg, 1.20 mmol, 3 equiv.)was introduced in a microwave flask equipped with a stirring bar and solubilized with water (150 μL).To this solution, the remaining reagents were added in the following order: solvent reaction (1 mL), 4-aminobenzenesulfonamide (16-22) (0.4 mmol, 1 equiv.)and tert-butyl nitrite (t-BuONO).The mixture was stirred and heated under microwave radiation at 150 W. The reaction was followed by TLC and after completion, the reaction mixture was partitioned between hexane and EtOAc.The aqueous phase was extracted with EtOAc (3 times), and the organic phase was dried over Na 2 SO 4 , filtered and concentrated.The product was obtained after flash chromatography on a Biotage Horizon, using 12 mm flash cartridge, flow 8 mL min -1 ; hexane/EtOAc; gradient 0-40% and 40%-40% (v/v).
S y n t h e s i s o f p r o t e c t e d g a l a c t o s y l -t r i a z o l obenzenesulfonamides (37-43) and final products (8-14)   General procedure 4-azidobenzenesulfonamides (30-36) (0.2 mmol, 1 equiv.)were diluted in DMF (0.3 mL) in a microwave sealed flask, equipped with a stirring bar, and treated with the propynyl sugar 15 (85.0 mg, 0.22 mmol, 1.1 equiv.),sodium ascorbate (3.96 mg, 0.02 mmol, 0.1 equiv.)and copper sulfate solution 0.1 mol.L -1 (60 μL).The mixture was stirred and heated under microwave radiation at 150 W varying time and temperature.The reaction was monitored by TLC [hexane/EtOAc 1:9 (v:v)], which showed the formation of only one product.The mixture was concentrated and coevaporated with toluene under reduced pressure to eliminate the solvent (DMF).Then, the product was extracted using EtOAc (3 portions of 5 mL) and washed with water (1 portion of 3 mL) to eliminate the remaining salts.After filtration, the organic phase was dried over Na 2 SO 4 , filtered and concentrated.The product was obtained after flash chromatography on a Biotage Horizon [12 mm flash cartridge, flow 8 mL min -1 ; hexane/EtOAc; gradient 0-50%, 50-100% and 100-100% (v/v)].The O-protected galactosyl-triazolobenzesulfonamides 37-43 (0.1 mmol) were dissolved in methanol (2 mL) and treated dropwise with NaOMe (1 mol L -1 ) at 0 °C until pH 9. The mixture was stirred until the completion of the reaction, followed by TLC (hexane/ EtOAc 1:3).Then, the mixture was neutralized with DOWEX resin (50WX8 H + ), filtered and concentrated under reduced pressure.The products 8-14 were obtained in quantitative yield.

Trans-sialidase inhibition assay
Trans-sialidase used in this study was a His-tagged 70 kDa recombinant material truncated to remove C-terminal repeats but retaining the catalytic N-terminal domain of the enzyme. 58Inhibition was assessed using the continuous fluorimetric assay described by Douglas and co-workers. 53Briefly, the assay was performed in triplicate in 96-well plates containing phosphate buffer solution at pH 7.4 (25 μL), recombinant enzyme solution (25 μL) and inhibitor solution (25 μL of a 4.0 mM solution).This mixture was incubated for 10 min at 26 °C followed by addition of MuNANA (Km = 0.68 mM; 53 25 μL of a 0.4 mM solution giving an assay concentration of 0.1 mM).The fluorescence of the released product (Mu) was measured after 10 min, with excitation and emission wavelengths of 360 and 460 nm, respectively, and the data were analyzed with GraphPad Prism software version 4.0 (San Diego, CA, USA).Inhibition percentages were calculated by the equation: % I = 100 × [1 -(V i /V 0 )], where V i is the velocity in the presence of inhibitor and V 0 is the velocity in absence of inhibitor.

Figure 2 .
Figure 2. TcTS inhibition promoted by galactosyl-triazolobenzenesulfonamides (8-14) using a continuous fluorimetric method.The results are based on three independent experiments, performed in triplicate.

Figure 4 .
Figure 4. Percentage of cell death caused by compounds 8-14 and benznidazole (Bz) evaluated against cultured mouse spleen cells for 24 h.Medium and Tween were used as negative and positive controls, respectively.The data are representative of three independent experiments, performed in duplicate.