Synthesis and tripanocidal activity of ferrocenyl and benzyl diamines against Trypanosoma brucei and Trypanosoma cruzi

Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of sleeping sickness and Chagas disease, respectively, two of the 17 preventable tropical infectious diseases (NTD) which have been neglected by governments and organizations working in the health sector, as well as pharmaceutical industries. High toxicity and resistance are problems of the conventional drugs employed against trypanosomiasis, hence the need for the development of new drugs with trypanocidal activity. In this work we have evaluated the trypanocidal activity of a series of N1,N2-dibenzylethane-1,2-diamine hydrochlorides (benzyl diamines) and N1-benzyl,N2-methyferrocenylethane-1,2-diamine hydrochlorides (ferrocenyl diamines) against T. brucei and T. cruzi parasite strains. We show that incorporation of the ferrocenyl group into the benzyl diamines increases the trypanocidal activity. The molecules exhibit potential trypanocidal activity in vitro against all parasite strains. Cytotoxicity assay was also carried out to evaluate the toxicity in HepG2 cells.     

Synthesis and in vitro antiprotozoal activities of 5-phenyliminobenzo[a]phenoxazine derivatives

A series of 5-phenyliminobenzo[a]phenoxazine derivatives were synthesized. The in vitro antiprotozoal activities were evaluated against Plasmodium falciparum K1, Trypanosoma cruzi, Leishmania donovani and Trypanosoma brucei rhodesiense. N,N-Diethyl-5-((4-methoxyphenyl)imino)-5H-benzo[a]phenoxazin-9-amine shows IC(50)=0.040 μmol L(-1) with a selective index of 1425 against Plasmodium falciparum K1.     

The in vitro activity of fatty diamines and amino alcohols against mixed amastigote and trypomastigote Trypanosoma cruzi forms

Four diamines and three amino alcohols derived from 1-decanol, 1-dodecanol and 1,2-dodecanediol were evaluated in an in vitro assay against a mixture of trypomastigote and intracellular amastigote forms of Trypanosoma cruzi. Two of these compounds (6 and 7) showed better activity against both proliferative stages of T. cruzi than the positive control benznidazole, three were of similar potency (1, 2 and 5) and two were less active (3 and 4).     

New bisphosphorothioates and bisphosphoroamidates: synthesis, molecular modeling and determination of insecticide and toxicological profile

A series of new compounds, N,N'-bis(dialkylphosphoryl)diamines and S,S'-bis(dialkylphosphoryl)-1,3-propanedithiols were prepared by a Todd-Atherton like reaction of dialkylphosphites with symmetrical diamines and 1,3-propanedithiols in a biphasic system [F.R. Athertoon, H.T. Howard, A.R. Todd, J. Chem. Soc. (1948) 1106-1111; F.R. Athertoon, H.T. Openshaw, A.R. Todd, J. Chem. Soc. (1945) 660-663]. The structures were characterized by IR, 1H NMR, 13C NMR and mass spectrometry. Compounds with butoxy, isobutoxy and isopropoxy groups linked in the phosphorus atom showed the lowest LD50 values when tested against Musca domestica and Stomoxys calcitrans. The pharmacological and toxicological evaluation of N,N'-bis(diisobutylphosphoryl)-1,3-propylenediamine and S,S'-bis(diisobutylphosphoryl)-1,3-propanedithiol, which were very active against M. domestica and S. calcitrans, demonstrated that these compounds present no toxicological effects against mice in a concentration of 200mg/kg. An explanation for the observed activity profile is presented based on results obtained in a molecular modeling study with insect and mammalian acetylcholinesterase models.     

Leishmanicidal and trypanocidal activities of 2-aminocyclohexanol and 1,2-cyclohexanediamine derivatives

A number of aminoalcohols, diamines and other related cycloanalogues of sphingosine have been synthesized and assayed in vitro against three Leishmania spp. and Trypanosoma cruzi. Most of the compounds were potent parasiticides, with IC50 values in the microM or lower range and potencies higher than those of pentamidine and benznidazol, the common therapeutic agents against these parasitoses.     

Synthesis and antimycobacterial activity of ferrocenyl ethambutol analogues and ferrocenyl diamines

A new series of ferrocenyl diamino alcohols and diamines were synthesized and their inhibitory potencies were probed with Mycobacterium tuberculosis. Interestingly, ferrocenyl diamines 6a and b display significant activities against M. tuberculosis H37Rv.     

Parallel synthesis of a series of potentially brain penetrant aminoalkyl benzoimidazoles

Alpha7 agonists were identified via GOLD (CCDC) docking in the putative agonist binding site of an alpha7 homology model and a series of aminoalkyl benzoimidazoles was synthesised to obtain potentially brain penetrant drugs. The array was prepared starting from the reaction of ortho-fluoronitrobenzenes with a selection of diamines, followed by reduction of the nitro group to obtain a series of monoalkylated phenylene diamines. N,N'-Carbonyldiimidazole (CDI) mediated acylation, followed by a parallel automated work-up procedure, afforded the monoacylated phenylenediamines which were cyclised under acidic conditions. Parallel work-up and purification afforded the array products in good yields and purities with a robust parallel methodology which will be useful for other libraries. Screening for alpha7 activity revealed compounds with agonist activity for the receptor.     

Trypanocidal properties, structure-activity relationship and computational studies of quinoxaline 1,4-di-N-oxide derivatives

Pyrazole and propenone quinoxaline derivatives were tested against intracellular forms of Leishmania peruviana and Trypanosoma cruzi. Both series were tested for toxicity against proliferative and non-proliferative cells. The pyrazole quinoxaline series was quite inactive against T. cruzi; however, the compound 2,6-dimethyl-3-f-quinoxaline 1,4-dioxide was found to inhibit 50% of Leishmania growth at 8.9 μM, with no impact against proliferative kidney cells and with low toxicity against THP-1 cells and murine macrophages. The compounds belonging to the propenone quinoxaline series were moderately active against T. cruzi. Among these compounds, two were particularly interesting, (2E)-1-(7-fluoro-3-methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-propenone and (2E)-3-(3,4,5-trimethoxy-phenyl)-1-(3,6,7-trimethyl-quinoxalin-2-yl)-propenone. The former possessed selective activity against proliferative cells (cancer and parasites) and was inactive against murine peritoneal macrophages; the latter was active against Leishmania and inactive against the other tested cells. Furthermore, insilico studies showed that both series respected Lipinski’s rules and that they confirmed a linear correlation between trypanocidal activities and LogP. Docking studies revealed that compounds of the second series could interact with the poly (ADP-ribose) polymerase protein of Trypanosoma cruzi.     

Thioureas differentially induce rat hepatic microsomal epoxide hydrolase and rGSTA2 irrespective of their oxygen radical scavenging effect: effects on toxicant-induced liver injury

Thioureas have been employed as potent hydroxyl radical scavengers and also inhibit production of oxygen free radicals. The in vitro oxygen radical scavenging effect by N,N'-substituted thioureas including dimethylthiourea (DMT), diethylthiourea (DET), tetramethylthiourea (TMT) and diphenylthiourea (DPT) was assessed by the conversion of phi x-174 DNA from supercoiled DNA to the open circular form or to fragmented DNA. Addition of the N,N'-substituted thioureas to the incubation mixture significantly prevented a single strand breakage of phi x-174 DNA induced by autooxidation of benzenetriol. These thioureas were also effective in preventing degradation of phi x-174 DNA induced by autooxidation of benzenetriol in the presence of ferrous iron. In view of the in vitro radical scavenging effect by the thioureas and the role of reactive oxygen species in the induction of phase II detoxifying enzymes, expression of microsomal epoxide hydrolase (mEH) and rGSTA2 in response to these agents was investigated in the rat liver. Rats treated with each of the alkylthioureas exhibited marked increases of mEH and rGSTA2 mRNA levels with TMT being the most effective. DPT an arylthiourea, however, was minimally active in increasing the mRNAs. Time-course studies revealed that DMT, DET and TMT increased the mRNA levels to the greatest extent at 24 h after a single dose of treatment. The levels of mEH and rGSTA2 mRNA were elevated in a dose-dependent manner by the alkylthioureas. Immunoblot analysis showed that the alkylthioureas induced mEH and rGSTA2 proteins in the liver (0.6 mmol/kg per day, 3 days), which was consistent with the increases in the mRNA levels. DMT, DET or TMT enhanced CCl4-induced liver toxicity, as monitored by plasma aminotransferase activity, although each of the agents alone caused only slight increase in the alanine aminotransferase activity. In contrast to the effects of the alkylthioureas, DPT protected the liver against the toxicant-induced injury. All of the thioureas prevented decreases in the hepatic glutathione level by CCl4. Expression of cytochrome P450 2E1 and P450 2B1/2, which are implicated with metabolic activation of CCl4, was assessed after treatment with the thioureas. P450 2E1 and P450 2B1/2 were differentially induced by the alkylthioureas with the expression of P450 2E1 being inversely related with that of P450 2B1/2. These results showed that N,N'-substituted alkylthioureas were capable of inducing mEH and rGSTA2 in the liver with elevation of the mRNAs, that induction of mEH and rGSTA2 by these alkylthioureas might be mediated by production of the reactive oxygens derived from metabolic activation of the agents irrespective of their radical scavenging effect and that the agents rather enhanced toxicant-induced liver injury with the induction of P450 2E1 or P450 2B1/2.     

Diaminopurine-acridine heterodimers for specific recognition of abasic site containing DNA. Influence on the biological activity of the position of the linker on the purine ring.

Three acridine-diaminopurine heterodimers tethered by a linker containing an N,N'-substituted guanidine were prepared. The molecules differ by the site of introduction of the linker on the 2,6-diaminopurine. The interactions of the new heterodimers with abasic site containing oligonucleotide were compared, and their cytotoxicity was measured in the presence or absence of the antitumor alkylating agent BCNU.     

Novel azabicyclo[3.2.2]nonane derivatives and their activities against Plasmodium falciparum K1 and Trypanosoma brucei rhodesiense

New diaryl substituted 2-azabicyclo[3.2.2]nonane derivatives have been synthesized in order to investigate the influence of the aromatic substitution and of N substitution on the antiprotozoal activities of those compounds. Following a manual method for the Hansch approach, different 4-substituted aryl rings were systematically inserted, and moieties with varying basicity and polarity were attached to the ring nitrogen. All compounds were investigated for their activities against Trypanosoma brucei rhodesiense (STIB 900) and the K(1) strain of Plasmodium falciparum (resistant to chloroquine and pyrimethamine) and for their cytotoxicity using microplate assays. Some of the new compounds are amongst the most active antitrypanosomal agents in this series, and the selectivity index of a single derivative is superior in the 2-azabicyclo-nonane series.     

Synthesis and SAR of alkanediamide-linked bisbenzamidines with anti-trypanosomal and anti-pneumocystis activity

A series of alkanediamide-linked bisbenzamidines was synthesized and tested in vitro against a drug-sensitive strain of Trypanosoma brucei brucei, a drug-resistant strain of Trypanosoma brucei rhodesiense and Pneumocystis carinii. Bisbenzamidines linked with longer alkanediamide chains were potent inhibitors of both strains of T. brucei. However, bisbenzamidines linked with shorter alkanediamide chains were the most potent compounds against P. carinii. N,N′-Bis[4-(aminoiminomethyl)phenyl] hexanediamide, 4 displayed potent inhibition (IC₅₀ = 2–3 nM) against T. brucei and P. carinii, and was non-cytotoxic in the A549 human lung carcinoma cell line. The inhibitory bioactivity was significantly reduced when the amidine groups in 4 were moved from the para to the meta positions or replaced with amides.     

Enantiomeric cisplatin analogues: an investigation on their activity towards tumors in mice

A series of enantiomeric cisplatin analogues of formula [diamPtCl₂] (diam = chiral chelating diamine) and the corresponding sulfato derivatives was prepared and tested for activity against tumors in mice, particularly P 388 leukemia. The configuration of the diamines has practically no influence on the antitumor activity. The effects of the leaving group and of the nature of the diamines are briefly discussed.     

Modelling the impact of geometric parameters on the redox potential of blue copper proteins

The synthesis and structure of a homologous series of cationic N(2)S(2) copper(I) Schiff base complexes constructed using o-tert-butylthiobenzaldehyde and a series of terminal diamines (ethane, propane, butane) are reported. The complexes differ only in the length of the methylene chain between the imine groups. This simple modification forces the copper centre to shift geometry from a planar (1,2-diaminoethane) to a more distorted tetrahedral motif (1,4-diaminobutane). The redox potentials of the three cations were measured using cyclic voltammetry in donor (acetonitrile) and non-donor solvents (dichloromethane). The S-Cu-N angles for each complex are correlated against the respective redox potential allowing an analysis of the geometric impact on the redox potential in soft copper centres. The redox potential is observed to increase as the metal centre moves from a planar towards a tetrahedral motif. Comparing this data with the reported structures of the blue copper proteins (rusticyanin and plastocyanin) allows an assessment of the contribution of the geometry of the metal binding site to the operating potential of these proteins to be made.     

Synthesis and biological evaluation of substrate-based inhibitors of 6-phosphogluconate dehydrogenase as potential drugs against African trypanosomiasis

The synthesis and biological evaluation of three series of 6-phosphogluconate (6PG) analogues is described. (2R)-2-Methyl-4,5-dideoxy, (2R)-2-methyl-4-deoxy and 2,4-dideoxy analogues of 6PG were tested as inhibitors of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver and also Trypanosoma brucei where the enzyme is a validated drug target. Among the three series of analogues, seven compounds were found to competitively inhibit 6PGDH from T. brucei and sheep liver enzymes at micromolar concentrations. Six inhibitors belong to the (2R)-2-methyl-4-deoxy series (6, 8, 10, 12, 21, 24) and one is a (2R)-2-methyl-4,5-dideoxy analogue (29b). The 2,4-dideoxy analogues of 6PG did not inhibit both enzymes. The trypanocidal effect of the compounds was also evaluated in vitro against T. brucei rhodesiense as well as other related trypanosomatid parasites (i.e., Trypanosoma cruzi and Leishmania donovani).     

Synthesis and antiprotozoan evaluation of new alkyl-linked bis(2-thioxo-[1,3,5]thiadiazinan-3-yl) carboxylic acids

Two new series of several alkyl-linked bis(2-thioxo-[1,3,5]thiadiazinan-3-yl) carboxylic acids were synthesized in a two step procedure from the corresponding alkyl bis-dithiocarbamic salt intermediary. The novel compounds were evaluated for their activity in vitro against Trypanosoma cruzi strain CL (clone CL B5) and Trichomonas vaginalis strain JH 31A.     

Synthesis, structure, and bioactivity of N'-substituted benzylidene-3,4,5-trimethoxybenzohydrazide and 3-acetyl-2-substituted phenyl-5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1,3,4-oxadiazole derivatives

Some 3-acetyl-2-substituted phenyl-5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1,3,4-oxadiazole derivatives were synthesized by cyclization reaction of N'-substituted benzylidene-3,4,5-trimethoxybenzohydrazide in acetic anhydride. Their structures were verified by elemental analysis, IR, (1)H NMR, and (13)C NMR. Compound 3i was provided with X-ray crystallographic data. The compounds were evaluated for their antiproliferative activities against some cancer cells in vitro by MTT method. Among them, 2a, 2b, 2c, 2f, 3l, and 3m were highly effective against PC3 cells and 2a, 2c, and 2f showed moderate activities against Bcap37 and BGC823 cells. The IC(50) values of high active compounds 2a, 2b, 2c, 2f, 3l, and 3m against PC3 cells were 0.2, 1.8, 0.2, 1.2, 1.7, and 0.3muM, respectively.     

Design, synthesis and evaluation of 2,4-diaminoquinazolines as inhibitors of trypanosomal and leishmanial dihydrofolate reductase

This paper describes the design, synthesis and evaluation of a series of 2,4-diaminoquinazolines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were designed by a generating virtual library of compounds and docking them into the enzyme active site. Following their synthesis, they were found to be potent and selective inhibitors of leishmanial dihydrofolate reductase. The compounds were also found to have potent activity against Trypanosoma brucei rhodesiense, a causative organism of African trypanosomiasis and also against Trypanosoma cruzi, the causative organism of Chagas disease. There was significantly lower activity against Leishmania donovani, one of the causative organisms of leishmaniasis.     

Lysine-69 plays a key role in catalysis by ornithine decarboxylase through acceleration of the Schiff base formation, decarboxylation, and product release steps.

Ornithine decarboxylase (ODC) is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the biosynthesis of the polyamine putrescine. Similar to other PLP-dependent enzymes, an active site Lys residue forms a Schiff base with PLP in the absence of substrate. The mechanistic role of this residue (Lys-69) in catalysis by Trypanosoma brucei ODC has been studied by analysis of the mutant enzymes, in which Lys-69 has been replaced by Arg (K69R ODC) and Ala (K69A ODC). Analysis of K69A ODC demonstrated that the enzyme copurified with amines (e.g. putrescine) that were tightly bound to the active site through a Schiff base with PLP. In contrast, on the basis of an absorption spectrum of K69R ODC, PLP is likely to be bound to this mutant enzyme in the aldehyde form. Pre-steady-state kinetic analysis of the reaction of K69R ODC with L-Orn and putrescine demonstrated that the rates of both the product release (k(off.Put) = 0.0041 s(-)(1)) and the decarboxylation (k(decarb) = 0.016 s(-)(1)) steps were decreased by10(4)-fold in comparison to wild-type ODC. Further, the rates of Schiff base formation between K69R ODC and either substrate or product have decreased by at least 10(3)-fold. Product release remains as the dominant rate-limiting step in the reaction (the steady-state parameters for K69R ODC are k(cat) = 0.0031 s(-)(1) and K(m) = 0.18 mM). The effect of mutating Lys-69 on the decarboxylation step suggests that Lys-69 may play a role in the proper positioning of the alpha-carboxylate for efficient decarboxylation. K69R ODC binds diamines and amino acids with higher affinity than the wild-type enzyme; however, Lys-69 does not mediate substrate specificity. Wild-type and K69R ODC have similar ligand specificity preferring to bind putrescine over longer and shorter diamines. Kinetic analysis of the binding of a series of diamines and amino acids to K69R ODC suggests that noncovalent interactions in the active site of K69R ODC promote selective ligand binding during Schiff base formation.