Antitrypanosomal Compounds Research Articles

Characterization of quinapyramine (Trypacide®) drugresistant Trypansoma evansi

Molecular karyotyping by pulsed field gel electrophoresis was used to characterize Trypanosoma evansi isolates. Ten T. evansi isolates from camels were collected in Eastern and Western Sudan. Isolates from Eastern Sudan which were kept under continuous prophylactic treatment with quinapyramine (Trypacide®), were found to bear a single pattern and belonged to one karyotype group. From Western Sudan where trypanosomosis management was done by individual treatment of proven parasitaemic cases, isolates with diverse karyotype patterns were obtained. This study concluded that the occurrence of karyotype homogeneity amongst T. evansi isolates from field situations where antitrypanosomal compounds have been used may infer the existence of drug-resistance

Characterization of quinapyramine (Trypacide®) drug- resistant Trypansoma evansi

Molecular karyotyping by pulsed field gel electrophoresis was used to characterizeTrypanosoma evansi isolates. Ten T. evansi isolates from camels were collected in Eastern and Western Sudan. Isolates from Eastern Sudan which were kept under continuous prophylactic treatment with quinapyramine (Trypacide®), were found to bear a single pattern and belonged to one karyotype group. From Western Sudan where trypanosomosis management was done by individual treatment of proven parasitaemic cases, isolates with diverse karyotype patterns were obtained. This study concluded that the occurrence of karyotype homogeneity amongst T. evansiisolates from field situations where anti-trypanosomal compounds have been used may infer the existence of drug-resistance. Key words: Trypanosoma evansi, drug resistance, Trypacide®, karyotyping, Sudan.

A novel non-stochastic quadratic fingerprints-based approach for the ‘in silico’ discovery of new antitrypanosomal compounds

A non-stochastic quadratic fingerprints-based approach is introduced to classify and design, in a rational way, new antitrypanosomal compounds. A data set of 153 organic chemicals, 62 with antitrypanosomal activity and 91 having other clinical uses, was processed by a k-means cluster analysis to design training and predicting data sets. Afterwards, a linear classification function was derived allowing the discrimination between active and inactive compounds. The model classifies correctly more than 93% of chemicals in both training and external prediction groups. The predictability of this discriminant function was also assessed by a leave-group-out experiment, in which 10% of the compounds were removed at random at each time and their activity predicted a posteriori. In addition, a comparison with models generated using four well-known families of 2D molecular descriptors was carried out. As an experiment of virtual lead generation, the present TOMOCOMD approach was finally satisfactorily applied on the virtual evaluation of 10 already synthesized compounds. The in vitro antitrypanosomal activity of this series against epimastigotes forms of Trypanosomal cruzi was assayed. The model was able to predict correctly the behaviour of these compounds in 90% of the cases.

Benzo[1,2- c]1,2,5-oxadiazole N-oxide derivatives as potential antitrypanosomal drugs. Part 3: Substituents-clustering methodology in the search for new active compounds

The results of a study on the use of Hansch’s series design, cluster methodology, for the generation of new benzo[1,2- c]1,2,5-oxadiazole N-oxide derivatives as antitrypanosomal compounds are described. In vitro activity of these compounds was tested against Tulahuen 2 strain of Trypanosoma cruzi. Clearly, the Hansch methodology allowed identifying two cluster-substituents suitable for further structural modifications. The most effective drugs, derivatives 11, 18, and 21, with 50% inhibitory concentration (IC 50) of the same order as that of the reference drug, represent an excellent structural point of chemical modifications for the design of future drugs. Preliminary results from the study of the mechanism of action of these benzofuroxans point to perturbation of the mitochondrial electron chain, inhibiting parasite respiration.

Inhibitory action of marine algae extracts on the Trypanosoma cruzi dihydroorotate dehydrogenase activity and on the protozoan growth in mammalian cells

Trypanosoma cruzi, the causative agent of Chagas' disease, replicates in mammalian cells and relies on the de novo pyrimidine biosynthetic pathway that supplies essential precursors for nucleic acid synthesis. The protozoan dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the pathway catalyzing production of orotate from dihydroorotate, markedly differs from the human enzyme. This study was thus aimed to search for potent inhibitors against T. cruzi DHOD activity, and a number of methanol extracts prepared from green, brown, and red algae were assayed. The extracts from two brown algae, Fucus evanescens and Pelvetia babingtonii, yielded 59 and 58% decrease in the recombinant DHOD activity, respectively, at the concentration of 50 μg/ml. Inhibition by these extracts was noncompetitive with respect to dihydroorotate, with apparent K i values of 35.3±5.9 and 10.3±4.4 μg/ml, respectively. Further, in an in vitro T. cruzi–HeLa cell infection system, ethanol-reconstituted F. evanescens and P. babingtonii extracts at the concentration of 1 μg/ml, respectively, decreased significantly the infection rate of host cells and the average parasite number per infected cell. These results imply that F. evanescens and P. babingtonii contain inhibitor(s) against the T. cruzi DHOD activity and against the protozoan infection and proliferation in mammalian cells. Identification of inhibitor(s) in these two brown algae and further screening of other marine algae may facilitate the discovery of new, anti-trypanosomal lead compounds.

Kola acuminata proanthocyanidins: a class of anti-trypanosomal compounds effective against Trypanosoma brucei

Human African trypanosomiasis is undergoing an alarming rate of recrudescence in many parts of sub-Saharan Africa. Yet, there is no successful chemotherapy for the disease due to a limited number of useful drugs, side effects and drawbacks of the existing medication, as well as the development of drug resistance by the parasite. Here we describe a new lead anti-trypanosomal compound isolated from Kola acuminata (Makasu). We purified a proanthocyanidin by chromatographic procedures and confirmed its homogeneity and structure by Nuclear Magnetic Resonance and Matrix-Assisted Laser Desorption Ionisation Time-of-Flight mass spectrometry, respectively. In vitro, this compound potently induced growth arrest and lysis of bloodstream form trypanosomes in a dose- and time-dependent manner. In a mouse model, it exhibited a trypanostatic effect that extended the life of infected, treated animals up to 8 days post-infection against the 4 days for infected, untreated animals. The proanthocyanidin showed a low cytotoxicity against mammalian cells, whereas treated-BF showed massive enlargement of their flagellar pocket and lysosome-like structures caused by an intense formation of multivesicular bodies and vesicles within these organelles. The observed ultrastructural alterations caused rupture of plasma membranes and the release of cell contents, indicative of a necrotic process rather than a programmed cell death. Interestingly, the proanthocyanidin acted against BF but not procyclic form trypanosomes. This new anti-trypanosomal compound should be further studied to determine its efficacy and suitability as an anti-trypanosomal drug and may be used as a tool to define novel specific drug targets in BF trypanosomes.

Bisguanidine, Bis(2-aminoimidazoline), and Polyamine Derivatives as Potent and Selective Chemotherapeutic Agents against Trypanosoma brucei rhodesiense. Synthesis and in Vitro Evaluation

The in vitro screening for trypanocidal activity against Trypanosoma brucei rhodesiense of an in-house library of 62 compounds [i.e. alkane, diphenyl, and azaalkane bisguanidines and bis(2-aminoimidazolines)], which were chosen for their structural similarity to the trypanocidal agents synthalin (1,10-decanediguanidine) and 4,4'-diguanidinodiphenylmethane and the polyamine N(1)-(3-amino-propyl)propane-1,3-diamine, respectively, is reported. The original synthetic procedure for the preparation of 21 of these compounds is also reported. Most compounds displayed low micromolar antitrypanosomal activity, with five of them presenting a nanomolar inhibitory action on the parasite: 1,9-nonanediguanidine (1c), 1,12-dodecanediguanidine (1d), 4,4'-bis[1,3-bis(tert-butoxycarbonyl)-2-imidazolidinylimino]diphenylamine (28a), 4,4'-bis(4,5-dihydro-1H-2-imidazolylamino)diphenylamine (28b), and 4,4'-diguanidinodiphenylamine (32b). Those molecules that showed an excellent in vitro activity as well as high selectivity for the parasite [e.g. 1c (IC(50) = 49 nM; SI > 5294), 28b (IC(50) = 69 nM; SI = 3072), 32b (IC(50) = 22 nM; SI = 29.5), 41b (IC(50) = 118 nM; SI = 881)] represent new antitrypanosomal lead compounds.

Selective antimicrotubule activity of N1-phenyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (GB-II-5) against kinetoplastid parasites.

Analogs of the antimitotic herbicide oryzalin (3,5-dinitro-N4,N4-di-n-propylsulfanilamide) were recently prepared that were more potent in vitro than the parent compound against the kinetoplastid parasite Leishmania donovani (Bioorg Med Chem Lett 12:2395-2398, 2002). In the present work, we show that the most active molecule in the group, N1-phenyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (GB-II-5), is a potent, selective antimitotic agent against kinetoplastid parasites. GB-II-5 possesses IC50 values of 0.41 and 0.73 microM in vitro against two strains of the related parasite Trypanosoma brucei but is much less toxic to J774 murine macrophages and PC3 prostate cancer cells, exhibiting IC50 values of 29 and 35 microM against these lines, respectively. Selectivity is also observed for GB-II-5 with purified leishmanial and mammalian tubulin. The assembly of 15 microM leishmanial tubulin is completely inhibited by 10 microM GB-II-5, whereas 40 microM GB-II-5 inhibits the assembly of 15 microM porcine brain tubulin by only 17%. In cultured L. donovani and T. brucei, treatment with 5 and 0.5 microM GB-II-5, respectively, causes a striking increase in the fraction of G2M cells compared with control. Given the potency and selectivity of this agent against kinetoplastid tubulin, GB-II-5 emerges as an exciting new antitrypanosomal and antileishmanial lead compound.

Synthesis and characterization of new ruthenium complexes with active ligands against Chagas' disease

Chagas' disease, considered incurable, is a major third world parasitosis that affects millions of people in Latin America. Previous work has shown that ruthenium clotrimazole complexes are more active against Trypanosoma cruzi, causative agent of Chagas' disease, than the corresponding free ligand. In this work, the synthesis and characterization of a series of new Ru(II) complexes with different antitrypanosomal active compounds is presented. Complexes of general formulae [Ru IICl 2(dmso) 2L], where dmso=dimethylsulfoxide and L=5-nitro-2-furaldehyde semicarbazone ( L1), N 4-n-butyl-5-nitro-2-furaldehyde semicarbazone ( L2) or 3-(5-nitrofuryl)acroleine semicarbazone ( L3), were prepared in good yields by reaction of [Ru IICl 2(dmso) 4] with L in ethanol or toluene solutions. Complexes were characterized by elemental analyses and electronic, FTIR, 1H and 13C NMR spectroscopies. Crystal and molecular structures of [RuCl 2(dmso) 2 L1] and [RuCl 2(dmso) 2 L2] were determined by X-ray diffraction methods. In both crystals the ruthenium metal atom is in a quite similar elongated octahedral environment, equatorially coordinated to the semicarbazone molecule, acting as a bidentate ligand through its azomethynic nitrogen and carbonylic oxygen atoms. The sixfold coordination is completed with the sulfur atoms of two dimethylsulfoxide ligands at cis positions and two chlorine ions at the axial positions. The proposed formula for L3 complex was supported by FTIR, NMR and theoretical studies. NOE–NMR experiments allowed to assign L 3 spatial distribution in the complex.

Monoterpene hydroperoxides with trypanocidal activity from Chenopodium ambrosioides.

Four monoterpene hydroperoxides were isolated from aerial parts of Chenopodium ambrosioides along with ascaridole (1), the anthelmintic principle of this plant, as anti-trypanosomal compounds. The structures of these monoterpenes were determined to be (-)-(2S,4S)- and (-)-(2R,4S)-p-mentha-1(7),8-dien-2-hydroperoxide (2a and 3a) and (-)-(1R,4S)- and (-)-(1S,4S)-p-mentha-2,8-dien-1-hydroperoxide (4a and 5a) on the basis of spectroscopic methods and chemical correlations. In vitro trypanocidal activities of ascaridole (1) and these hydroperoxides (2a-5a) against epimastigotes of Trypanosoma cruzi were 23, 1.2, 1.6, 3.1, and 0.8 microM, respectively. Fresh leaves of C. ambrosioides also contained isomeric hydroperoxides 6a and 7a, and the content ratio of 2a-7a suggested that these hydroperoxides were formed through the singlet-oxygen oxidation of limonene.

Computer assisted design of potentially active anti-trypanosomal compounds

A computer assisted molecular modeling was used to design molecules having a shape complementary to the active site of glutathione reductase (GR) and trypanothione reductase (TR). The designed 5-nitro compound derivatives, were obtained from structural knowledge gleaned on glutathione (GSSG), trypanothione (T[S]2) and GSP disulfide (glutathionylspermidine disulfide). These molecules form complexes with the enzymes GR and TR. The theoretical lead compound was: N1-[1-(5-nitro-2-furyl)methylidene]-N4-{4-[3-(2,2,2-trifluoroacetyl)hexahydro-1-1-pyrimidynil]butyl} semicarbazide (NPIPCO). A multi-disciplinary team developed around the efforts to synthesize this lead. In this work we report on eight compounds that were synthesized in the pathway to NPIPCO: 4-(2-methoxyethyl)-1-, 4-butyl-1-, 4-hexyl-1- and 2-methoxphenyl-1-(5-nitrofurfurilidene) semicarbazides and the corresponding 5-nitrothiophenes. These substances are expected to act as pro-transition state analogues. Enzymologic studies proved that many of these compounds are inhibitors of TR. Furthermore, they showed inhibitory activity on Tripanosoma cruzi growth in vitro.

ChemInform Abstract: Squalamine Analogues as Potential Anti-Trypanosomal and Anti-Leishmanial Compounds.

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Squalamine analogues as potential anti-trypanosomal and anti-leishmanial compounds

This paper concerns the synthesis of various simplified analogues of the novel anti-microbial agent, squalamine. The compounds were then investigated for activity against Trypanosoma brucei, the causative agent of African trypanosomiasis, Trypanosoma cruzi, the causative agent of Chagas disease and Leishmania donovani, the causative agent of visceral leishmaniasis. Several compounds showed in vitro activity, especially against T. brucei and L. donovani. However, one compound showed poor in vivo activity.

Stimulation of tumor growth in vitro and in vivo by suramin on the VX2 model.

Suramin is an antitrypanosomal compound with confirmed efficacy against several human malignancies. It is generally assumed that its mechanism of action includes the interaction with different growth factors, unlike most of the anticancer drugs. Its anticancer activity has not been tested in vivo against squamous cell carcinoma. The purpose of this study was to assess the efficacy and toxicity of suramin in vivo and in vitro on the VX2 tumor model at therapeutic monitored plasma concentrations. We determined the pharmacokinetics of suramin in rabbits, and modelized its administration in order to obtain plasma concentrations between 150 and 300 micrograms/ml throughout the treatment course of 3 weeks. Under these conditions, antitumor effects of suramin were evaluated in vivo by comparing liver tumor involvement in suramin-treated and control rabbits. Liver involvement was quantified by image analysis and in vitro effects were also determined at the same concentrations. In vivo, suramin promoted liver tumor growth significantly (p < 0.05), compared to untreated controls. In vitro, suramin significantly stimulated tumor cell growth at concentrations above 200 micrograms/ml (p < 0.01). Suramin may have stimulatory effects on tumor growth in squamous cell carcinoma at relevant plasma drug concentrations. Caution should be taken in further trials in patients with squamous cell carcinomas.

Antitrypanosomal Activity of Phosphonylmethoxyalkylpurines

Phosphonylmethoxyalkylpurines and -pyrimidines exhibit potent activity against a broad spectrum of DNA viruses. We evaluated some of these nucleotide analogues for antitrypanosomal activity in vitro and in mice. The most active compounds were (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine (HPMPDAP), which inhibited growth of Trypanosoma brucei brucei by 50% (EC50 value) when incubated in vitro for 24 hr with 0.23-5.69 micrograms drug/ml. Both compounds completely eliminated multidrug-resistant T. b. brucei in culture at 1 microgram/ml after 4-5 days exposure. Mice infected with drug-susceptible T. b. brucei were cured with 2 doses of 10 mg/kg HPMPDAP. Two or 5 doses of 50 mg/kg 9-(2-phosphonylmethoxyethyl) adenine (PMEA) or 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), respectively, were necessary to eliminate T. b. brucei infections in mice. Mice infected with multidrug-resistant T. b. brucei were not cured with the above dosages. The most active compound against Trypanosoma congolense was PMEDAP with an EC50 value of 3.21-11.63 micrograms/ml. Thus, some of the phosphonylmethoxyalkyl purines showed potential as antitrypanosomal compounds at dosages that are below those toxic for mice.

Generation of Monoclonal Antibodies to the Anti-Trypanosomal Drug Isometamidium

Mice were immunized with either an isometamidium-human serum albumin (HSA) conjugate or an isometamidium-porcine thyroglobulin conjugate (PTG). Thereafter, monoclonal antibodies (MAbs) IL-A 1001, IL-A 1002, IL-A 1003, 5F7.B7, and 5F7.C9 were generated and selected on the basis that they recognized conjugated and unconjugated isometamidium, but lacked cross-reactivity with the carrier molecules. All five MAbs were of the IgG1 isotype. Each of the five MAbs was assessed in a competitive ELISA for isometamidium; in each case, the minimum level of detection was approximately 10 ng/ml. Each MAb exhibited approximately 0.1% cross-reactivity with the anti-trypanosomal compound diminazene. However, based on their cross-reactivity with the anti-trypanosomal compound homidium, the MAbs could be divided into two groups; IL-A 1001, IL-A 1002, and IL-A 1003, produced using an isometamidium-HSA conjugate as an immunogen, exhibited low levels of cross-reactivity (approximately 0.1%). In contrast, 5F7.B7 and 5F7.C9, produced using an isometamidium-PTG conjugate as an immunogen, exhibited high levels of cross-reactivity.

Synthesis and antitrypanosomal evaluation of some thiazole-containing amino acids and peptides

Several amino acids and peptides containing thiazole and thiazolidine residues were prepared. They were tested in vivo and in vitro as possible antitrypanosomal compounds. Some derivatives showed a slight activity. As they are structurally related to glutathione, their inhibitory properties towards glutathionylspermidine synthetase, trypanothione synthetase and trypanothione reductase were determined. No inhibitory activity was found.

Anti-trypanosomal compounds. I. In vivo and in vitro activity of inhibitors of glycolysis in trypanosomes

Carboxylated and phosphorylated esters of glycerol, possible inhibitors of the atypical glycolysis in trypanosomes have been synthesised and tested on Trypanosoma equiperdum in vitro and Trypanosoma brucei, on mice, and some of them also on glycerol phosphate dehydrogenase (GDH). Two of these compounds which showed no in vitro activity behaved as good competitive inhibitors of GDH; 3 other compounds were shown to have a good activity on cultures of trypanosomes, but owing to their weak solubility, tests on the enzyme could not be conclusive. Indications for the design of other compounds have been given.

Existence of an extended series of antitumor compounds which bind to deoxyribonucleic acid by nonintercalative means.

Viscometric titrations of bacteriophage PM2 closed circular DNA, in addition to spectrophotometric and fluorometric methods, were used to investigate the mode of DNA binding of a number of antitrypanosomal and antitumor compounds. Several classes of compounds were identified which failed to unwind PM2 DNA, which appeared to have a large DNA binding site of at least 4 base pairs and which often showed considerable selectivity of binding to poly[d(AT)] as opposed to poly[d(GC)]. The classes included the antiviral antibiotics distamycin and netropsin, bisamidines such as the trypanocidal drug berenil, phthalanilide bisamidines, aromatic bis(guanylhydrazones), and the bisquaternary ammonium heterocycles. It is proposed that the compounds all bind in the minor groove of the DNA double helix.