N-n-alkyl-3,4-dihydroxybenzamides as inhibitors of the trypanosome alternative oxidase: activity in vitro and in vivo

Abstract

On the basis of our previous demonstration of the high inhibitory activity of a series of p-n-alkyloxybenzhydroxamic acids and n-alkyl esters of 3,4-dihydroxybenzoic acid against the trypanosome alternative oxidase in a cell-free mitochondrial preparation of Tiypanosoma brucei brucei, we synthesized a series of N-n-alkyl-3,4-dihydroxybenzamides for evaluation as inhibitors of this enzyme. This class of compounds was selected with the expectation of their having similar inhibitory activity to but greater solubility than the esters and hydroxamic acids noted above and greater resistance to serum hydrolases in vivo. We predicted that such properties would allow an inhibitor of the trypanosome alternative oxidase to be coadministered with glycerol as a means of providing treatment for infections by African trypanosomes. As expected, such benzamides were both more soluble and more stable, some being more active against the target enzyme than the corresponding ester. One, N-n-butyl-3,4-dihydroxybenzamide, was selected for evaluation in vivo against T. brucei brucei.When combined with glycerol, this benzamide was found to be curative. A regimen wherein 450 mg of N-n-butyl-3,4-dihydroxybenzamide per kg and 15 g of glycerol per kg were given hourly in three divided doses cured 17 of 19 mice with established T. brucei brucei infections. This combination is more active in vivo than any other designed to block simultaneously both the unique respiratory electron transport system and the anaerobic glycolytic pathways of these pathogenic protozoa.The Trypanosoma brucei complex, the causative agent of human and animal trypanosomiasis in Africa, has several unusual biochemical pathways, including a mitochondrial terminal oxidase known as the trypanosome alternative oxidase (TAO) (1). This enzyme is the sole terminal oxidase functioning in the life cycle stages found in the mammalian host and, by the following criteria, is similar to the alternative oxidase found in some plants. Both are ubiquinoloxygen oxidoreductases (1). With a monoclonal antibody against the plant enzyme, Western blots (immunoblots) of Trypanosoma brucei brucei show a single band of the same size (35 kDa) as the plant alternative oxidase (3). Furthermore, the two enzymes are sensitive to the same range of specific inhibitors (2).Since the TAO is an enzyme with no homolog in the mammalian host, it has been the target of a rational approach to the design of drugs for the treatment of African trypanosomiasis. Early work showed that the TAO is sensitive to substituted hydroxamic acids (6). In combination with glycerol, which blocks a glycerol-producing pathway by mass action, salicylhydroxamic acid (SHAM) is trypanocidal (4), although the concentrations required for a cure are toxic to the host (9). In a search for alternatives to SHAM, we designed, synthesized, and tested compounds against TAO activity as measured in a crude mitochondrial preparation of T. brucei brucei (5,7,8). The most active compounds were p-n-alkyloxybenzhydroxamic acids and n-alkyl 3,4-dihy...

Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity

“…More potent antitrypanosomal TAO inhibitors have been developed, however [42,43,44]. Our finding, therefore, that aqp2 -deficiency is associated with TAO-inhibitor sensitivity, has implications for potential future therapeutic strategies.…”

Alternative oxidase inhibitors: Mitochondrion‐targeting as a strategy for new drugs against pathogenic parasites and fungi

“…The replacement of the benzoate linkage with a benzamide group ( 145 ‐ 149 ) produced inhibitors that were more soluble and more stable to serum hydrolases in vivo than the 3,4‐dihydroxybenzoate analogues, and several were more active ( 146 and 149 ) against the target enzyme than the corresponding ester ( 136 and 143 ) . In fact, the n ‐butyl‐3,4‐dihydroxybenzamide ( 146 ) was found to be curative in T. brucei brucei –infected mice (450 mg/kg by intraperitoneal dosage) when coadministrated with glycerol (15 g/kg per oral in three doses at hourly intervals).…”

“…The in vivo toxicity of the benzamide ( 145‐149 ) and benzoate compounds ( 136 and 141 ‐ 143 ) decreased when the alkyl chain contained more than six methylene units. Of note, the amides had slightly lower LD 50 values than the ester counterparts . Importantly, 3,4‐dihydroxy aromatic compounds ( 153 ) are inactive if the carboxyl (eg, acid) group is not linked directly to the phenyl ring (Figure ).…”

Human antiprotozoal therapy: past, present, and future

“…In an experimental mouse model of T. brucei infection, combinations of suramin and eflornithine, melarsoprol and eflornithine, and nifurtimox and melarsoprol have shown promise; suramin plus eflornithine is now being evaluated in humans (67). Other antitrypanosomal compounds with efficacy in the mouse model are the spiroarsoranes (pentavalent arsenicals with less toxicity than trivalent arsenicals) (81) and some newly synthesized dihydroxybenzamides with specific inhibitory activity against trypanosomal alternative oxidase (52).…”