An iterative process produces oxamniquine derivatives that kill the major species of schistosomes infecting humans.

Currently there is only one method of treatment for human schistosomiasis, the drug praziquantel. Strong selective pressure has caused a serious concern for a rise in resistance to praziquantel leading to the necessity for additional pharmaceuticals, with a distinctly different mechanism of action, to be used in combination therapy with praziquantel. Previous treatment of Schistosoma mansoni included the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The oxamniquine activating enzyme was identified as a S. mansoni sulfotransferase (SmSULT-OR). Structural data have allowed for directed drug development in reengineering oxamniquine to be effective against S. haematobium and S. japonicum. Guided by data from X-ray crystallographic studies and Schistosoma worm killing assays on oxamniquine, our structure-based drug design approach produced a robust SAR program that tested over 300 derivatives and identified several new lead compounds with effective worm killing in vitro. Previous studies resulted in the discovery of compound CIDD-0066790, which demonstrated broad-species activity in killing of schistosome species. As these compounds are racemic mixtures, we tested and demonstrate that the R enantiomer CIDD-007229 kills S. mansoni, S. haematobium and S. japonicum better than the parent drug (CIDD-0066790). The search for derivatives that kill better than CIDD-0066790 has resulted in a derivative (CIDD- 149830) that kills 100% of S. mansoni, S. haematobium and S. japonicum adult worms within 7 days. We hypothesize that the difference in activation and thus killing by the derivatives is due to the ability of the derivative to fit in the binding pocket of each sulfotransferase (SmSULT-OR, ShSULT-OR, SjSULT-OR) and to be efficiently sulfated. The purpose of this research is to develop a second drug to be used in conjunction with praziquantel to treat the major human species of Schistosoma. Collectively, our findings show that CIDD-00149830 and CIDD-0072229 are promising novel drugs for the treatment of human schistosomiasis and strongly support further development and in vivo testing.

Molecular basis for hycanthone drug action in schistosome parasites.

Hycanthone (HYC) is a retired drug formerly used to treat schistosomiasis caused by infection from Schistosoma mansoni and S. haematobium. Resistance to HYC was first observed in S. mansoni laboratory strains and in patients in the 1970s and the use of this drug was subsequently discontinued with the substitution of praziquantel (PZQ) as the single antischistosomal drug in the worldwide formulary. In endemic regions, multiple organizations have partnered with the World Health Organization to deliver PZQ for morbidity control and prevention. While the monotherapy reduces the disease burden, additional drugs are needed to use in combination with PZQ to stay ahead of potential drug resistance. HYC will not be reintroduced into the schistosomiasis drug formulary as a combination drug because it was shown to have adverse properties including mutagenic, teratogenic and carcinogenic activities. Oxamniquine (OXA) was used to treat S. mansoni infection in Brazil during the brief period of HYC use, until the 1990s. Its antischistosomal efficacy has been shown to work through the same mechanism as HYC and it does not possess the undesirable properties linked to HYC. OXA demonstrates cross-resistance in Schistosoma strains with HYC resistance and both are prodrugs requiring metabolic activation in the worm to toxic sulfated forms. The target activating enzyme has been identified as a sulfotransferase enzyme and is currently used as the basis for a structure-guided drug design program. Here, we characterize the sulfotransferases from S. mansoni and S. haematobium in complexes with HYC to compare and contrast with OXA-bound sulfotransferase crystal structures. Although HYC is discontinued for antischistosomal treatment, it can serve as a resource for design of derivative compounds without contraindication.

Ferrocenyl, Ruthenocenyl, and Benzyl Oxamniquine Derivatives with Cross-Species Activity against Schistosoma mansoni and Schistosoma haematobium.

Schistosomiasis is a parasitic disease that affects more than 250 million people annually, mostly children in poor, tropical, rural areas. Only one treatment (praziquantel) is available, putting control efforts at risk should resistance occur. In pursuit of treatment alternatives, we derivatized an old antischistosomal agent, oxamniquine (OXA). Four organometallic derivatives of OXA were synthesized and tested against Schistosoma mansoni in vitro and in vivo. Of these, a ferrocenyl derivative, 1, killed larval and adult worms 24 h postexposure in vitro, in contrast to OXA, which lacks in vitro activity against adult worms. A dose of 200 mg/kg of 1 completely eliminated the worm burden in mice. Subsequently, a ruthenocenyl (5) and a benzyl derivative (6) of OXA were synthesized to probe the importance of the ferrocenyl group in 1. Compounds 1, 5, and 6 were lethal to both S. mansoni and S. haematobium adults in vitro. In vivo, at 100 mg/kg, all three compounds revealed S. mansoni worm burden reductions of 76 to 93%, commensurate with OXA. Our findings present three compounds with activity against S. mansoni in vitro, comparable activity in vivo, and high activity against S. haematobium in vitro. These compounds may possess a different binding mode or mode of action compared to OXA and present excellent starting points for further SAR studies.

Synthesis and evaluation of new oxamniquine derivatives.

Oxamniquine derivatives were synthesized and evaluated as novel schistosomicide agents. Oxamniquine (1,2,3,4-tetrahydro-2-[[(1-methylethyl)amino]methyl]-7-nitro-6-quinolinemethanol) was submitted to the Mannich reaction, using formaldehyde, paraformaldehyde and acetaldehyde as reagents, and gave three unexpected products: two of them were cyclized on the alkylamine side chain and another etherified on the aminequinolinemethanol group. The three compounds were biologically evaluated using mice infected with Schistosoma mansoni and showed promising activities, but had higher toxicities. For studies on structure-activity relationships, results demonstrate that the side alkylamine group can be modified with preserved activity, but that this modification is associated with increased toxicity.

Action of new organometallic complexes against Leishmania donovani.

The action of 16 newly synthesized metal complexes having the general structure cis-Pt-(II)-Xn-Ln have been tested in vitro against the promastigote forms of Leishmania donovani. The metal complexes at 24 h and maximum dosages inhibited growth from 0%, e.g. in cis-Pt-nifurtimox, to 100%, e.g. in cis-Pt-(2,3,4,5,6-pentafluoroaniline)2Br2 or cis-Pt-pentamidine-I2. A study of the cytotoxicty of these latter complexes on the phagocytic cell line J-774 showed neither high cytotoxicity nor cytolysis. At the maximum dosage after 24 h of permanent contact with the cells (extreme, non-physiological conditions), cytolysis did not exceed 30%. For most of the compounds, cytolysis ranged from 0%, for cis-Pt-oxamniquine-Cl2 to 27.7%, for cis-Pt-pentamidine-I2. The compound cis-Pt-(2,3,4,5,6-pentafluoroaniline)2-Br2 caused up to 1.4% cytolysis under the above conditions. Parasites exposed to cis-Pt-pentamidine-I2 showed notably reduced DNA, RNA and protein synthesis, unlike those exposed to other compounds. Parasites examined by electron microscopy showed effects mainly on the nucleus, though in some cases the mitochondria were affected, altering the internal membranes of the cytoplasmic organelles. The in-vivo activity of the complex cis-Pt-guanethidine-Cl2 was evaluated in parasitized Wistar rats, in which the number of amastigotes per gram of spleen was reduced by 75% compared with controls.

Preliminary investigations of some derivatives of oxamniquine.

Oxamniquine is a potent schistosomicide used clinically in the treatment of infections due to Schistosoma mansoni. Although relatively well tolerated, some central nervous system (CNS) effects characterised by convulsions have been reported in a small proportion of the population receiving this drug. Oxamniquine, the major metabolite and the secondary alcohol have been screened for convulsant activity by assessing their ability to potentiate catechol induced seizures in urethane anaesthetised mice. Significant (p < 0.05) potentiation was observed with subconvulsive doses (1.5 mg/kg) of strychnine. In contrast, oxamniquine and the secondary alcohol, each at 200 mg/kg ip, both produced significant (p < 0.05) depressions of seizures in this model whereas no effect was seen following 140 mg/kg ip of the acid derivative. These results indicate anticonvulsant rather than convulsant activity in oxamniquine and the alcohol derivative. The failure to observe any effect with the acid derivative may have been due to poorer CNS penetration.

Oxamniquine: a labeling procedure with technetium-99m and a biodistribution study in mice.

Oxamniquine (OXY), a tetrahydroquinoline derivative, is used as an antischistosomal drug and generally has been labeled with carbon-14 and tritium. We decided instead to label it with technetium-99 (99mTc). In order to determine the optimal conditions, different concentrations of this drug were incubated with various stannous chloride solutions. We then added 99mTc, and chromatography was performed using 0.9% NaCl solution, acetone and 1.2N HCl as the mobile phase. Using a solution of 1.0 mg/mL stannous chloride and 0.5 mg/mL oxamniquine, over 94% of the radioactivity bound to oxamniquine (99mTc-OXY). In the biodistribution study, 99mTc-OXY was administered in mice intramuscularly, orally and intravenously. When the intramuscular route was used, the main uptake (after 30 minutes) of the labeled drug was in the kidneys, liver and intestines; after 240 minutes the labeled drug was still found in the liver and kidneys, but at increased levels in the intestines. It was also present in the faeces. When the oral route was employed, labeled OXY was mainly found in the stomach after 30 minutes, but there was a decrease after 240 minutes. During this period radioactivity increased in the intestines. When the intravenous route was employed the labeled OXY was found in the liver and spleen. The radioactivity decreased with time in these organs. Using infected animals, radioactivity was found in isolated worms.

In vitro metabolism studies on oxamniquine and related compounds by chiral liquid chromatography.

A previously developed method based on alpha 1-acid glycoprotein for the resolution of the enantiomers of the Pfizer antischistosomal drug oxamniquine was used to examine possible enantioselectivity in the in vitro microsomal hydroxylation of a metabolic precursor, UK-3883, but was found to be limited by the poor operational stability of the analytical column ("EnantioPac") employed. As an alternative approach, a "Pirkle" covalently-bonded dinitrobenzoyl leucine column was used, with simple precolumn solute derivatization to the carbamate to improve chromatographic performance. The method allowed preliminary examination of the stereochemistry of the in vitro biotransformation, hydroxylation of UK-3883 to oxaminquine, which yielded evidence for substrate enantioselectivity in favour of the dextrorotatory enantiomer of UK-3883.

Studies on some derivatives of oxamniquine.

On the basis of the remarkable biological similarities between hycanthone and oxamniquine and as a sequel to our finding that some esters of hycanthone are active against hycanthone-resistant schistosomes, we prepared oxamniquine acetate, oxamniquine N-methylcarbamate, and four substituted phenylsulfonohydrazones of oxamniquine aldehyde. These compounds were tested for their effect on survival of and on [3H]uridine incorporation into hycanthone-sensitive and -resistant Schistosoma mansoni. All of these derivatives were effective to a greater or lesser degree in killing worms and in inhibiting [3H]uridine incorporation in the sensitive strain, but none was effective in the resistant strain.

Lack of correlation between schistosomicidal and anticholinergic properties of hycanthone and related drugs.

Visual observation of the motor activity of Schistosoma mansoni kept in vitro showed an increase of activity in the presence of hycanthone (HC). In addition, HC caused a delay in the paralytic effects of carbachol. Similar results were observed in the presence of oxamniquine (OXA). The same pattern of motor activity, however, was shown by HC-resistant worms, by Schistosoma japonicum, and by worms exposed to drug precursors (lucanthone and UK-3883), which are not schistosomicidal in vitro. Other analogs with in vitro killing activity (IA-4 and IA-4 N-oxide) showed minimal anticholinergic effects. The anticholinergic effects of HC and OXA were quickly reversible in vitro and in vivo, whereas their antischistosomal effects are irreversible and delayed. Incubation of schistosomes with high concentrations of carbachol or with anticholinergic drugs failed to compete with the schistosomicidal effects of HC. These results are viewed as contradictory to the hypothesis that HC kills schistosomes by blocking their acetylcholine receptors.

Studies on the mode of action of oxamniquine and related schistosomicidal drugs.

Adult Schistosoma mansoni were incubated for 1 hour in vitro with various drugs and then returned into the mesenteric veins of permissive animal hosts. Survival of schistosomes was assessed 3-4 weeks later by portal perfusion. Under these conditions, oxamniquine and hycanthone proved effective in killing S. mansoni, whereas UK-3883, lucanthone and lucanthone-4-desmethyl had no lethal activity. The same drugs which were schistosomicidal in vitro also persistently inhibited DNA, RNA, and protein synthesis in S. mansoni, whereas they were only transiently inhibitory against Schistosoma japonicum, against hycanthone-resistant S. mansoni and against immature worms. When drugs were administered in vivo to infected mice and the synthesis of macromolecules was assayed in vitro on worms obtained 1 or 3 days after treatment, not only oxamniquine and hycanthone, but also UK-3883 and lucanthone, proved effective in inhibiting the synthesis of macromolecules in sensitive--but not in resistant--S. mansoni. It is suggested that oxamniquine, like hycanthone, may exert its schistosomicidal activity by inhibiting nucleic acid synthesis in the parasite.

The metabolism of oxamniquine - a new schistosomicide.

The metabolism of oxamniquine, 6-hydroxymethyl-7-nitro-2-isopropylaminomethyl-1,2,3,4-tetrahydroquinoline, has been studied in the mouse, rat, hamster, rabbit, rhesus monkey, dog and man. Urinary excretion is a major route of elimination in man. The compound is converted to two metabolites, the major one arising from oxidation of the 6-hydroxymethyl group to a carboxyl group and the other by oxidation of the side chain to give the 2-carboxylic acid. There is a species at the dose levels used since both acidic metabolites were found in appreciable quantities only in the urine of mouse, rabbit, hamster and dog. The 2-carboxylic acid was not found in the urine of rhesus monkey and rat and occurred in only trace amounts in human urine.