PAGES: 141-144 DOI: Full paper
In Vitro Evaluation of the Activity of Aromatic Nitrocompounds against Trypanosoma cruzi

Renata B Oliveira, Ana Paula F Passos, Rosana O AlvesI, Alvaro J RomanhaI, Maria Auxiliadora F Prado, José Dias de Souza FilhoII, Ricardo J Alves +

Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Olegário Maciel 2360, 30180-112 Belo Horizonte, MG, Brasil
ICentro de Pesquisas René Rachou-Fiocruz, Belo Horizonte, MG, Brasil
IIDepartamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil


Fourteen compounds were evaluated for their activity against Trypanosoma cruzi blood stream forms at the concentration of 500 µg/ml. Six compounds were active and re-tested at lower concentrations.

Nifurtimox 1, Benznidazole 2 and Megazol 3 (Fig. 1) are nitroheterocycles which exhibit, as do other ni-trocompounds, activity against Trypanosoma cruzi, the causative agent of Chagas disease (Albuquerque & Perie 1999). Their activity is due at least in part to the well-documented sensitivity of trypanosomes and particularly T. cruzi towards oxidative stress. Upon one electron reduction these compounds generate radical anions (R-NO2/R-NO2 ), which interfere with oxygen metabolism (La-Scalea 1998, Viodé et al. 1999). For Benznidazole, the generation of reduced reactive species that react with parasite macromolecules is also proposed (Urbina 1999).

Differently from mammalian host cells, T. cruzi is deficient in antioxidant enzymes, which are essential to prevent oxidative damage. Trypanothione reductase (TR) is the key enzyme involved in the protection of T. cruzi against the oxidative stress (Zhang et al. 1996). The radical anions generated by the nitrocompounds upon reduction lead to TR depletion and, as consequence, toxicity to T. cruzi.

Taking into account the low efficacy and the side effects of Benznidazole (Neves et al. 1995, Urbina 1999), the current clinically available drug, development of new drugs for the treatment of Chagas disease is mandatory.

In this work, we report the evaluation of in vitro activity of nitroaromatic derivatives 4-13 against trypo-mastigotes of T. cruzi. Compounds 14 and 15 were included for comparison with the corresponding nitrocom-pounds 5 and 8. Some secondary products (16 and 17) were also evaluated.

The rationale behind the choice of the compounds was that the o-nitrobenzyl halides and acetates can generate the alkylating agents quinonimine methide upon reduction to the corresponding hydroxylamino or amino derivatives (Teicher & Sartorelli 1980, Wakselman et al. 1990). These reactive species could alkylate important biomolecules of the parasite, as nucleic acids and proteins (Fig. 2c). As the reduction of nitroaromatics generate superoxide anion (Fig. 2a), these compounds may potentially have a dual mode of action, through oxidative stress and/or alkylation. The o-nitrobenzyl halides can also lead to the reactive benzyl radicals formed by one-electron reduction and subsequent fragmentation (Fig 2b), which is sufficient to expel a good leaving group such as Br- but not a acetate anion (Kirkpatrick et al. 1986). The carboxyl group in the aromatic nucleus allows the preparation of derivatives (esters and amides) for structure-activity relationship purposes. The presence these electron-withdrawing group (e.g., COOH, CONHR and COOMe) is expected to facilitate the reduction of the nitro group (Palmer et al. 1992). The carbohydrate moiety (compounds 11 and 12) was incorporated to improve solubility properties. The piperazino derivative 13 constitutes a potential bis-alkylating agent (Delgado & Remer 1991).



Chemistry - The compounds 4-10 and 13-17 were synthesised from the 4-methylbenzoic acid. The compounds 4-7 and 15 were prepared according to literature procedures (Rich & Gurwara 1975, Barany & Albericio 1985, Lloyd-Williams et al. 1991). The carbohydrate derivatives 11 and 12 were prepared in six steps from commercially available methyl-a-D-glucopyranoside. All compounds were fully characterised by IR, 1H and 13C NMR (Oliveira 1998).

Biological assays - Blood of mice infected with trypomastigotes of the Y strain of T. cruzi was used in the experiments. Drug tests were made in 96 well plates. To each well containing 195 µl of infected blood (2 x 106 
parasites/ml), 5 µl of the drug solution in DMSO was added, resulting in a final concentration of 500 µg/ml. The plates were then maintained at 4ºC for 24 h. Afterwards, the parasite concentration was evaluated using an optical microscope with a 400 X magnification. The parasite concentration reduction (parasite lysis) was determined in comparison with untreated parasites. Positive (7.5 µg/ml crystal violet) and solvent controls (2.5% v/v DMSO) were performed in all experiments. In the specified concentration crystal violet causes 50% reduction of parasite concentration (IC50). DMSO and almost all compounds tested cause no morphological alterations or lysis either of the parasites, erythrocytes or leukocytes. The only exception was compound 8 that presented a moderated erythrocyte lysis at higher concentrations. The IC100 of crystal violet is 30 µg/ml. The experiments were made in duplicate and repeated. The active compounds, those that presented reduction in parasite concentration of approximately 100% (5, 8 and 14-17), were re-tested at lower concentrations.



As shown in the Table, the most active compounds are benzyl bromide derivatives, with one exception (compound 17). As compounds 14 and 15 bearing no nitro substituent were highly active, their activity might be associated to their intrinsic alkylating properties. The introduction of a nitro group in the aromatic ring resulted in an increase of the activity, as seen for compound 5 and 8. This is supposed to be due to the presence of both nitro and benzylic bromine substituents, acting probably, as initially rationalized (Fig. 2). Nitrocompound 4 bearing no benzylic bromine has less than 50% of the activity of the above compounds, in the highest concentration tested. The very low activity of compound 11 was unexpected and can be related to inadequate physicochemical properties of the hydrophilic carbohydrate moiety.

Since benzyl acetates cannot generate the nitrobenzyl radical as do benzyl bromide, the benzyl acetates 6, 9, 10, 12 and 13 need to be reduced to the corresponding hydroxylamino or amino derivatives to act as alkylating agents via the formation of the highly reactive quinonimine methide species. The inefficient reduction of the nitro group by T. cruzi bloodstream forms may explain the low activities of these compounds. Further experiments are underway testing these same compounds against tissue culture forms (amastigotes) of the parasite. The hydrolysis of the esters before reduction to give the poor active benzyl alcohol 7 cannot be ruled out.

Surprisingly, compound 17, bearing no alkylating or nitro functionalities, was one of the most active. In consequence, the mode of action of 17 might differ from that of the other compounds of this series. This compound was obtained as a by-product in the synthesis of 16.

In conclusion, 2-nitrobenzyl bromides and acetates bearing either carboxyl, ester or amide derivatives were prepared and assayed in vitro against the bloodstream forms of T. cruzi. Some compounds displayed significant activity. Compound 5, bearing an orto-nitrobenzyl bromide moiety was the most active. Compound 17, bearing none of those functional groups, was also very active, suggesting the involvement of other(s) mechanism(s) of action against T. cruzi bloodstream forms. Further studies are necessary to assess the real potential of these compounds or derivatives thereof as drug leads. Work in this direction is in progress.



Albuquerque NC, Perie JJ 1999. Nova rota de síntese do megazol [1-metil-2-(5-amino-1,3,4-tiadiazol-2-il)-5-nitroimidazol], um antiparasitário ativo contra o T. cruziRev Bras Cien Farm 35:57-64.

Barany G, Albericio F 1985. A three-dimensional orthogonal protection scheme for solid-phase peptide synthesis under mild conditions. J Am Chem Soc 107:4936-4942.

Delgado JN, Remers WA 1991. Wilson and Gisvold's Textbook of Organic Medicinal and Pharmaceutical Chemistry, 9th ed., Lippincott, Philadelphia, 350 pp.

Kirkpatric DL, Johnson KE, Sartorelli AC 1986. Nitrobenzyl derivatives as bioreductive alkylating agents: evidence for the reductive formation of a reactive intermediate. J Med Chem 29: 2048-2052.

La-Scalea MA 1998. Comportamento voltamétrico e mecanismo de ação biológica de nitroimidazóis.Rev Farm Bioquím Univ São Paulo 34: 59-75.

Lloyd-Williams P, Gairí M, Albericio F, Giralt E 1991. Convergent solid-phase peptide synthesis. X. Synthesis and purification of protected peptide fragments using the photolabile Nbb-resin.Tetrahedron 47:9867-9880.

Neves DP, Melo AL, Genaro O, Linardi PM 1995. Parasitologia Humana, 9. ed., Atheneu, São Paulo, p. 82-114.

Oliveira RB 1998. Síntese de 2,6-bis-[(4-acetoximetil-3-nitrobenzoil)amino]-2,6-didesoxi- a-D-glicopiranosídeo de metila, Potencial Agente Antineoplásico, a Partir da D-glicosamina, Thesis, Universidade Federal de Minas Gerais, Belo Horizonte, 196 pp.

Palmer BD, Wilson WR, Cliffe S, Denny WA 1992. Hypoxic-selective antitumor agents. 5. Synthesis of water-soluble nitroaniline mustards with selective cytotoxicity for hypoxic mammalian cells. J Med Chem 35:3214-3222.

Rich DH, Gurwara SK 1975. Preparation of a new o-nitrobenzyl resin for solid-phase synthesis of tert-butyloxycarbonyl protected peptide acids. J Am Chem Soc 97:1575-1579.

Teicher BA, Sartorelli AC 1980. Nitrobenzyl halides and carbamates as prototype bioreductive alkylating agents. J Med Chem 23:955-960.

Urbina JA 1999. Parasitological cure of Chagas disease: is it possible? Is it relevant? Mem Inst Oswaldo Cruz 94 (Suppl. I): 349-355.

Viodé C, Bettache N, Cenas N, Krauth-Siegel RL, Chauvière G, Bakalara N, Périé J 1999. Enzymatic reduction studies of nitroheterocycles. Biochem Pharmacol 57: 549-557.

Wakselman M, Cerutti I, Chany C 1990. Nitrobenzyl esters as potential conjugated alkylating and differentiation promoting agents: antitumor effect in vivoEur J Med Chem 25:519-526.

Zhang Y, Bond CS, Bailey S, Cunningham ML, Fairlamb AH, Hunter WN 1996. The crystal structure of trypanothione reductase from the human pathogen Trypanosoma cruzi at 2.3 Å resolution. Protein Sci 5: 52-61.


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