Synthesis and Biophysical and Biological Studies of N-Phenylbenzamide Derivatives Targeting Kinetoplastid Parasites.

The AT-rich mitochondrial DNA (kDNA) of trypanosomatid parasites is a target of DNA minor groove binders. We report the synthesis, antiprotozoal screening, and SAR studies of three series of analogues of the known antiprotozoal kDNA binder 2-((4-(4-((4,5-dihydro-1H-imidazol-3-ium-2-yl)amino)benzamido)phenyl)amino)-4,5-dihydro-1H-imidazol-3-ium (1a). Bis(2-aminoimidazolines) (1) and bis(2-aminobenzimidazoles) (2) showed micromolar range activity against Trypanosoma brucei, whereas bisarylimidamides (3) were submicromolar inhibitors of T. brucei, Trypanosoma cruzi, and Leishmania donovani. None of the compounds showed relevant activity against the urogenital, nonkinetoplastid parasite Trichomonas vaginalis. We show that series 1 and 3 bind strongly and selectively to the minor groove of AT DNA, whereas series 2 also binds by intercalation. The measured pKa indicated different ionization states at pH 7.4, which correlated with the DNA binding affinities (ΔTm) for series 2 and 3. Compound 3a, which was active and selective against the three parasites and displayed adequate metabolic stability, is a fine candidate for in vivo studies.

Methyl N-(tert-butoxycarbonyl)pyridine-2-carbimidothioate: A new reagent for the synthesis of N-phenylpyridinecarboxamidine ("arylimidamide") DNA-minor groove binders from poorly nucleophilic amines.

We report the synthesis and use of methyl N-(tert-butoxycarbonyl)pyridine-2-carbimidothioate as new reagent for the preparation of N-phenylpyridinecarboxamidines ("arylimidamides"), a class of DNA minor groove binding molecules with antiprotozoal activity. This versatile reagent allowed the access to electron-deficient halogen-containing bis(arylimidamides) that could not be obtained with the classical methods reported in the literature. With this two-step protocol, the N-Boc-protected arylimidamide intermediate, which is soluble in organic solvents, can be purified by centrifugal preparative thin layer chromatography on silica and/or by reverse-phase (C-18) chromatography. The target N-phenylpyridinecarboxamidines are obtained as salts by smooth hydrolysis of the Boc-protecting group with TFA. This methodology allows the synthesis of a pharmaceutically important class of antiparasitic compounds otherwise inaccessible.

Imidazoline- and Benzamidine-Based Trypanosome Alternative Oxidase Inhibitors: Synthesis and Structure-Activity Relationship Studies.

The trypanosome alternative oxidase (TAO), a mitochondrial enzyme involved in the respiration of the bloodstream form trypomastigotes of Trypanosoma brucei, is a validated drug target against African trypanosomes. Earlier series of TAO inhibitors having a 2,4-dihydroxy-6-methylbenzoic acid scaffold ("head") and a triphenylphosphonium or quinolin-1-ium cation as a mitochondrion-targeting group ("tail") were shown to be nanomolar inhibitors in enzymatic and cellular assays. We investigated here the effect of different mitochondrion-targeting cations and other scaffold modifications on the in vitro activity of this class of inhibitors. Low micromolar range activities were obtained, and the structure-activity relationship studies showed that modulation of the tail region with polar substituents is generally detrimental to the enzymatic and cellular activity of TAO inhibitors.

Synthesis, biological, and photophysical studies of molecular rotor-based fluorescent inhibitors of the trypanosome alternative oxidase.

We have recently reported on the development and trypanocidal activity of a class of inhibitors of Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to lipophilic cations via C14 linkers to enable optimal interaction with the enzyme's active site. This strategy resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has not been formally established. We therefore synthesized a series of fluorescent analogues to visualize accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and their photophysical and biological properties were studied. These probes display a red emission, with a high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver) mitochondria was estimated to 66 ± 20% of the total. The compounds inhibited recombinant TAO enzyme in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity (SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected, presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins, whereas the probe was located, virtually exclusively, in the trypanosome's mitochondrion.

Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei.

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

African sleeping sickness is a potentially deadly illness caused by the parasite Trypanosoma brucei. The disease is treatable, but many of the current treatments are old and are becoming increasingly ineffective. For instance, resistance is growing against pentamidine, a drug used in the early stages in the disease, as well as against melarsoprol, which is deployed when the infection has progressed to the brain. Usually, cases resistant to pentamidine are also resistant to melarsoprol, but it is still unclear why, as the drugs are chemically unrelated. Studies have shown that changes in a water channel called aquaglyceroporin 2 (TbAQP2) contribute to drug resistance in African sleeping sickness; this suggests that it plays a role in allowing drugs to kill the parasite. This molecular 'drain pipe' extends through the surface of T. brucei, and should allow only water and a molecule called glycerol in and out of the cell. In particular, the channel should be too narrow to allow pentamidine or melarsoprol to pass through. One possibility is that, in T. brucei, the TbAQP2 channel is abnormally wide compared to other members of its family. Alternatively, pentamidine and melarsoprol may only bind to TbAQP2, and then 'hitch a ride' when the protein is taken into the parasite as part of the natural cycle of surface protein replacement. Alghamdi et al. aimed to tease out these hypotheses. Computer models of the structure of the protein were paired with engineered changes in the key areas of the channel to show that, in T. brucei, TbAQP2 provides a much broader gateway into the cell than observed for similar proteins. In addition, genetic analysis showed that this version of TbAQP2 has been actively selected for during the evolution process of T. brucei. This suggests that the parasite somehow benefits from this wider aquaglyceroporin variant. This is a new resistance mechanism, and it is possible that aquaglyceroporins are also larger than expected in other infectious microbes. The work by Alghamdi et al. therefore provides insight into how other germs may become resistant to drugs.

Aqueous pKa prediction for tautomerizable compounds using equilibrium bond lengths.

The accurate prediction of aqueous pKa values for tautomerizable compounds is a formidable task, even for the most established in silico tools. Empirical approaches often fall short due to a lack of pre-existing knowledge of dominant tautomeric forms. In a rigorous first-principles approach, calculations for low-energy tautomers must be performed in protonated and deprotonated forms, often both in gas and solvent phases, thus representing a significant computational task. Here we report an alternative approach, predicting pKa values for herbicide/therapeutic derivatives of 1,3-cyclohexanedione and 1,3-cyclopentanedione to within just 0.24 units. A model, using a single ab initio bond length from one protonation state, is as accurate as other more complex regression approaches using more input features, and outperforms the program Marvin. Our approach can be used for other tautomerizable species, to predict trends across congeneric series and to correct experimental pKa values.

Discovery and Pharmacological Studies of 4-Hydroxyphenyl-Derived Phosphonium Salts Active in a Mouse Model of Visceral Leishmaniasis.

We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in the submicromolar range (EC50 from 0.04 to 0.28 µM, SI > 10) against the protozoan parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of compound 1 [(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium bromide] in a mouse model of visceral leishmaniasis were established. Compound 1 reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an oral dosage of four daily doses of 1.5 mg/kg. Mode of action studies showed that compound 1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of Leishmania parasites. Disruption of the energetic metabolism, with a decrease of intracellular ATP levels as well as mitochondrial depolarization together with a significant reactive oxygen species production, contributes to the leishmanicidal effect of 1. Importantly, this compound was equally effective against antimonials and miltefosine-resistant clinical isolates of Leishmania infantum, indicating its potential as antileishmanial lead.

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

The alternative oxidase (AOX) is a ubiquitous terminal oxidase of plants and many fungi, catalyzing the four-electron reduction of oxygen to water alongside the cytochrome-based electron transfer chain. Unlike the classical electron transfer chain, however, the activity of AOX does not generate adenosine triphosphate but has functions such as thermogenesis and stress response. As it lacks a mammalian counterpart, it has been investigated intensely in pathogenic fungi. However, it is in African trypanosomes, which lack cytochrome-based respiration in their infective stages, that trypanosome alternative oxidase (TAO) plays the central and essential role in their energy metabolism. TAO was validated as a drug target decades ago and among the first inhibitors to be identified was salicylhydroxamic acid (SHAM), which produced the expected trypanocidal effects, especially when potentiated by coadministration with glycerol to inhibit anaerobic energy metabolism as well. However, the efficacy of this combination was too low to be of practical clinical use. The antibiotic ascofuranone (AF) proved a much stronger TAO inhibitor and was able to cure Trypanosoma vivax infections in mice without glycerol and at much lower doses, providing an important proof of concept milestone. Systematic efforts to improve the SHAM and AF scaffolds, aided with the elucidation of the TAO crystal structure, provided detailed structure-activity relationship information and reinvigorated the drug discovery effort. Recently, the coupling of mitochondrion-targeting lipophilic cations to TAO inhibitors has dramatically improved drug targeting and trypanocidal activity while retaining target protein potency. These developments appear to have finally signposted the way to preclinical development of TAO inhibitors.

SAR of 4-Alkoxybenzoic Acid Inhibitors of the Trypanosome Alternative Oxidase.

The SAR of 4-hydroxybenzaldehyde inhibitors of the trypanosome alternative oxidase (TAO), a critical enzyme for the respiration of bloodstream forms of trypanosomes, was investigated. Replacing the aldehyde group with a methyl ester resulted in a 10-fold increase in TAO inhibition and activity against T. brucei. Remarkably, two analogues containing the 2-hydroxy-6-methyl scaffold (9e and 16e) displayed single digit nanomolar TAO inhibition, which constitute the most potent 4-alkoxybenzoic acid derivatives described to date. 9e was 50-times more potent against TAO and 10-times more active against T. brucei compared to its benzaldehyde analogue 1. The farnesyl derivative 16e was as potent a TAO inhibitor as ascofuranone with IC50 = 3.1 nM. Similar to ascofuranone derivatives, the 2-hydroxy and 6-methyl groups seemed essential for low nanomolar TAO inhibition of acid derivatives, suggesting analogous binding interactions with the TAO active site.

Automated techniques in pKa determination: Low, medium and high-throughput screening methods.

Drug discovery programs that generate hundreds of new molecular entities need efficient methodologies for physicochemical profiling. Several high-throughput methods for pKa screening have been developed in the last 15 years to determine this key physicochemical parameter. Separation techniques such as HPLC-MS or capillary electrophoresis are particularly well-suited due to their high throughput and capacity to deal with impure or complex samples. In addition, potentiometric and (mostly) UV-metric-based methods (plate-based and automated systems), find their place as very precise methodologies for pKa determination despite of somewhat lower throughput. Finally, pKa prediction software packages are useful estimator tools but, to date, they cannot replace experimental measurements when accurate pKa values are required.

Inhibition of trypanosome alternative oxidase without its N-terminal mitochondrial targeting signal (ΔMTS-TAO) by cationic and non-cationic 4-hydroxybenzoate and 4-alkoxybenzaldehyde derivatives active against T. brucei and T. congolense.

African trypanosomiasis is a neglected parasitic disease that is still of great public health relevance, and a severe impediment to agriculture in endemic areas. The pathogens possess certain unique metabolic features that can be exploited for the development of new drugs. Notably, they rely on an essential, mitochondrially-localized enzyme, Trypanosome Alternative Oxidase (TAO) for their energy metabolism, which is absent in the mammalian hosts and therefore an attractive target for the design of safe drugs. In this study, we cloned, expressed and purified the physiologically relevant form of TAO, which lacks the N-terminal 25 amino acid mitochondrial targeting sequence (ΔMTS-TAO). A new class of 32 cationic and non-cationic 4-hydroxybenzoate and 4-alkoxybenzaldehyde inhibitors was designed and synthesized, enabling the first structure-activity relationship studies on ΔMTS-TAO. Remarkably, we obtained compounds with enzyme inhibition values (IC50) as low as 2 nM, which were efficacious against wild type and multidrug-resistant strains of T. brucei and T. congolense. The inhibitors 13, 15, 16, 19, and 30, designed with a mitochondrion-targeting lipophilic cation tail, displayed trypanocidal potencies comparable to the reference drugs pentamidine and diminazene, and showed no cross-resistance with the critical diamidine and melaminophenyl arsenical classes of trypanocides. The cationic inhibitors 15, 16, 19, 20, and 30 were also much more selective (900 - 344,000) over human cells than the non-targeted neutral derivatives (selectivity >8-fold). A preliminary in vivo study showed that modest doses of 15 and 16 reduced parasitaemia of mice infected with T. b. rhodesiense (STIB900). These compounds represent a promising new class of potent and selective hits against African trypanosomes.

Prediction of Aqueous pK a Values for Guanidine-Containing Compounds Using Ab Initio Gas-Phase Equilibrium Bond Lengths.

In this work, we demonstrate the existence of linear relationships between gas-phase equilibrium bond lengths of the guanidine skeleton of 2-(arylamino)imidazolines and their aqueous pK a value. For a training set of 22 compounds, in the most stable conformation of their lowest energy tautomeric form, three bonds were found to exhibit r 2 and q 2 values >0.95 and root-mean-squared-error of estimation values ≤0.25 when regressed individually against pK a. The equations describing these one-bond-length linear relationships, in addition to a multiple linear regression model using all three bond lengths, were then used to predict the experimental pK a values of an external test set of further 27 derivatives. The optimal protocol we derive here shows an overall mean absolute error (MAE) of 0.20 and standard deviation of errors of 0.18 for the test set. Predictions for a second test set of diphenyl-based bis(2-iminoimidazolidines) yielded an MAE of 0.27 and a standard deviation of 0.10. The predictive power of the optimal model is further demonstrated by its ability to correct erroneously reported experimental values. Finally, a previously established guanidine model is recalibrated at a new level of theory, and predictions are made for novel phenylguanidine derivatives, showing an MAE of just 0.29. The protocols established and tested here pass both of Roy's modern and stringent MAE-based criteria for a "good" quantitative structure-activity relationship/quantitative structure-property relationship model predictivity. Notably, the ab initio bond length high correlation subset protocol developed in this work demonstrates lower MAE values than the Marvin program by ChemAxon for all test sets.

Functional and structural analysis of AT-specific minor groove binders that disrupt DNA-protein interactions and cause disintegration of the Trypanosoma brucei kinetoplast.

Trypanosoma brucei, the causative agent of sleeping sickness (Human African Trypanosomiasis, HAT), contains a kinetoplast with the mitochondrial DNA (kDNA), comprising of >70% AT base pairs. This has prompted studies of drugs interacting with AT-rich DNA, such as the N-phenylbenzamide bis(2-aminoimidazoline) derivatives 1 [4-((4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide dihydrochloride] and 2 [N-(3-chloro-4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)-4-((4,5-dihydro-1H-imidazol-2-yl)amino)benzamide] as potential drugs for HAT. Both compounds show in vitro effects against T. brucei and in vivo curative activity in a mouse model of HAT. The main objective was to identify their cellular target inside the parasite. We were able to demonstrate that the compounds have a clear effect on the S-phase of T. brucei cell cycle by inflicting specific damage on the kinetoplast. Surface plasmon resonance (SPR)-biosensor experiments show that the drug can displace HMG box-containing proteins essential for kDNA function from their kDNA binding sites. The crystal structure of the complex of the oligonucleotide d[AAATTT]2 with compound 1 solved at 1.25 Å (PDB-ID: 5LIT) shows that the drug covers the minor groove of DNA, displaces bound water and interacts with neighbouring DNA molecules as a cross-linking agent. We conclude that 1 and 2 are powerful trypanocides that act directly on the kinetoplast, a structure unique to the order Kinetoplastida.

Bis(2-aminoimidazolines) and Bisguanidines: Synthetic Approaches, Antiparasitic Activity and DNA Binding Properties.

BACKGROUND: Parasitic diseases caused by protozoan parasites of the genus Trypanosoma and Plasmodium cause some of the deadliest and disabling human infections in tropical and subtropical areas. Diphenyl-based bis(2-phenylimino)imidazolidines and bisguanidines are extremely potent antiparasitic agents against Trypanosoma brucei (etiological agent of African trypanosomiasis) and Plasmodium falciparum (etiological agent of severe malaria). Many of these compounds are also curative in mouse models of stage 1 African trypanosomiasis representing promising leads for the development of antitrypanosomal drugs. In addition, different classes of bis(2-iminoimidazolidines) and bisguanidines have been shown to have antimicrobial activity against other pathogens (e.g. bacteria, fungi, parasitic worms). Due to their structural and physicochemical properties, these dibasic compounds, which are dications at physiological pH, are prone to bind to the minor groove of DNA at AT-rich sites. In several cases, such interaction is thought to be responsible for their antimicrobial activity. RESULTS: In this review, we give a comprehensive view of the synthetic methods used to introduce the 2-aminoimidazoline scaffold in a molecule. Synthetic routes that give access to these cyclic guanidines (i.e. unsubstituted, 1-, 4-, and 5-substituted 2-aminoimidazolines) are detailed. The in vitro and in vivo antiprotozoal activity of bis(2-aminoimidazolines) and bisguanidines against kinetoplastid parasites (T. brucei, T. cruzi, Leishmania), Plasmodium spp. and other pathogens (e.g. ESKAPE bacteria, Candida spp., M. tuberculosis, E. multilocularia) is also reviewed. Finally, the targets that are involved in the antimicrobial activity (e.g. DNA) or other biological activities (e.g. α-adrenergic receptors, imidazoline binding sites, kinases) of this class of dicationic compounds are discussed.

Conjugates of 2,4-Dihydroxybenzoate and Salicylhydroxamate and Lipocations Display Potent Antiparasite Effects by Efficiently Targeting the Trypanosoma brucei and Trypanosoma congolense Mitochondrion.

We investigated a chemical strategy to boost the trypanocidal activity of 2,4-dihydroxybenzoic acid (2,4-DHBA)- and salicylhydroxamic acid (SHAM)-based trypanocides with triphenylphosphonium and quinolinium lipophilic cations (LC). Three series of LC conjugates were synthesized that were active in the submicromolar (5a-d and 10d-f) to low nanomolar (6a-f) range against wild-type and multidrug resistant strains of African trypanosomes (Trypanosoma brucei brucei and T. congolense). This represented an improvement in trypanocidal potency of at least 200-fold, and up to >10 000-fold, compared with that of non-LC-coupled parent compounds 2,4-DHBA and SHAM. Selectivity over human cells was >500 and reached >23 000 for 6e. Mechanistic studies showed that 6e did not inhibit the cell cycle but affected parasite respiration in a dose-dependent manner. Inhibition of trypanosome alternative oxidase and the mitochondrial membrane potential was also studied for selected compounds. We conclude that effective mitochondrial targeting greatly potentiated the activity of these series of compounds.

Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei.

Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH) was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1) was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect) forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase.

Lowering the pKa of a bisimidazoline lead with halogen atoms results in improved activity and selectivity against Trypanosoma brucei in vitro.

Diphenyl-based bis(2-iminoimidazolidines) are promising antiprotozoal agents that are curative in mouse models of stage 1 trypanosomiasis but devoid of activity in the late-stage disease, possibly due to poor brain penetration caused by their dicationic nature. We present here a strategy consisting in reducing the pKa of the basic 2-iminoimidazolidine groups though the introduction of chlorophenyl, fluorophenyl and pyridyl ring in the structure of the trypanocidal lead 4-(imidazolidin-2-ylideneamino)-N-(4-(imidazolidin-2-ylideneamino)phenyl)benzamide (1). The new compounds showed reduced pKa values (in the range 1-3 pKa units) for the imidazolidine group linked to the substituted phenyl ring. In vitro activities (EC50) against wild type and resistant strains of T. b. brucei (s427 and B48, respectively) were in the submicromolar range with four compounds being more active and selective than 1 (SI > 340). In particular, the two most potent compounds (3b and 5a) acted approximately 6-times faster than 1 to kill trypanosomes in vitro. No cross-resistance with the diamidine and melaminophenyl class of trypanocides was observed indicating that these compounds represent interesting leads for further in vivo studies.

Antiprotozoal activity and DNA binding of dicationic acridones.

Dicationic acridone derivatives were synthesized and their antiparasitic activity was evaluated. Acridones displayed in vitro nanomolar IC50 values against Trypanosoma brucei rhodesiense STIB900 with selectivity indices >1000. Compounds 1b, 3a, and 3b were as potent as the reference drug melarsoprol in this assay. Submicromolar-range activities were observed against wild-type (NF54) and resistant (K1) strains of Plasmodium falciparum, whereas no significant activity was detected against Trypanosoma cruzi or Leishmania donovani. Compounds 1a and 1b were curative in the STIB900 mouse model for human African trypanosomiasis. UV spectrophotometric titrations and circular dichroism (CD) experiments with fish sperm (FS) DNA showed that these compounds form complexes with DNA with binding affinities in the 10(4) M(-1) range. Biological and biophysical data show that antiparasitic activity, toxicity, and DNA binding of this series of acridones are dependent on the relative position of both imidazolinium cations on the heterocyclic scaffold.

Observation of diastereotopic signals in (15) N†NMR spectroscopy.

The first example in the literature of a compound showing anisochronous (15) N atoms resulting from diastereotopicity is described. Racemic 1,3-dimethyl-2-phenyloctahydro-1H-benzimidazole was prepared and studied by (1) H, (13) C and (15) N NMR spectroscopy. If convenient conditions were used (monitored by theoretical calculations of (2) JN-H spin-spin coupling constants), two (15) N NMR signals were observed and corresponded to the diastereotopic atoms. GIAO/density-functional calculations of chemical shifts were not only in good agreement with the experimental values but also served as prediction tools. This study suggests that (15) N NMR spectroscopy could be used to probe chirality.

Influence of ornithine decarboxylase antizymes and antizyme inhibitors on agmatine uptake by mammalian cells.

Agmatine (4-aminobutylguanidine), a dicationic molecule at physiological pH, exerts relevant modulatory actions at many different molecular target sites in mammalian cells, having been suggested that the administration of this compound may have therapeutic interest. Several plasma membrane transporters have been implicated in agmatine uptake by mammalian cells. Here we report that in kidney-derived COS-7 cell line, at physiological agmatine levels, the general polyamine transporter participates in the plasma membrane translocation of agmatine, with an apparent Km of 44 ± 7 µM and Vmax of 17.3 ± 3.3 nmol h(-1) mg(-1) protein, but that at elevated concentrations, agmatine can be also taken up by other transport systems. In the first case, the physiological polyamines (putrescine, spermidine and spermine), several diguanidines and bis(2-aminoimidazolines) and the polyamine transport inhibitor AMXT-1501 markedly decreased agmatine uptake. In cells transfected with any of the three ornithine decarboxylase antizymes (AZ1, AZ2 and AZ3), agmatine uptake was dramatically reduced. On the contrary, transfection with antizyme inhibitors (AZIN1 and AZIN2) markedly increased the transport of agmatine. Furthermore, whereas putrescine uptake was significantly decreased in cells transfected with ornithine decarboxylase (ODC), the accumulation of agmatine was stimulated, suggesting a trans-activating effect of intracellular putrescine on agmatine uptake. All these results indicate that ODC and its regulatory proteins (antizymes and antizyme inhibitors) may influence agmatine homeostasis in mammalian tissues.