Trypanocidal Activity of Nitroaromatic Prodrugs

Current Topics in Medicinal Chemistry, 2011, 11, 000-000

1

Trypanocidal Activity of Nitroaromatic Prodrugs: Current Treatments and Future Perspectives Shane R. Wilkinson1,*, Christopher Bot1, John M. Kelly2 and Belinda S Hall1 1

Queen Mary Pre-Clinical Drug Discovery Group, School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK; 2Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT UK Abstract: Chagas disease and African sleeping sickness are trypanosomal infections that represent important public health problems in Latin America and Africa, respectively. The restriction of these diseases to the poorer parts of the world has meant that they have been largely neglected and limited progress has been made in their treatment. The nitroheterocyclic prodrugs nifurtimox and benznidazole, in use against Chagas disease for >40 years, remain the only agents available for this infection. In the case of African sleeping sickness, nifurtimox has recently been added to the arsenal of medicines, with the nitroheterocycle fexinidazole currently under evaluation. For a long time, the cytotoxic mechanism of these drugs was poorly understood: nifurtimox was thought to act via production of superoxide anions and nitro radicals, while the mode of benznidazole action was more obscure. The trypanocidal activity of nitroheterocyclic drugs is now known to depend on a parasite type I nitroreductase (NTR). This enzyme is absent from mammalian cells, a difference that forms the basis for the drug selectivity. The role of this enzyme in drug activation has been genetically and biochemically validated. It catalyses the 2-electron reduction of nitroheterocyclic compounds within the parasite, producing toxic metabolites without significant generation of superoxide. Recognition that this enzyme is responsible for activation of nitroheterocyclic prodrugs has allowed screening for compounds that preferentially target the parasite. This approach has led to the identification of two new classes of anti-trypanosomal agents, nitrobenzylphosphoramide mustards and aziridinyl nitrobenzamides, and promises to yield new, safer, more effective drugs.

Keywords: Aziridinyl nitrobenzamide, benznidazole, neglected diseases, nifurtimox, nitrobenzylphosphoramide mustard, trypanosomal infections, type I nitroreductase. TYPANOSOMAL DISEASES Protozoan parasites belonging to the genus Trypanosoma are responsible for two major insect-transmitted diseases in humans, Chagas disease and African sleeping sickness (Table 1). More than 10 million people are infected by the causative agents, Trypanosoma cruzi and Trypanosoma brucei respectively, resulting in over 60,000 deaths per year [1, 2]. As a result of concerted surveillance, treatment, insect vector control and improved housing programmes, there has been a significant decline in the prevalence of both infections [3-7]. However, with African sleeping sickness, history has shown that localised epidemics can readily arise following political and socioeconomic disruption, killing tens of thousands of people [6, 7]. In the case of Chagas disease, while infection rates are falling in some endemic areas, migration, tourism, illicit drug usage and modern medical practices have all contributed to the disease becoming problematic in other regions [8-11]: recent estimates suggest that more than 300,000 people living in the USA are infected and blood banks are now routinely screened for the parasite. With no immediate prospect of a vaccine, drugs are essential to combat these diseases. Current therapies (see *Address correspondence to this author at the Queen Mary Pre-Clinical Drug Discovery Group, School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK; Tel: +44 (0)20 7882 8285; Fax: +44 (0)20 8983 0973; E-mail: [email protected]

1568-0266/11 $58.00+.00

Table 2 and 3) are controversial: some are toxic, others mutagenic, most have limited efficacy and clinically resistant or refractory strains have been reported. There is an urgent need for new, cost effective treatments. However, the economic costs associated with bringing a new drug to market are estimated to be more than $500 million dollars [12]. As trypanosomal parasites primarily affect people living in the poorest regions of the world, research has not been commercially attractive to pharmaceutical companies and trypanosomal infections are referred to as ‘neglected diseases’. Recently, this situation has improved with syndicates such as the Drugs for Neglected Diseases Initiative (DNDi) and the Consortium for Parasitic Drug Development, coupled with financial support from organisations such as the Bill and Melinda Gates Foundation and the Wellcome Trust, providing a necessary interface between academia and industry. As examples of this new impetus, nifurtimox-eflornithine combination therapy (NECT) has been recommended as the treatment of choice for late stage West African sleeping sickness and several other compounds are at various stages in clinical trials (e.g. anti-sterol agents, fexinidazole, pafuramidine maleate) (www.dndi.org; www.thecpdd.org). NITROHETEROCYCLIC PANOCIDAL AGENTS

PRODRUGS

AS

TRY-

Nitroheterocyclic compounds encompass a range of molecules characterised by one or more nitro-groups linked to an aromatic ring [13]. Some, such as chloramphenicol and © 2011 Bentham Science Publishers Ltd.

2 Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 16

Table 1.

Wilkinson et al.

Characteristics of Human Trypanosomal Infections

African Sleeping Sickness

Chagas Disease

Distribution

Sub-Saharan Africa.

Central and South America.

Causative agent

Trypanosoma brucei gambiense (West Africa). Trypanosoma brucei rhodesiense (East Africa).

Trypanosoma cruzi.

Vector

Tsetse fly (Glossina spp).

Triatomine bugs (Reduviidae)

Mode of transmission

Vector bite. Mother-to-child (placental). Contaminated needles.

Vector faeces (at site of bite or in contaminated food and drink). Blood transfusion or transplantation. Contaminated needles. Mother-to-child via placenta or breast milk.

Site of infection

Parasites free living, initially found in blood and lymph (haemolymphatic phase), but later invade CNS (neurological phase).

Parasites live intracellularly and can infect any nucleated cell, but have a preference for muscle tissue, particularly smooth and cardiac.

Pathology

Haemolymphatic phase: swelling at site of infection, headache, lymphadenopathy, fever. Neurological phase: slurred speech, confusion, seizures, sleep cycle disturbances, coma. Progression to neurological stage is rapid for T.b rhodesiense infection (weeks) and slow for T.b gambiense (months to years). Both fatal without treatment.

Acute stage (weeks): typically asymptomatic, occasionally mild to severe fever with ECG abnormalities. Indeterminate stage (months to years): no symptoms. Chronic stage (years) - (approximately 30% of infected individuals) : cardiac - enlargement of the heart, thinning of ventricular walls, arrhythmias, risk of sudden death; megasyndromes - gross enlargement of oesophagus or colon, difficulty swallowing, weight loss, malnutrition; dementia.

Table 2.

Drugs Used to Treat African Sleeping Sickness. Modified from [2, 7, 123-126]

Drug

Structure

Suramin

Use

Problems

Negative charge at physiological Ineffective against early stage of T. b. gambiense. pH restricts its use to early stage of Ineffective against late stage of both T. b. rhodesiense and T. b. gambiense. disease. Effective against T. b. rhodesiense. Side effects include nausea, vomiting, albuminuria, haematuria, hypersensitivity reactions, peripheral neuropathy. symmetrical polysulphonated naphthalene, based on urea

pentamidine

aromatic diamidine melarsoprol

trivalent melaminophenyl arsenical eflornithine

Derivative of ornithine

Positive charge at physiological Ineffective against early stage of T. b. rhodepH restricts its use to early stage of siense. disease. Ineffective against stage of both T. b. rhodesiense and T. b. gambiense. Effective against T. b. gambiense. Side effects include hypoglycaemia, injection site pain, diarrhoea, nausea, vomiting. Resistance observed in field. Can cross the blood-brain barrier. Used to target late stage of disease. Side effects including reactive arsenical encephaEffective against both T. b. rhode- lopathy, Jarisch-Herxheimer-like reaction, phlebisiense and T. b. gambiense. tis, peripheral neuropathy, skin reactions. Toxic (kills up to 5% of patients). Can cross the blood-brain barrier. Ineffective against late stage of T. b. rhodesiense. Used to target late stage of disease. Difficult dosing scheme, cost of hospitalisation. Effective against T. b. gambiense. Side effects include diarrhoea, nausea, vomiting, convulsions, anaemia, leucopenia, thrombocytopenia.

Trypanocidal Activity of Nitroaromatic Prodrugs

Table 3.

Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 16

3

Drugs Used to Treat Chagas Disease. Modified from [2, 30, 33, 34, 127-135]

Drug

Structure

Nifurtimox

Use

Problems

Acute stage Chagas disease.

The therapeutic benefit of this agent against chronic stage Chagas disease is uncertain. Variation in strain susceptibility. Side effects include CNS toxicity, including disorientation, disturbances of equilibrium such as ataxia and nystagmus, excitation, forgetfulness, insomnia, irritability, psychosis, seizures, tremors, eosinophilia, impotence, leukopenia, muscle weakness, peripheral neuropathy. Reported to have mutagenic and carcinogenic properties, although this has been questioned

Acute stage Chagas disease.

The therapeutic benefit of this agent against chronic stage Chagas disease is uncertain. Currently under evaluation (BENEFIT program). Variation in strain susceptibility. Side effects include agranulosis, peripheral neuropathy, progressive purpuric dermatitis, seizures. Reported to have carcinogenic properties, although this has been questioned.

5-nitrofuran

Benznidazole 2-nitroimidazole

azomycin, have been isolated as natural products, but most are generated synthetically during the production of plastics, dyes, solvents, pesticides, explosives and pharmaceuticals [14-17]. Those of medicinal or veterinarian use include the broad spectrum nitrofuran and nitroimidazole antibiotics that are effective against a variety of microbial urinary or gastrointestinal tract (GI) infections (Tables 4 and 5). In Europe and USA, the use of certain nitroheterocycles, particularly those based on nitrofuran, has been discontinued since they have been reported to be mutagenic [18]. Elsewhere, these drugs are commonly prescribed. These concerns have led to “nitro-scepticism” across the whole class of compounds, even to agents whose toxicology has never been evaluated. Despite this, there has been a renaissance in the use of nitroaromatic compounds to treat a range of diseases. Several are currently undergoing evaluation as anti-cancer therapies while others are being screened for anti-microbial properties [19-22]. Additionally, as the predominance of strains refractory to other antibiotics increases, there have been proposals to reinstate the use of “problematic” nitroheterocycles to treat specific bacterial infections [23, 24]. The nitroimidazopyrans are of particular interest for the treatment of tuberculosis [22]. These prodrugs display an intriguing activation mechanism that results in production of nitric oxide, a reaction catalysed by a F420-dependent oxidoreductase pathway [25, 26]. This system is absent from eukaryotes and the trypanocidal activity observed using PA-824, the lead nitroimidazopyran structure, occurs via a mechanism distinct from that reported in bacteria [27; Hall and Wilkinson, unpublished]. Interest in using nitroheterocycles to treat African sleeping sickness and Chagas disease began in the 1950’s after a trypanocidal screening programme identified nitrofurazone as a lead compound [28, 29]. The effectiveness of this agent in humans was demonstrated, but failure to totally eradicate parasitaemia, coupled with neuropathological toxicity resulted in these trials being suspended (Packchanian, 1957; cited in [30]). However, these findings led to investigations into the activity of other nitroaromatic compounds, with ni-

furtimox and benznidazole emerging as the most promising candidates [31, 32]. Nifurtimox is a 5-nitrofuran marketed under the brand name Lampit™ (Tables 3 and 5). It was initially used throughout Latin America as the front-line treatment against acute Chagas disease. However, due to GI tract and CNS side effects, coupled with the refractory nature of some T. cruzi strains, its limited efficacy and genotoxicity, nifurtimox use has now been discontinued in Brazil, Argentina, Chile and Uruguay, and it is not available for use in the USA except via the CDC [30, 33-35]. Despite these problems, nifurtimox, as part of NECT, has been successfully trialled as a treatment for late stage West African African sleeping sickness and has recently been added to the WHO Essential Medicines List (www.who.int) [36-39]. Additionally, it is currently undergoing assessment as a treatment for pediatric neuroblastoma [20, 40]. Benznidazole is a 2-nitroimidazole marketed under the name Radanil™ or Rochagan™ (Tables 3 and 4). It is currently the drug of choice against early stage Chagas disease. There is evidence to suggest that it may be of use in the chronic stages of an infection, but the data are generally observational, with relatively few well designed randomized clinical trials [41-43]. To evaluate whether benznidazole treatment against late stage disease is an appropriate course of action, especially in patients with cardiovascular complications, a large scale, multicentre, international trial (BENEFIT project) is currently underway [44]. Although benznidazole is considered a safer treatment than nifurtimox, it is also genotoxic and can cause a range of side effects ranging from dermatitis and polyneuropathy to agranulocytosis [30]. Nifurtimox and benznidazole are both prodrugs that must undergo activation before mediating their cytotoxic effects. A key step in this process involves reduction of the nitrogroup located on the furan or imidazole ring in reactions catalysed by nitroreductases (NTR). Based on their oxygensensitivity and flavin co-factors, NTRs can be divided into two groups [45]. Type I NTRs are FMN containing enzymes


Table 4.

Wilkinson et al.

Anti-Infective Nitroimidazole Drugs

*INN

Structure

Target infection

Benznidazole

Chagas disease

Metronidazole

Obligate anaerobic bacterial infections, peptic ulcers, gingivitis, bacterial vaginosis, giardiasis, amoebiasis, trichomoniasis

Tinidazole

Obligate anaerobic bacterial infections, bacterial vaginosis, giardiasis, amoebiasis, trichomoniasis

Secnidazole

Giardiasis, amoebiasis, trichomoniasis

Ornidazole

Obligate anaerobic bacterial infections, bacterial vaginosis, giardiasis, trichomoniasis

Satranidazole

Periodontitis, giardiasis, amoebiasis, trichomoniasis

Nimorazole

Obligate anaerobic bacterial infections, bacterial vaginosis, giardiasis, trichomoniasis

*INN corresponds to International Non-proprietary Name.

Table 5.

Anti-Infective Nitrofuran Drugs. INN

Structure

Target infection

Nifurtimox

Chagas disease, African sleeping sickness

Nitrofurantoin

Bacterial urinary tract infections

Furazolidone

Bacterial GI tract infections, giardiasis

Nitrofurazone

Topical agents for wounds, burns, ulcers, skin infections

Nifuratel

Bacterial genitourinary tract infections, trichomoniasis



5

(Table 5) contd….

INN

Structure

Target infection

Nifurtoinol

Bacterial urinary tract infections

Nifuroxazide

Bacterial GI tract infections

Nifurzide

Bacterial GI tract infections

*INN corresponds to International Non-proprietary Name.

mainly found in bacteria and absent from most eukaryotes, with a subset of protozoan parasites, including trypanosomes, being major exceptions [46-49]. They function by reducing the nitro-group, through a nitroso intermediate, to a hydroxylamine derivative via a series of 2-electron transfers using NAD(P)H as the source of reducing equivalents. For nitrofurans, the hydroxylamine form can then be processed further to generate cytotoxic metabolites either by: (i) forming nitrenium cations that promote DNA breakage [50, 51], (ii) generating DNA crosslinking adducts [19], or (iii) cleavage of the aromatic ring to yield potentially toxic open chain nitriles [45, 52] Fig. (1). In the case of 2-nitroimidazoles, the hydroxylamine metabolite can undergo rearrangement and hydration to produce a dihydro-dihydroxyimidazole that can then decompose to release glyoxal Fig. (2) [53]. Both of these products are capable of interacting with a range of biomolecules, in particular forming adducts with DNA and thiols [54, 55]. As type I NTR mediated nitro reduction does not involve oxygen and does not result in the production of reactive oxygen species (ROS), this activity is said to be “oxygen insensitive”. In contrast, the ubiquitous type II NTRs contain FAD or FMN as a co-factor and are “oxygen sensitive”. These mediate a 1-electron reduction of the nitrogroup leading to the production of an unstable nitro-radical Fig. (1) [45, 56-58]. In the presence of oxygen, the radical undergoes futile cycling resulting in the production of superoxide anions and regeneration of the parent nitro-compound. Most nitroheterocyclic prodrugs can undergo both kinds of NTR mediated activation event. This has led to debate as to which pathway is the more important in terms of cytotoxicity. Initial observations indicated that nifurtimox could induce oxidative stress in trypanosomes, suggesting that a type II mechanism was key to its trypanocidal activity [57, 59-62]. Subsequently, several parasite FAD-containing enzymes including trypanothione reductase, lipoamide dehydrogenase and cytochrome P450 reductase were shown to reduce nifurtimox [62-64]. This mechanism did prove attractive, since trypanosomes were believed to have a limited enzymatic capacity to metabolise ROS [65, 66]. However, it is now clear that these pathogens do possess a complex antioxidant defence system, with the pathways being distinct from those found in most other eukaryotes [67-70]. In trypanosomes, benznidazole does not induce a significant level of oxygen consumption and free radical production, but interestingly, this does occur in mammalian systems [71]. To

date the only direct link between drug-induced ROS formation and trypanocidal activity stems from functional studies on one of the iron superoxide dismutases from T. brucei. Parasites in which one copy of this gene (TbSODB1) had been deleted displayed hypersensitity to both nifurtimox and benznidazole [72], the latter observation apparently contradicting the earlier findings. Functional analysis of other oxidative defence pathways indicates that they do not play a major role in protection against the trypanocidal activity of nitroheterocyclic drugs [68, 73-77]. Bacteria and trypanosomes selected for resistance to nitroheterocyclic drugs frequently have mutations in, or reduced expression of, their type I NTR complement, suggesting that these enzymes play a major role in activation [48, 78, 79]. Two trypanosomal enzymes with a type I NTR activity have been reported [48, 80]. In vitro analysis of one of these, a prostaglandin F2 synthase (PGFS), demonstrated that it could mediate the 2-electron reduction of nifurtimox, but not benznidazole, and only under anaerobic conditions [80]. However, over expression of this enzyme in infective T. brucei does not alter parasite susceptibility to either agent (Hall and Wilkinson, unpublished). Therefore, it is implicit that within the cell, PGFS does not play a major role in cytotoxicity toward nitroheterocycles. The second type I NTR oxidoreductase displays all the characteristics shown by many of its bacterial counterparts [48]: it is active under both anaerobic and aerobic conditions, it contains FMN as a cofactor and it has a wide substrate range, including nifurtimox and benznidazole. Moreover, the metabolites generated from both trypanocidal prodrugs by the action of T. brucei type I NTR confirms the 2-electron reduction pathway outlined in Figs. (1 and 2): Nifurtimox is converted to its open chain nitrile form, whereas benznidazole forms a dihydrodihydroxy derivative (Hall and Wilkinson, unpublished). A clear link between the trypanosomal type I NTR activity and nitroheterocyclic drug metabolism has now been established using genetically modified T. cruzi and T. brucei lines where the enzyme levels have been altered [48]: null mutant/ heterozygous cells display resistance to various nitroheterocyclic agents, including nifurtimox and benznidazole, while over expression confers hypersensitivity. Consistent with this, parasites selected for resistance to nifurtimox were found to have lost one copy of the chromosome containing the type I NTR gene [48]. Additionally, a whole ge-


Wilkinson et al. R O

.

NH

nitrenium ion

type I NTR R

O-

O

type I NTR

R

2e-

2e-

O

N+

R

N

O

O

NHOH

O

nitrofuran

R

2e-

O

NH2

hydroxylamine

nitroso

-.

amine

-H2O

O2

type II NTR

1e-

R O

O2

N

R

-

2e-

.

R

O

O

N

O

O

nitro anion radical

N

open chain nitrile

Fig. (1). Reduction of nitrofurans by type I and type II nitroreductases. In reactions mediated by type I nitroreductases (NTRs), the conserved nitro group on the nitrofuran prodrug undergoes a series of two-electron reductions to generate the hydroxylamine derivative via an unstable nitroso intermediate. The hydroxylamine can then be metabolised further with several pathways proposed. These include: 1. formation of a nitrenium ion, 2. reduction to the amine form, or 3. cleavage of the furan ring forming unsaturated then saturated open chain nitriles [19, 45, 50-52]. In contrast, the ubiquitous type II NTRs carry out a one-electron reduction of the conserved nitro group, leading to the formation of an unstable nitro radical. In the presence of oxygen, this then reacts via a futile cycle leading to the generation of superoxide anions and the regeneration of the nitrofuran prodrug. type I NTR

R N N

ON+ O

2-nitroimidazole

2e-

type I NTR

R

2e-

N N nitroso

N O

R

N

R

OH

N

H2O

N NH OH

hydroxylamine

N hydroxyl

NH

R

OH N HO

NH2

N

dihydro-dihydroxyl

R

O

+ O

HN HN

NH2

glyoxal

Fig. (2). Reduction of 2-nitroimidazoles by type I nitroreductases. As described in Figure 1, the conserved nitro group on the imidazole ring undergoes a series of two-electron reductions, mediated by type I NTRs, to generate the hydroxylamine derivative, via an unstable nitroso intermediate. At physiological pH, the hydroxylamine forms a nitrenium ion that spontaneously decomposes to a hydroxy metabolite. This undergoes hydration to the dihydro-dihydroxy derivative that can fragment to release glyoxal [53].

nome “loss of function” screen using nifurtimox or benznidazole against an induced T. brucei RNAi library generated hits targeting only the type I NTR transcript (Alsford, Baker and Horn, pers. commun.). Together, these experiments clearly implicate the trypanosomal type I NTR as a key player in the activation of nitroheterocyclic agents.

EMPIRICAL SCREENING IDENTIFIES TRYPANOCIDAL NITROHETEROCYCLICS The finding that trypanosomal infections can be treated with nifurtimox or benznidazole triggered a series of screening programmes to explore the parasite killing activities of other nitrofurans and nitroimidazoles. Due to the availability


of appropriate chemical libraries, analysis of nitroimidazoles was conducted using an empirical strategy [81-91]. In contrast, various nitroheterocycles including nitrofuran, nitrothiophene and nitrobenzamide derivatives were tested in small scale screens using compounds designed to exploit parasite specific pathways such as trypanothione homeostasis, sterol biosynthesis and purine uptake mechanisms [92101]. These approaches identified several lead structures, notably fexinidazole and megazol Fig. (3A and B) respectively), both of which are effective against T. brucei and T. cruzi [91, 102, 103], although fexinidazole never progressed past animal studies. In contrast, a concerted effort was made to determine how the trypanocidal activity of megazol is mediated. Studies clearly demonstrated that it is taken up by passive diffusion, and once in the cell, undergoes nitroreduction, with both type I and II NTR mechanisms proposed [48, 62, 104]. However, following reports that megazol displays genotoxic properties by mediating DNA damage, further trials as a trypanocidal agent were suspended [105-107]. Recently, there has been renewed interest in megazol derivatives, which are reported to show lower mutagenic properties [108-112]. A DNDi sponsored retrospective study analysing the trypanocidal activities of nitroimidazoles has resulted in a reassessment of fexinidazole as a potential treatment for African sleeping sickness (www.dndi.org). DNDi has signed an agreement with Sanofi-Aventis for the development, manufacture and distribution of this drug and clinical trials are underway. According to Executive Director Dr Bernard Pécoul, “Fexinidazole is the first compound to be advanced by DNDi all the way from discovery into clinical development, and is currently the only compound in clinical development for the treatment of sleeping sickness. Thus, this project holds great promise for the patients and the practitioners in the field.". It will be of interest to determine whether this nitroimidazole also functions as a prodrug and to assess how it undergoes activation, given that fexinidazole is reportedly not mutagenic. Intriguingly, BSF T. brucei selected on nifurtimox or fexinidazole display reciprocal cross-resistance, indicating that these two nitroheterocyclic drugs have a common activity, presumably at the level of activation [27]. TARGETING NITROHETEROCYCLIC PRODRUG ACTIVATION TO DEVELOP NEW TRYPANOCIDAL AGENTS A clear link between type I NTR and the antitrypanosomal selectivity of nitroheterocyclic prodrugs has been established [48]. To identify novel parasitic agents, we have exploited this finding and established the structure activity relationship of a range of nitroaromatic compounds, using cellular and biochemical screens. Initial studies focused on the aziridinyl nitrobenzamide (ANB) and nitrobenzylphosphoramide mustard (NBPM) prodrugs, agents originally developed for use in the two-step anti-cancer treatment GDEPT (gene directed enzyme-linked prodrug therapy) [19, 21]. In the first phase of GDEPT, a gene for a type I NTR, generally E. coli nfsB, is targeted to and expressed within a tumour cell. The second stage involves administration of the nitroheterocyclic prodrug, which is then converted to cytotoxic moieties by the bacterial enzyme. During this activation, the electron-withdrawing nitro group is converted to an


7

electron-donating hydroxylamine derivative. The subsequent rearrangement of electrons within the nitroaromatic backbone acts as an electronic switch, facilitating presentation of cytotoxic moieties within the cell (reviewed in [21]). As trypanosomes express a type I NTR, the first step of GDEPT is not required and these compounds can be used directly against the parasite. NBPMs consist of a nitrobenzyl group linked to a phosphoramide mustard moiety Fig. (3) [113, 114]. Two distinct groups of NBPMs have been generated, differing in the linkage between the nitrobenzyl and the phosphoramide mustard units [115]. In one class, the phosphoramide is part of a cyclic structure analogous to cyclophosphamide. During the NTR-mediated reduction of the nitro group to its hydroxylamine derivative, a C-O bond in the cyclophosphamide moiety is broken, leading to linearization of this ring, exposing the cytotoxic mustard Fig. (4). Biochemical screens with recombinant T. brucei NTR (TbNTR) revealed that although this class of NBPM function as substrates for the parasite enzyme, albeit poorly, they do not display activity against bloodstream form (BSF) T. brucei [115]. In the second NBPM group, the phosphoramide is part of a linear structure. It has been proposed that following NTR-mediated nitro-activation, the parental acyclic compound fragments, again through the C-O bond found in the linear phosphoramide side chain, releasing the cytotoxic mustard Fig. (4). Although the initial structure, LH7 Fig. (3), is a poor substrate for TbNTR, it is reduced at a rate similar to that of the cyclic analogues, and displays trypanocidal activities [115]. Using LH7 as lead, the biochemical and parasite killing activities of a library of acyclic NBPMs have now been evaluated [115]. This revealed that inclusion of a halogenated group at the 2-position on the phenyl ring dramatically increases the rate at which they are activated by TbNTR and significantly improves the trypanocidal properties Fig. (3C). One explanation is that these effects may be due to an enhancement of the electronic rearrangement on the aromatic ring. Prior to prodrug activation, the phenyl ring contains two electron withdrawing substituents, a nitro group and a halogen. These both vie for electrons on the heterocycle, consequently making the nitro group more prone to reduction by TbNTR. Following conversion of this nitro substituent to its hydroxylamine derivative, electrons are pushed onto the aromatic ring. This electronic switch facilitates cleavage of the benzyl C-O bond found on the phosphoramide mustard containing side chain, promoting fragmentation of this linkage and release of the cytotoxic moiety. Therefore, halogenated NBPMs are activated more readily by TbNTR than non-halogenated counterparts resulting in a potent activity. We have also carried out a similar screening strategy to evaluate whether ANBs have potential against trypanosomes. The archetypal compound of this class is CB1954 (also known as tretrazicar), an agent being trialled as part of a co-therapy targeting liver cancer, under the trade name Prolarix™ [116,117]. It consists of a 2, 4-dinitrobenzylamide ring linked at the 5-position to an aziridinyl substituent Fig. (3D). Both nitro groups are prone to reduction by bacterial type I NTR generating an equimolar mixture of 2- and 4hydroxylamine metabolites Fig. (5) [118]. These products


A O

Wilkinson et al.

B

ON+

N

O

N

O

S

C

S

N

N N

O

NH2

-O

Cl

N+ O

N

i

O O P N NH2

N+ O

O

Cl

Cl

N

O

Cl

N+

N+ O

NH2

F

O

O

-O

Cl

N

ii O-

O

O NH

O P N -O

O-

N+ O

O

N+

NH -O

ii F

O NH2

-O

i Cl

O-

N+

Cl

O

N+ O

NH2

N

D O P N

-O

ON+

O

N+ O

iii

N

iii

Fig. (3). Structure of nitroheterocyclic agents with trypanocidal activity. Compounds A and B correspond to the fexinidazole and megazol, respectively. Compounds in panel C represent three acyclic nitrobenzylphosphoramide mustards (NBPMs), including LH7 (i) and two halogenated derivatives (ii) and (iii). The latter two agents are extremely potent against BSF T. brucei [115]. The structures in panel D are aziridinyl nitrobenzamides (ANBs) corresponding to CB1954 (i) and two derivatives, (ii) and (iii). All three ANBs significantly inhibit growth of the BSF T. brucei and T. cruzi amastigotes (Bot, Hall and Wilkinson, unpublished).

-O

O N+

Cl O

O P N NH

NTR reduction

NH2 O P N O

acyclical NBPM

H O

O P N NH

spontaneous

Cl

HO

N O

Cl

hydroxylamine

O N+

H N

Cl

cyclical NBPM

-O

OH

Cl

Cl

NTR reduction

NH2 O P N O

hydroxylamine

Cl

Cl

exposed mustard

OH HN

O P N NH

Cl

OH

spontaneous H

N

O O P N NH2

Cl

Cl

Cl

released mustard

Fig. (4). Proposed activation mechanisms for nitrobenzylphosphoramide mustards. The type I NTR mediated conversion of the nitro group to the hydroxylamine derivative causes a redistribution of electrons within the aromatic ring. This promotes cleavage of a C-O bond in the phosphoramide side chain facilitating the presentation of the cytotoxic mustard (shaded region). This triggers DNA damage by acting as an alkylating agent [113, 114]. For compounds containing a cyclophosphamide (cyclical) arrangement, the structure undergoes ring opening, exposing the cytotoxic mustard. For agents where the phosphoramide substituent is part of a linear (acyclical) structure, the backbone is postulated to fragment, releasing the cytotoxic phosphoramide mustard.


Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 16 HN

NH2 O

OH O

NH2 NH2

-O

N+ O

O

N+

O-

2eO NH2

-O

N+ O

N

CB1954

9

-O

2e-

N+ O

2-amine

N

N

2-hydroxylamine O

NTR reduction

N+

O-

O NH2

2e-

4-amine

H2N N O

2e-

ON+ O NH2

NH HO

N

acetyl CoA O

4-hydroxylamine

N+

O-

O NH2

O

N-acetoxy

HN

O

N

Fig. (5). Proposed mechanisms of reductive activation of the aziridinyl dinitrobenzamide CB1954. Reduction of CB1954 can occur at either the 2- or 4-nitro position to generate the corresponding hydroxylamine and then amine derivatives. Additionally, the 4-hydroxylamine derivative can react with acetyl CoA to form the DNA cross-linking N-acetoxy species [121].

have cytotoxic activities in mammalian systems, either directly, or through the formation of downstream amine or acetoxy derivatives Fig. (5) [119-122]. In vitro analysis has shown that CB1954 functions as a substrate for both T. brucei and T. cruzi NTR and that it blocks replication of the mammalian forms of the parasites. Interestingly, only the 2hydroxylamine derivative prevents parasite growth, with the 4- product having no observable effect. When we extended these screens to other ANBs, only compounds with a 2-nitro group were found to be NTR substrates, and this translated into trypanocidal activity. Taken together, these findings confirm that the 2-nitro group has a major role in the inhibition of parasite growth. To confirm that the most potent NBPMs and ANBs are activated in vivo by the trypanosomal NTR, we evaluated the susceptibility of genetically modified parasites with altered levels of the enzyme. In all cases, BSF T. brucei with lowered levels of the NTR exhibited increased relative resistance, while parasites over expressing the reductase displayed hypersensitivity, mirroring observations with nifurtimox and benznidazole [48, 115]. The selective nature of drug activity was determined by comparative toxicity studies which showed that the most potent drugs displayed a selective index of >1200 toward the parasite [115]. Thus, the strategy of using the trypanosomal NTR to screen for nitroheterocyclic prodrug activation is a highly effective method for identifying compounds that specifically target the parasites.

age. Nifurtimox is now used against African sleeping sickness as part of the recently approved NECT and is being trialled against pediatric neuroblastoma. Benznidazole is being evaluated as a treatment for late stage Chagas disease. With the molecular mechanism by which these prodrugs selectively kill parasites having been established, it has been possible to take a more systematic approach to investigate the trypanocidal activity of other nitroheterocycles. As mammals lack type I NTR activity, they should be less susceptible to agents that act by this cytotoxic mechanism. In preliminary screens, this appears to be the case with the relative toxicities of the most potent NBPM and ANB trypanocidal agents displaying significant selectively toward the parasite. It is encouraging that by specifically targeting type I NTR with a relatively small number of compounds, a significant number (7 out of 30) have shown promise that warrants further investigation. ACKNOWLEDGEMENTS The authors would like to thank Dr Longqin Hu (Rutgers, The State University of New Jersey, USA) and Dr Nuala Helsby (The University of Auckland, New Zealand) for supplying the NBPM and ANB compounds described in this review and Dr David Horn, Ms Nicola Baker and Dr Sam Alsford (all London School of Hygiene and Tropical Medicine, UK) for providing preliminary data. This work is supported by The Welcome Trust. ABBREVIATIONS

SUMMARY

ANB

=

Aziridinyl nitrobenzamide

Nifurtimox and benznidazole have been used for nearly forty years to treat acute Chagas disease. Despite problems, trials have been undertaken to evaluate extending their us-

BSF

=

Bloodstream form

CNS

=

Central nervous system


Wilkinson et al.

CPR

=

Cytochrome P450 reductase

DNDi

=

Drugs for Neglected Diseases Initiative

[21]

FAD

=

Flavin adenine dinucleotide

[22]

FMN

=

Flavin adenine mononucleotide

GDEPT =

Gene directed enzyme-linked prodrug therapy

GI

=

Gastro-intestinal

INN

=

International nonproprietary name

[23]

NBPM =

Nitrobenzylphosphoramide mustard

[24]

NECT

=

Nifurtimox-eflornithine combinational therapy

[25]

NTR

=

Nitroreductase

RNAi

=

RNA interference

ROS

=

Reactive oxygen species

PGFS

=

Prostaglandin F2 synthase.

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

[17] [18] [19] [20]

Barrett, M. P.; Burchmore, R. J.; Stich, A.; Lazzari, J. O.; Frasch, A. C.; Cazzulo, J. J.; Krishna, S. The trypanosomiases. Lancet, 2003, 362, 1469-1480. Wilkinson, S. R.; Kelly, J. M. Trypanocidal drugs: mechanisms, resistance and new targets. Exp. Rev. Mol. Med., 2009, 11, e31. Barrett, M. P. The rise and fall of sleeping sickness. Lancet, 2006, 367, 1377-1378. Schofield, C. J.; Jannin, J.; Salvatella, R. The future of Chagas disease control. Trends Parasitol., 2006, 22, 583-588. Simarro, P. P.; Jannin, J.; Cattand, P. Eliminating human African trypanosomiasis: where do we stand and what comes next? PLoS Med., 2008, 5, e55. Steverding, D. The history of African trypanosomiasis. Parasit Vectors, 2008, 1, 3. Brun, R.; Blum, J.; Chappuis, F.; Burri, C. Human African trypanosomiasis. Lancet, 2010, 375, 148-159. Bern, C.; Montgomery, S. P.; Katz, L.; Caglioti, S.; Stramer, S. L. Chagas disease and the US blood supply. Curr. Opin. Infect. Dis., 2008, 21, 476-482. Bern, C.; Montgomery, S. P. An estimate of the burden of Chagas disease in the United States. Clin. Infect. Dis., 2009, 49, e52-54. Gascon, J.; Bern, C.; Pinazo, M. J. Chagas disease in Spain, the United States and other non-endemic countries. Acta Trop., 2010, 115, 22-27. Rassi, A. Jr.; Rassi, A.; Marin-Neto, J.A. Chagas disease. Lancet, 2010, 375, 1388-1402. Adams, C. P.; Brantner, V. V. Estimating the cost of new drug development: is it really 802 million dollars? Health Aff., (Millwood) 2006, 25, 420-428. Grunberg, E.; Titsworth, E. H. Chemotherapeutic properties of heterocyclic compounds: monocyclic compounds with fivemembered rings. Annu. Rev. Microbiol., 1973, 27, 317-346. Spain, J. C. Biodegradation of nitroaromatic compounds. Annu. Rev. Microbiol., 1995, 49, 523-555. Peres, C. M.; Agathos, S. N. Biodegradation of nitroaromatic pollutants: from pathways to remediation. Biotechnol. Annu. Rev., 2000, 6, 197-220. Rieger, P. G.; Meier, H. M.; Gerle, M.; Vogt, U.; Groth, T.; Knackmuss, H. J. Xenobiotics in the environment: present and future strategies to obviate the problem of biological persistence. J. Biotechnol., 2002, 94, 101-123. Roldan, M. D.; Perez-Reinado, E.; Castillo, F.; Moreno-Vivian, C. Reduction of polynitroaromatic compounds: the bacterial nitroreductases. FEMS Microbiol. Rev., 2008, 32, 474-500. McCalla, D. R. Mutagenicity of nitrofuran derivatives: review. Environ Mutagen, 1983, 5, 745-765. Denny, W. A. Prodrugs for gene-directed enzyme-prodrug therapy (suicide gene therapy). J. Biomed. Biotechnol., 2003, 2003, 48-70. Saulnier Sholler, G. L.; Kalkunte, S.; Greenlaw, C.; McCarten, K.; Forman, E. Antitumor activity of nifurtimox observed in a patient

[26]

[27]

[28] [29]

[30] [31]

[32] [33] [34]

[35] [36]

[37] [38]

[39]

[40]

with neuroblastoma. J. Pediatr. Hematol. Oncol., 2006, 28, 693695. Chen, Y.; Hu, L. Design of anticancer prodrugs for reductive activation. Med. Res. Rev., 2009, 29, 29-64. Stover, C. K.; Warrener, P.; VanDevanter, D. R.; Sherman, D. R.; Arain, T. M.; Langhorne, M. H.; Anderson, S. W.; Towell, J. A.; Yuan, Y.; McMurray, D. N.; Kreiswirth, B. N.; Barry, C. E.; Baker, W. R. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature, 2000, 405, 962-966. Kashanian, J.; Hakimian, P.; Blute, M. Jr.; Wong, J.; Khanna, H.; Wise, G.; Shabsigh, R. Nitrofurantoin: the return of an old friend in the wake of growing resistance. B4U Int., 2008, 102, 1634-1637. Arya, S. C.; Agarwal, N. Nitrofurantoin: the return of an old friend in the wake of growing resistance. BJU Int., 2009, 103, 994-995. Manjunatha, U.H.; Boshoff, H.; Dowd, C. S.; Zhang, L.; Albert, T. J.; Norton, J. E,; Daniels, L.; Dick, T.; Pang, S. S.; Barry, C. E. Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA, 2006, 103, 431-436. Singh, R.; Manjunatha, U.; Boshoff, H. I. M.; Ha, Y. H.; Niyomrattanakit, P.; Ledwidge, R.; Dowd, C. S.; Lee, I. Y.; Kim, P.; Zhang, L.; Kang, S.; Keller, T. H.; Jiricek, J.; Barry, C. E. PA-824 kills nonreplicating mycobacterium tuberculosis by intracellular no release. Science, 2008, 322, 1392-1395 Sokolova, A. Y.; Wyllie, S.; Patterson, S.; Oza, S. L.; Read, K. D.; Fairlamb, A. H. Cross-resistance to nitro drugs and implications for treatment of human African trypanosomiasis. Antimicrob. Agents Chemother., 2010, 54, 2893-2900. Evens, F.; Niemegeers, K.; Packchanian, A. Nitrofurazone therapy of Trypanosoma gambiense sleeping sickness in man. Am. J. Trop. Med. Hyg., 1957, 6, 665-678. Packchanian, A. Chemotherapy of African sleeping sickness. I. Chemotherapy of experimental Trypanosoma gambiense infection in mice (Mus musculus) with nitrofurazone. Am. J. Trop. Med. Hyg., 1955, 4, 705-711. Rodriques Coura, J.; de Castro, S. L. A critical review on Chagas disease chemotherapy. Mem Inst. Oswaldo Cruz, 2002, 97, 3-24. Grunberg, E.; Beskid, G.; Cleeland, R.; DeLorenzo, W. F.; Titsworth, E.; Scholer, H. J.; Richle, R.; Brener, Z. Antiprotozoan and antibacterial activity of 2-nitroimidazole derivatives. Antimicrob. Agents Chemother., (Bethesda) 1967, 7, 513-519. Bock, M.; Gonnert, R.; Haberkorn, A. Studies with Bay 2502 on animals. Bol. Chil. Parasitol., 1969, 24, 13-19. Ferreira, R. C.; Ferreira, L. C. Mutagenicity of nifurtimox and benznidazole in the Salmonella/microsome assay. Braz J. Med. Biol. Res., 1986, 19, 19-25. Alejandre-Duran, E.; Claramunt, R. M.; Sanz, D.; Vilaplana, M. J.; Molina, P.; Pueyo, C. Study on the mutagenicity of nifurtimox and eight derivatives with the L-arabinose resistance test of Salmonella typhimurium. Mutat. Res., 1988, 206, 193-200. Moraga, A. A.; Graf, U. Genotoxicity testing of antiparasitic nitrofurans in the Drosophila wing somatic mutation and recombination test. Mutagenesis, 1989, 4, 105-110. Priotto, G.; Fogg, C.; Balasegaram, M.; Erphas, O.; Louga, A.; Checchi, F.; Ghabri, S.; Piola, P. Three drug combinations for latestage Trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in Uganda. PLoS Clin. Trials, 2006, 1, e39. Checchi, F.; Piola, P.; Ayikoru, H.; Thomas, F.; Legros, D.; Priotto, G. Nifurtimox plus eflornithine for late-stage sleeping sickness in uganda: A case series. PLoS Negl. Trop. Dis., 2007, 1, e64. Priotto, G.; Kasparian, S.; Ngouama, D.; Ghorashian, S.; Arnold, U.; Ghabri, S.; Karunakara, U. Nifurtimox-eflornithine combination therapy for second-stage Trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in Congo. Clin. Infect. Dis., 2007, 45, 1435-1442. Priotto, G.; Kasparian, S.; Mutombo, W.; Ngouama, D.; Ghorashian, S.; Arnold, U.; Ghabri, S.; Baudin, E.; Buard, V.; Kazadi-Kyanza, S.; Ilunga, M.; Mutangala, W.; Pohlig, G.; Schmid, C.; Karunakara, U.; Torreele, E.; Kande, V. Nifurtimoxeflornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. Lancet, 2009, 374, 56-64. Saulnier Sholler, G. L.; Brard, L.; Straub, J. A.; Dorf, L.; Illeyne, S.; Koto, K.; Kalkunte, S.; Bosenberg, M.; Ashikaga, T.; Nishi, R. Nifurtimox induces apoptosis of neuroblastoma cells in vitro and in vivo. J. Pediatr. Hematol. Oncol., 2009, 31, 187-193.

Trypanocidal Activity of Nitroaromatic Prodrugs [41]

[42]

[43]

[44]

[45] [46]

[47]

[48]

[49]

[50] [51] [52]

[53]

[54] [55] [56]

[57] [58]

[59] [60]

Viotti, R.; Vigliano, C.; Armenti, H.; Segura, E. Treatment of chronic Chagas' disease with benznidazole: clinical and serologic evolution of patients with long-term follow-up. Am. Heart J., 1994, 127, 151-162. Reyes, P. A.; Vallejo, M. Trypanocidal drugs for late stage, symptomatic Chagas disease (Trypanosoma cruzi infection). Cochrane Database Syst Rev., 2005, CD004102. Available from http://www.cochrane.org/reviews/en/ab004102.html Perez-Molina, J. A.; Perez-Ayala, A.; Moreno, S.; FernandezGonzalez, M. C.; Zamora, J.; Lopez-Velez, R. Use of benznidazole to treat chronic Chagas' disease: a systematic review with a metaanalysis. J. Antimicrob. Chemother., 2009, 64, 1139-1147. Marin-Neto, J. A.; Rassi, A. Jr.; Avezum, A. Jr.; Mattos, A. C.; Rassi, A.; Morillo, C. A.; Sosa-Estani, S.; Yusuf, S. The BENEFIT trial: testing the hypothesis that trypanocidal therapy is beneficial for patients with chronic Chagas heart disease. Mem Inst. Oswaldo Cruz, 2009, 104 (Suppl 1), 319-324. Peterson, F. J.; Mason, R. P.; Hovsepian, J.; Holtzman, J. L. Oxygen-sensitive and -insensitive nitroreduction by Escherichia coli and rat hepatic microsomes. J. Biol. Chem., 1979, 254, 4009-4014. Nixon, J. E.; Wang, A.; Field, J.; Morrison, H. G.; McArthur, A. G.; Sogin, M. L.; Loftus, B. J.; Samuelson, J. Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica. Eukaryot Cell, 2002, 1, 181-190. Muller, J.; Wastling, J.; Sanderson, S.; Muller, N.; Hemphill, A. A novel Giardia lamblia nitroreductase, GlNR1, interacts with nitazoxanide and other thiazolides. Antimicrob. Agents Chemother., 2007, 51, 1979-1986. Wilkinson, S. R.; Taylor, M. C.; Horn, D.; Kelly, J. M.; Cheeseman, I. A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes. Proc. Natl. Acad. Sci. USA, 2008, 105, 5022-5027. Pal, D.; Banerjee, S.; Cui, J.; Schwartz, A.; Ghosh, S. K.; Samuelson, J. Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases). Antimicrob. Agents Chemother., 2009, 53, 458-464. McCalla, D. R.; Reuvers, A.; Kaiser, C. Breakage of bacterial DNA by nitrofuran derivatives. Cancer Res., 1971, 31, 2184-2188. Streeter, A. J.; Hoener, B. A. Evidence for the involvement of a nitrenium ion in the covalent binding of nitrofurazone to DNA. Pharm. Res., 1988, 5, 434-436. Gavin, J. J.; Ebetino, F. F.; Freedman, R.; Waterbury, W. E. The aerobic degradation of 1-(5-nitrofurfurylideneamino)-2imidazolidinone (NF-246) by Escherichia coli. Arch. Biochem. Biophys., 1966, 113, 399-404. Panicucci, R.; McClelland, R.A. 4,5-dihydro-4,5dihydroxylimidazoles as products of the reduction of 2nitroimidazoles, HPLC assay and demonstration of equilibrium transfer of glyoxal to guanine. Can. J. Chem., 1989, 67, 2128-2135. Whitmore, G. F.; Varghese, A. J. The biological properties of reduced nitroheterocyclics and possible underlying biochemical mechanisms. Biochem. Pharmacol., 1986, 35, 97-103. Whitmore, G. F.; Varghese, A. J.; Gulyas, S. Reaction of 2nitroimidazole metabolites with guanine and possible biological consequences. IARC Sci. Publ., 1986, (70), 185-196. Mason, R. P.; Holtzman, J. L. The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates. Biochemistry, 1975, 14, 1626-1632. Docampo, R.; Stoppani, A. O. Generation of superoxide anion and hydrogen peroxide induced by nifurtimox in Trypanosoma cruzi. Arch. Biochem. Biophys., 1979, 197, 317-321. Moreno, S. N.; Mason, R. P.; Muniz, R. P.; Cruz, F. S.; Docampo, R. Generation of free radicals from metronidazole and other nitroimidazoles by Tritrichomonas foetus. J. Biol. Chem., 1983, 258, 4051-4054. Docampo, R.; Mason, R. P.; Mottley, C.; Muniz, R. P. Generation of free radicals induced by nifurtimox in mammalian tissues. J. Biol. Chem., 1981, 256, 10930-10933. Giulivi, C.; Turrens, J. F.; Boveris, A. Chemiluminescence enhancement by trypanocidal drugs and by inhibitors of antioxidant enzymes in Trypanosoma cruzi. Mol. Biochem. Parasitol., 1988, 30, 243-251.

Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 16 [61] [62] [63]

[64]

[65] [66] [67] [68]

[69] [70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78] [79]

[80]

11

Docampo, R. Sensitivity of parasites to free radical damage by antiparasitic drugs. Chem. Biol. Interact, 1990, 73, 1-27. Viode, C.; Bettache, N.; Cenas, N.; Krauth-Siegel, R. L.; Chauviere, G.; Bakalara, N.; Perie, J. Enzymatic reduction studies of nitroheterocycles. Biochem. Pharmacol., 1999, 57, 549-557. Henderson, G. B.; Ulrich, P.; Fairlamb, A. H.; Rosenberg, I.; Pereira, M.; Sela, M.; Cerami, A. "Subversive" substrates for the enzyme trypanothione disulfide reductase: alternative approach to chemotherapy of Chagas disease. Proc. Natl. Acad. Sci. USA, 1988, 85, 5374-5378. Blumenstiel, K.; Schoneck, R.; Yardley, V.; Croft, S. L.; KrauthSiegel, R. L. Nitrofuran drugs as common subversive substrates of Trypanosoma cruzi lipoamide dehydrogenase and trypanothione reductase. Biochem. Pharmacol., 1999, 58, 1791-1799. Boveris, A.; Sies, H.; Martino, E. E.; Docampo, R.; Turrens, J. F.; Stoppani, A. O. Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi. Biochem. J., 1980, 188, 643-648. Fairlamb, A. H.; Cerami, A. Metabolism and functions of trypanothione in the Kinetoplastida. Annu. Rev. Microbiol., 1992, 46, 695-729. Flohe, L.; Hecht, H. J.; Steinert, P. Glutathione and trypanothione in parasitic hydroperoxide metabolism. Free Radic. Biol. Med., 1999, 27, 966-984. Wilkinson, S. R.; Obado, S. O.; Mauricio, I. L.; Kelly, J. M. Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA, 2002, 99, 13453-13458. Wilkinson, S. R.; Kelly, J. M. The role of glutathione peroxidases in trypanosomatids. Biol. Chem., 2003, 384, 517-525. Irigoin, F.; Cibils, L.; Comini, M. A.; Wilkinson, S. R.; Flohe, L.; Radi, R. Insights into the redox biology of Trypanosoma cruzi: Trypanothione metabolism and oxidant detoxification. Free Radic. Biol. Med., 2008, 45, 733-742. Moreno, S. N.; Docampo, R.; Mason, R. P.; Leon, W.; Stoppani, A. O. Different behaviors of benznidazole as free radical generator with mammalian and Trypanosoma cruzi microsomal preparations. Arch. Biochem. Biophys., 1982, 218, 585-591. Prathalingham, S. R.; Wilkinson, S. R.; Horn, D.; Kelly, J. M. Deletion of the Trypanosoma brucei superoxide dismutase gene sodb1 increases sensitivity to nifurtimox and benznidazole. Antimicrob. Agents Chemother., 2007, 51, 755-758. Kelly, J. M.; Taylor, M. C.; Smith, K.; Hunter, K. J.; Fairlamb, A. H. Phenotype of recombinant Leishmania donovani and Trypanosoma cruzi which over-express trypanothione reductase. Sensitivity towards agents that are thought to induce oxidative stress. Eur. J. Biochem., 1993, 218, 29-37. Wilkinson, S. R.; Temperton, N. J.; Mondragon, A.; Kelly, J. M. Distinct mitochondrial and cytosolic enzymes mediate trypanothione-dependent peroxide metabolism in Trypanosoma cruzi. J. Biol. Chem., 2000, 275, 8220-8225. Wilkinson, S. R.; Meyer, D. J.; Taylor, M. C.; Bromley, E. V.; Miles, M. A.; Kelly, J. M. The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin. J. Biol. Chem., 2002, 277, 17062-17071. Wilkinson, S. R.; Horn, D.; Prathalingam, S. R.; Kelly, J. M. RNA interference identifies two hydroperoxide metabolizing enzymes that are essential to the bloodstream form of the african trypanosome. J. Biol. Chem., 2003, 278, 31640-31646. Wilkinson, S. R.; Prathalingam, S. R.; Taylor, M. C.; Ahmed, A.; Horn, D.; Kelly, J. M. Functional characterisation of the iron superoxide dismutase gene repertoire in Trypanosoma brucei. Free Radic. Biol. Med., 2006, 40, 198-209. McCalla, D. R.; Kaiser, C.; Green, M. H. Genetics of nitrofurazone resistance in Escherichia coli. J. Bacteriol., 1978, 133, 10-16. Whiteway, J.; Koziarz, P.; Veall, J.; Sandhu, N.; Kumar, P.; Hoecher, B.; Lambert, I. B. Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli. J. Bacteriol., 1998, 180, 5529-5539. Kubata, B. K.; Kabututu, Z.; Nozaki, T.; Munday, C. J.; Fukuzumi, S.; Ohkubo, K.; Lazarus, M.; Maruyama, T.; Martin, S. K.; Duszenko, M.; Urade, Y. A key role for old yellow enzyme in the metabolism of drugs by Trypanosoma cruzi. J. Exp. Med., 2002, 196, 1241-1251.

12 Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 16 [81] [82] [83]

[84] [85]

[86] [87] [88] [89] [90] [91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

De Carneri, I. Antiprotozoan activity of nitroimidazoles. Arzneimittelforschung, 1969, 19, 382-386. Ross, W. J.; Jamieson, W. B.; McCowen, M. C. Antiparasitic nitroimidazoles. 1. Some 2-styryl-5-nitroimidazoles. J. Med. Chem., 1972, 15, 1035-1040. Ross, W. J.; Jamieson, W. B.; McCowen, M. C. Antiparasitic nitroimidazoles. 3. Synthesis of 2-(4-carboxystyryl)-5-nitro-1vinylimidazole and related compounds. J. Med. Chem., 1973, 16, 347-352. Ross, W. J.; Todd, A. Antiparasitic nitroimidazoles. 7. Some 4- and 5-styrylnitroimidazoles. J. Med. Chem., 1973, 16, 863-865. Ross, W. J.; Jamieson, W. B. Antiparasitic nitroimidazoles. 9. Synthesis of some 2-(4-dialkylaminomethylstyryl)-and 2-(4amidinostyryl)5-nitro-1-vinylimidazoles. J. Med. Chem., 1975, 18, 430-432. Ross, W. J.; Jamieson, W. B. Antiparasitic nitroimidazoles. 8. Derivatives of 2-(4-formylstyryl)-5-nitro-1-vinylimidazole. J. Med. Chem., 1975, 18, 158-161. Winkelmann, E.; Raether, W.; Gebert, U.; Sinharay, A. Chemotherapeutically active nitro compounds. 4. 5-Nitroimidazoles (Part I). Arzneimittelforschung, 1977, 27, 2251-2263. Winkelmann, E.; Raether, W.; Gebert, U. Chemotherapeutically active nitro-derivatives. 4. 5-Nitroimidazoles (Part IV). Arzneimittelforschung, 1978, 28, 1682-1684. Winkelmann, E.; Raether, W.; Sinharay, A. Chemotherapeutically active nitro compounds. 4.5-Nitroimidazoles (Part II). Arzneimittelforschung, 1978, 28, 351-366. Winkelmann, E.; Raether, W. Chemotherapeutically acitve nitro compounds. 4. 5-Nitroimidazoles (Part III). Arzneimittelforschung, 1978, 28, 739-749. Raether, W.; Seidenath, H. The activity of fexinidazole (HOE 239) against experimental infections with Trypanosoma cruzi, trichomonads and Entamoeba histolytica. Ann. Trop. Med. Parasitol., 1983, 77, 13-26. Cerecetto, H.; Di Maio, R.; Ibarruri, G.; Seoane, G.; Denicola, A.; Peluffo, G.; Quijano, C.; Paulino, M. Synthesis and antitrypanosomal activity of novel 5-nitro-2-furaldehyde and 5nitrothiophene-2-carboxaldehyde semicarbazone derivatives. Farmaco, 1998, 53, 89-94. Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Risso, M.; Sagrera, G.; Seoane, G.; Denicola, A.; Peluffo, G.; Quijano, C.; Stoppani, A. O.; Paulino, M.; Olea-Azar, C.; Basombrio, M. A. Synthesis and antitrypanosomal evaluation of E-isomers of 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives. structure-activity relationships. Eur. J. Med. Chem., 2000, 35, 343350. Aguirre, G.; Cabrera, E.; Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Seoane, G.; Duffaut, A.; Denicola, A.; Gil, M. J.; Martinez-Merino, V. Design, synthesis and biological evaluation of new potent 5nitrofuryl derivatives as anti-Trypanosoma cruzi agents. Studies of trypanothione binding site of trypanothione reductase as target for rational design. Eur. J. Med. Chem., 2004, 39, 421-431. Stewart, M. L.; Bueno, G. J.; Baliani, A.; Klenke, B.; Brun, R.; Brock, J. M.; Gilbert, I. H.; Barrett, M. P. Trypanocidal activity of melamine-based nitroheterocycles. Antimicrob. Agents Chemother., 2004, 48, 1733-1738. Baliani, A.; Bueno, G. J.; Stewart, M. L.; Yardley, V.; Brun, R.; Barrett, M. P.; Gilbert, I. H. Design and synthesis of a series of melamine-based nitroheterocycles with activity against Trypanosomatid parasites. J. Med. Chem., 2005, 48, 5570-5579. Aguirre, G.; Boiani, M.; Cabrera, E.; Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Denicola, A.; Sant'anna, C. M.; Barreiro, E. J. New potent 5-nitrofuryl derivatives as inhibitors of Trypanosoma cruzi growth. 3D-QSAR (CoMFA) studies. Eur. J. Med. Chem., 2006, 41, 457-466. Gerpe, A.; Odreman-Nunez, I.; Draper, P.; Boiani, L.; Urbina, J. A.; Gonzalez, M.; Cerecetto, H. Heteroallyl-containing 5nitrofuranes as new anti-Trypanosoma cruzi agents with a dual mechanism of action. Bioorg. Med. Chem., 2008, 16, 569-577. Gerpe, A.; Alvarez, G.; Benitez, D.; Boiani, L.; Quiroga, M.; Hernandez, P.; Sortino, M.; Zacchino, S.; Gonzalez, M.; Cerecetto, H. 5-Nitrofuranes and 5-nitrothiophenes with anti-Trypanosoma cruzi activity and ability to accumulate squalene. Bioorg. Med. Chem., 2009, 17, 7500-7509. Baliani, A.; Peal, V.; Gros, L.; Brun, R.; Kaiser, M.; Barrett, M. P.; Gilbert, I. H. Novel functionalized melamine-based nitroheterocy-

Wilkinson et al.

[101]

[102] [103]

[104] [105]

[106]

[107] [108]

[109]

[110]

[111]

[112]

[113]

[114]

[115] [116]

[117]

[118]

cles: synthesis and activity against trypanosomatid parasites. Org. Biomol. Chem., 2009, 7, 1154-1166. Hwang, J. Y.; Smithson, D.; Connelly, M.; Maier, J.; Zhu, F.; Guy, K. R. Discovery of halo-nitrobenzamides with potential application against human African trypanosomiasis. Bioorg. Med. Chem. Lett., 2010, 20, 149-152. Filardi, L. S.; Brener, Z. A nitroimidazole-thiadiazole derivative with curative action in experimental Trypanosoma cruzi infections. Ann. Trop. Med. Parasitol., 1982, 76, 293-297. Bouteille, B.; Marie-Daragon, A.; Chauviere, G.; de Albuquerque, C.; Enanga, B.; Darde, M. L.; Vallat, J. M.; Perie, J.; Dumas, M. Effect of megazol on Trypanosoma brucei brucei acute and subacute infections in Swiss mice. Acta Trop., 1995, 60, 73-80. Barrett, M. P.; Fairlamb, A. H.; Rousseau, B.; Chauviere, G.; Perie, J. Uptake of the nitroimidazole drug megazol by African trypanosomes. Biochem. Pharmacol., 2000, 59, 615-620. Poli, P.; Aline de Mello, M.; Buschini, A.; Mortara, R. A.; Northfleet de Albuquerque, C.; da Silva, S.; Rossi, C.; Zucchi, T. M. Cytotoxic and genotoxic effects of megazol, an anti-Chagas' disease drug, assessed by different short-term tests. Biochem. Pharmacol., 2002, 64, 1617-1627. Enanga, B.; Ariyanayagam, M. R.; Stewart, M. L.; Barrett, M. P. Activity of megazol, a trypanocidal nitroimidazole, is associated with DNA damage. Antimicrob. Agents, Chemother., 2003, 47, 3368-3370. Nesslany, F.; Brugier, S.; Mouries, M. A.; Le Curieux, F.; Marzin, D. In vitro and in vivo chromosomal aberrations induced by megazol. Mutat. Res., 2004, 560, 147-158. Chauviere, G.; Bouteille, B.; Enanga, B.; de Albuquerque, C.; Croft, S. L.; Dumas, M.; Perie, J. Synthesis and biological activity of nitro heterocycles analogous to megazol, a trypanocidal lead. J. Med. Chem., 2003, 46, 427-440. Carvalho, S. A.; da Silva, E. F.; Santa-Rita, R. M.; de Castro, S. L.; Fraga, C. A. Synthesis and antitrypanosomal profile of new functionalized 1,3,4-thiadiazole-2-arylhydrazone derivatives, designed as non-mutagenic megazol analogues. Bioorg. Med. Chem. Lett., 2004, 14, 5967-5970. Carvalho, A. S.; Menna-Barreto, R. F.; Romeiro, N. C.; de Castro, S. L.; Boechat, N. Design, synthesis and activity against Trypanosoma cruzi of azaheterocyclic analogs of megazol. Med. Chem., 2007, 3, 460-465. Carvalho, S. A.; Lopes, F. A.; Salomao, K.; Romeiro, N. C.; Wardell, S. M.; de Castro, S. L.; da Silva, E. F.; Fraga, C. A. Studies toward the structural optimization of new brazilizone-related trypanocidal 1,3,4-thiadiazole-2-arylhydrazone derivatives. Bioorg. Med. Chem., 2008, 16, 413-421. Salomao, K.; de Souza, E. M.; Carvalho, S. A.; da Silva, E. F.; Fraga, C. A.; Barbosa, H. S.; de Castro, S. L. In vitro and in vivo activity of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol on Trypanosoma cruzi. Antimicrob. Agents Chemother., 2010, 54, 2023-2031. Hu, L.; Yu, C.; Jiang, Y.; Han, J.; Li, Z.; Browne, P.; Race, P. R.; Knox, R. J.; Searle, P. F.; Hyde, E. I. Nitroaryl phosphoramides as novel prodrugs for E. coli nitroreductase activation in enzyme prodrug therapy. J. Med. Chem., 2003, 46, 4818-4821. Jiang, Y.; Han, J.; Yu, C.; Vass, S. O.; Searle, P. F.; Browne, P.; Knox, R. J.; Hu, L. Design, synthesis, and biological evaluation of cyclic and acyclic nitrobenzylphosphoramide mustards for E. coli nitroreductase activation. J. Med. Chem., 2006, 49, 4333-4343. Hall, B. S.; Wu, X.; Hu, L.; Wilkinson, S. R. Exploiting the drugactivating properties of a novel trypanosomal nitroreductase. Antimicrob. Agents Chemother., 2010, 54, 1193-1199. Chung-Faye, G.; Palmer, D.; Anderson, D.; Clark, J.; Downes, M.; Baddeley, J.; Hussain, S.; Murray, P. I.; Searle, P.; Seymour, L.; Harris, P. A.; Ferry, D.; Kerr, D. J. Virus-directed, enzyme prodrug therapy with nitroimidazole reductase: a phase I and pharmacokinetic study of its prodrug, CB1954. Clin. Cancer Res., 2001, 7, 2662-2668. Patel, P.; Young, J. G.; Mautner, V.; Ashdown, D.; Bonney, S.; Pineda, R. G.; Collins, S. I.; Searle, P. F.; Hull, D.; Peers, E.; Chester, J.; Wallace, D. M.; Doherty, A.; Leung, H.; Young, L. S.; James, N. D. A phase I/II clinical trial in localized prostate cancer of an adenovirus expressing nitroreductase with CB1954 [correction of CB1984]. Mol. Ther., 2009, 17, 1292-1299. Knox, R. J.; Friedlos, F.; Sherwood, R. F.; Melton, R. G.; Anlezark, G. M. The bioactivation of 5-(aziridin-1-yl)-2,4-


[119]

[120]

[121]

[122] [123] [124]

[125] [126]


dinitrobenzamide (CB1954)--II. A comparison of an Escherichia coli nitroreductase and Walker DT diaphorase. Biochem. Pharmacol., 1992, 44, 2297-2301. Knox, R. J.; Friedlos, F.; Biggs, P. J.; Flitter, W. D.; Gaskell, M.; Goddard, P.; Davies, L.; Jarman, M. Identification, synthesis and properties of 5-(aziridin-1-yl)-2-nitro-4-nitrosobenzamide, a novel DNA crosslinking agent derived from CB1954. Biochem. Pharmacol., 1993, 46, 797-803. Helsby, N. A.; Wheeler, S. J.; Pruijn, F. B.; Palmer, B. D.; Yang, S.; Denny, W. A.; Wilson, W. R. Effect of nitroreduction on the alkylating reactivity and cytotoxicity of the 2,4-dinitrobenzamide-5aziridine CB 1954 and the corresponding nitrogen mustard SN 23862: distinct mechanisms of bioreductive activation. Chem. Res. Toxicol., 2003, 16, 469-478. Helsby, N. A.; Ferry, D. M.; Patterson, A. V.; Pullen, S. M.; Wilson, W. R. 2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT. Br. J. Cancer, 2004, 90, 1084-1092. Tang, M. H.; Helsby, N. A.; Wilson, W. R.; Tingle, M. D. Aerobic 2- and 4-nitroreduction of CB 1954 by human liver. Toxicology, 2005, 216, 129-139. Barrett, S. V.; Barrett, M. P. Anti-sleeping sickness drugs and cancer chemotherapy. Parasitol. Today, 2000, 16, 7-9. Legros, D.; Ollivier, G.; Gastellu-Etchegorry, M.; Paquet, C.; Burri, C.; Jannin, J.; Buscher, P. Treatment of human African trypanosomiasis--present situation and needs for research and development. Lancet Infect. Dis., 2002, 2, 437-440. Docampo, R.; Moreno, S. N. Current chemotherapy of human African trypanosomiasis. Parasitol. Res., 2003, 90 (Supp 1), S1013. Nok, A. J. Arsenicals (melarsoprol), pentamidine and suramin in the treatment of human African trypanosomiasis. Parasitol. Res., 2003, 90, 71-79.

Received: May 10, 2010

Accepted: June 30, 2010

[127] [128] [129] [130]

[131] [132] [133]

[134]

[135]

13

Filardi, L. S.; Brener, Z. Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease. Trans. R Soc. Trop. Med. Hyg., 1987, 81, 755-759. Castro, J. A.; Diaz de Toranzo, E. G. Toxic effects of nifurtimox and benznidazole, two drugs used against American trypanosomiasis (Chagas' disease). Biomed. Environ. Sci., 1988, 1, 19-33. Ferreira, R. C.; Schwarz, U.; Ferreira, L. C. Activation of antiTrypanosoma cruzi drugs to genotoxic metabolites promoted by mammalian microsomal enzymes. Mutat. Res., 1988, 204, 577-583. Gorla, N. B.; Ledesma, O. S.; Barbieri, G. P.; Larripa, I. B. Thirteenfold increase of chromosomal aberrations non-randomly distributed in chagasic children treated with nifurtimox. Mutat. Res., 1989, 224, 263-267. Teixeira, A. R.; Cordoba, J. C.; Souto Maior, I.; Solorzano, E. Chagas' disease: lymphoma growth in rabbits treated with Benznidazole. Am. J. Trop. Med Hyg., 1990, 43, 146-158. Teixeira, A. R.; Calixto, M. A.; Teixeira, M. L. Chagas' disease: carcinogenic activity of the antitrypanosomal nitroarenes in mice. Mutat. Res., 1994, 305, 189-196. Murta, S. M.; Gazzinelli, R. T.; Brener, Z.; Romanha, A. J. Molecular characterization of susceptible and naturally resistant strains of Trypanosoma cruzi to benznidazole and nifurtimox. Mol. Biochem. Parasitol., 1998, 93, 203-214. Iatropoulos, M. J.; Wang, C. X.; von Keutz, E.; Williams, G. M. Assessment of chronic toxicity and carcinogenicity in an accelerated cancer bioassay in rats of Nifurtimox, an antitrypanosomiasis drug. Exp. Toxicol. Pathol., 2006, 57, 397-404. Castro, J. A.; de Mecca, M. M.; Bartel, L. C. Toxic side effects of drugs used to treat Chagas' disease (American trypanosomiasis). Hum. Exp. Toxicol., 2006, 25, 471-479.