Natural Purine Research Articles

Recent Developments and Future Perspectives of Purine Derivatives as a Promising Scaffold in Drug Discovery.

Numerous purine-containing compounds have undergone extensive investigation for their medical efficacy across various diseases. The swift progress in purine-based medicinal chemistry has brought to light the therapeutic capabilities of purine-derived compounds in addressing challenging medical conditions. Defined by a heterocyclic ring comprising a pyrimidine ring linked with an imidazole ring, purine exhibits a diverse array of therapeutic attributes. This review systematically addresses the multifaceted potential of purine derivatives in combating various diseases, including their roles as anticancer agents, antiviral compounds (anti-herpes, anti-HIV, and anti-influenzae), autoimmune and anti-inflammatory agents, antihyperuricemic and anti-gout solutions, antimicrobial agents, antitubercular compounds, anti-leishmanial agents, and anticonvulsants. Emphasis is placed on the remarkable progress made in developing purine-based compounds, elucidating their significant target sites. The article provides a comprehensive exploration of developments in both natural and synthetic purines, offering insights into their role in managing a diverse range of illnesses. Additionally, the discussion delves into the structure-activity relationships and biological activities of the most promising purine molecules. The intriguing capabilities revealed by these purine-based scaffolds unequivocally position them at the forefront of drug candidate development. As such, this review holds potential significance for researchers actively involved in synthesizing purine-based drug candidates, providing a roadmap for the continued advancement of this promising field.

Dihydroazolopyrimidines: Past, Present and Perspectives in Synthesis, Green Chemistry and Drug Discovery.

Dihydroazolopyrimidines are an important class of heterocycles that are isosteric to natural purines and are therefore of great interest primarily as drug-like molecules. In contrast to the heteroaromatic analogs, synthetic approaches to these compounds were developed much later, and their chemical properties and biological activity have not been studied in detail until recently. In the review, different ways to build dihydroazolopyrimidine systems from different building blocks are described - via the initial formation of a partially hydrogenated pyrimidine ring or an azole ring, as well as a one-pot assembly of azole and azine fragments. Special attention is given to modern approaches: multicomponent reactions, green chemistry, and the use of non-classical activation methods. Information on the chemical properties of dihydroazolopyrimidines and the prospects for their use in the design of drugs of various profiles are also summarized in this review.

Nanomaterial-Doped Xerogels for Biosensing Measurements of Xanthine in Clinical and Industrial Applications.

First-generation amperometric xanthine (XAN) biosensors, assembled via layer-by-layer methodology and featuring xerogels doped with gold nanoparticles (Au-NPs), were the focus of this study and involved both fundamental exploration of the materials as well as demonstrated usage of the biosensor in both clinical (disease diagnosis) and industrial (meat freshness) applications. Voltammetry and amperometry were used to characterize and optimize the functional layers of the biosensor design including a xerogel with and without embedded xanthine oxidase enzyme (XOx) and an outer, semi-permeable blended polyurethane (PU) layer. Specifically, the porosity/hydrophobicity of xerogels formed from silane precursors and different compositions of PU were examined for their impact on the XAN biosensing mechanism. Doping the xerogel layer with different alkanethiol protected Au-NPs was demonstrated as an effective means for enhancing biosensor performance including improved sensitivity, linear range, and response time, as well as stabilizing XAN sensitivity and discrimination against common interferent species (selectivity) over time-all attributes matching or exceeding most other reported XAN sensors. Part of the study focuses on deconvoluting the amperometric signal generated by the biosensor and determining the contribution from all of the possible electroactive species involved in natural purine metabolism (e.g., uric acid, hypoxanthine) as an important part of designing XAN sensors (schemes amenable to miniaturization, portability, or low production cost). Effective XAN sensors remain relevant as potential tools for both early diagnosis of diseases as well as for industrial food monitoring.

Gut Metabolites and Breast Cancer: The Continuum of Dysbiosis, Breast Cancer Risk, and Potential Breast Cancer Therapy

The complex association between the gut microbiome and cancer development has been an emerging field of study in recent years. The gut microbiome plays a crucial role in the overall maintenance of human health and interacts closely with the host immune system to prevent and fight infection. This review was designed to draw a comprehensive assessment and summary of recent research assessing the anticancer activity of the metabolites (produced by the gut microbiota) specifically against breast cancer. In this review, a total of 2701 articles were screened from different scientific databases (PubMed, Scopus, Embase and Web of Science) with 72 relevant articles included based on the predetermined inclusion and exclusion criteria. Metabolites produced by the gut microbial communities have been researched for their health benefits and potential anticancer activity. For instance, the short-chain fatty acid, butyrate, has been evaluated against multiple cancer types, including breast cancer, and has demonstrated anticancer potential via various molecular pathways. Similarly, nisin, a bacteriocin, has presented with a range of anticancer properties primarily against gastrointestinal cancers, with nominal evidence supporting its use against breast cancer. Comparatively, a natural purine nucleoside, inosine, though it has not been thoroughly investigated as a natural anticancer agent, has shown promise in recent studies. Additionally, recent studies demonstrated that gut microbial metabolites influence the efficacy of standard chemotherapeutics and potentially be implemented as a combination therapy. Despite the promising evidence supporting the anticancer action of gut metabolites on different cancer types, the molecular mechanisms of action of this activity are not well established, especially against breast cancer and warrant further investigation. As such, future research must prioritise determining the dose-response relationship, molecular mechanisms, and conducting animal and clinical studies to validate in vitro findings. This review also highlights the potential future directions of this field.

Recent Advances in the Development of Pyrazolopyridines as Anticancer Agents.

Cancer, especially malignant tumor, is a serious threat to people's life and health. It is recognized as an enormous challenge in the 21st century. Continuous efforts are needed to overcome this problem. Pyrazolopyridine nucleus, similar in structure to purine, shows a variety of biological activities, which is mainly attributed to the antagonistic nature towards the natural purines in many biological processes. This has aroused enormous attention for many researchers. At present, a large number of new chemical entities containing pyrazolopyridine nucleus have been found as anticancer agents. In this review we summarize novel pyrazolopyridine-containing derivatives with biological activities. Furthermore, we outline the relationships between the structures of variously modified pyrazolopyridines and their anticancer activity.

Application of diazonium chemistry in purine modifications: A focused review

Deaminative modifications of purines via diazonium chemistry have long been the cornerstone in modified nucleoside synthesis. The purpose of this review is to bring into focus the impact of dediazoniative methods such as non‐aqueous Sandmeyer halogenations, fluorination, arylation, and carbon‐heteroatom bond formations on natural purines and their nucleosides as versatile tools for purine modifications en route to various nucleoside drugs, drug discovery scaffolds, and adducted oligonucleotides of biomedical interests. Metal free reactions of arenediazonium salts with purine nucleosides for the synthesis of 8‐aryl purines, in particular, 8‐aryl guanosines and photochromic 8‐arylazo purines are also highlighted. The treatise is expected to create broad interest among organic and biological chemists for further explorations in this area.

Intrinsic drug potential of oxazolo[5,4-d]pyrimidines and oxazolo[4,5-d]pyrimidines.

The oxazole and pyrimidine rings are widely displayed in natural products and synthetic molecules. They are known as the prime skeletons for drug discovery. On the account of structural and chemical diversity, oxazole and pyrimidine-based molecules, as central scaffolds, not only provide different types of interactions with various receptors and enzymes, showing broad biological activities, but also occupy a core position in medicinal chemistry, showing their importance for development and discovery of newer potential therapeutic agents (Curr Top Med Chem, 16, 2016, 3133; Int J Pharm Pharm Sci, 8, 2016, 8; BMC Chem, 13, 2019, 44). For a long time, relatively little attention has been paid to their fused rings that are oxazolopyrimidines, whose chemical structure is similar to that of natural purines because probably none of these compounds were found in natural products or their biological activities turned out to be unexpressed (Bull Chem Soc Jpn, 43, 1970, 187). Recently, however, a significant number of studies have been published on the biological properties of oxazolo[5,4-d]pyrimidines, showing their significant activity as agonists and antagonists of signaling pathways involved in the regulation of the cell life cycle, whereas oxazolo[4,5-d]pyrimidines, on the contrary, represent a poorly studied class of compounds. Limited access to this scaffold has resulted in a corresponding lack of biological research (Eur J Organ Chem, 18, 2018, 2148). Actually, oxazolo[5,4-d]pyrimidine is a versatile scaffold used for the design of bioactive ligands against enzymes and receptors. This review focuses on biological targets and associated pathogenetic mechanisms, as well as pathological disorders that can be modified by well-known oxazolopyrimidines that have been proven to date. Many molecular details of these processes are omitted here, which the interested reader will find in the cited literature. This work also does not cover the methods for the synthesis of the oxazolopyrimidines, which are exhaustively described by De Coen et al. (Eur J Organ Chem, 18, 2018, 2148). The review as well does not discuss the structure-activity relationship, which is described in detail in the original works and deliberately, whenever possible, cites not primary sources, but mostly relevant review articles, so that the reader who wants to delve into a particular problem will immediately receive more complete information. It is expected that the information presented in this review will help readers better understand the purpose of the development of oxazolopyrimidines and the possibility of their development as drugs for the treatment of a wide range of diseases.

Synthesis, Antitumor and Antiviral In Vitro Activities of New Benzotriazole-Dicarboxamide Derivatives.

Cancer and viral infections continue to threaten humankind causing death worldwide. Hence, the discovery of new anticancer and antiviral agents still represents a major scientific goal. Heterocycles designed to mimic the chemical structure of natural pyrimidines and purines have been designed over the years, exerting their activity acting as false substrates on several different targets. We reported a series of bis-benzotriazole-dicarboxamide derivatives which inhibit viral helicase of poliovirus, and hence we planned structure modifications to obtain different series of new dicarboxamides. Here, the synthesis and characterization of 56 new compounds: 31 bis-benzotriazole dicarboxamides and 25 mono-substituted acidic derivatives are reported. The synthesized compounds were tested for their antiviral and antitumor activity. Mostly, compounds 4a, 4c and 4d showed antiviral activity against tested Picornaviruses, Coxsackievirus B5 and Poliovirus-1. Likewise, four derivatives (3b, 3d, 4d, 9b) showed notable antiproliferative activity inhibiting cell growth in two distinct antitumor screenings. Compound 3b was selected as the antitumor lead compound for the wide range of activity and the potency proved. The lead compound was proved to induce apoptosis in SK-MES1 tumor cells, in a dose-dependent manner.

Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms

Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors reduced but not abolished it. Importantly, GUO-mediated neuron-like cell differentiation was independent of adenosine receptor activation as it was not altered by the blockade of these receptors. Noteworthy, the neuritogenic activity of GUO was not affected by blocking the phosphoinositide 3-kinase pathway, while it was reduced by inhibitors of protein kinase C or soluble guanylate cyclase. Furthermore, the inhibitor of the enzyme heme oxygenase-1 but not that of nitric oxide synthase reduced GUO-induced neurite outgrowth. Interestingly, we found that GUO was largely metabolized into guanine by the purine nucleoside phosphorylase (PNP) enzyme released from cells. Taken together, our results suggest that GUO, promoting neuroblastoma cell differentiation, may represent a potential therapeutic agent; however, due to its spontaneous extracellular metabolism, the role played by the GUO-PNP-guanine system needs to be further investigated.

Ru complexes containing Cp, mPTA and natural purine bases (mPTA = methyl-N-1,3,5-triaza-7-phosphaadamantane): Evaluation of their antiproliferative activity, solubility and redox properties

Complexes [RuCp(Adeninate–κN9)(mPTA)2](Cl0.5)(CF3SO3)2.5·H2O (1·H2O), [RuCp(Guaninate-κN7)(mPTA)2](CF3SO3)2·H2O (2·H2O), [RuCp(Theophyllinate-κN7)(mPTA)2](CF3SO3)2·1.5H2O (3·1.5H2O) and [RuCp(Pur-κN)(mPTA)(PPh3)](CF3SO3) (4–6) (Pur = Adeninate, Guaninate, Teophyllinate; mPTA = N-methyl −1,3,5-triaza-7-phosphaadamantane) have been synthesized and characterized. Structure of complexes 1·H2O and 3·1.5H2O were determined by single-crystal X-ray diffraction. Solubility in water, Log P, electrochemical properties and antiproliferative activities of the complexes (against cisplatin-sensitive T2 and cisplatin-resistant SKOV3 cell lines) have been assessed and discussed.

Preclinical in vitro screening of newly synthesised amidino substituted benzimidazoles and benzothiazoles

Newly synthesised benzimidazole/benzotiazole derivatives bearing amidino, namely 3,4,5,6-tetrahydropyrimidin-1-ium chloride, substituents have been evaluated for their potential antitumor activity in vitro. Compounds and standard drugs (doxorubicin, staurosporine and vandetanib) were tested on three human lung cancer cell lines A549, HCC827 and NCI-H358. We tested compounds in MTS citotoxicity assay and in BrdU proliferative assay performed on 2 D and 3 D assay format. Because benzmidazole scaffold is similar to natural purines, we tested the most active compounds for ability to induce cell apoptosis of A549 by binding to DNA in comparison with doxorubicin and saturosporine. Additionally, the ADME properties of the most active benzothiazole/benzimidazole and non-active compounds were determined to see if the different ADME properties are the cause of different activity in 2 D and 3 D assays, as well as to see if the tested active compounds have drug like properties and potency for further profilation. ADME characterisation included solubility, lipophilicity, permeability, metabolic stability and binding to plasma proteins. In general, the benzothiazole derivatives were more active in comparison to their benzimidazole analogues. The exception was 2-phenyl substituted benzimidazole 6a being active with very pronounced activity especially towards HCC827 cells. All active compounds have similar mode of action on A549 cell line as standard compound doxorubicin, which binds to nucleic acids with the DNA double helix. Tested active benzothiazole compounds were characterised by moderate to good solubility, good metabolic stability, low permeability and high binding to plasma proteins. One tested active benzimidazole derivative showed ADME properties, but lower lipophilicity resulted in low PPB and higher metabolic instability. In addition, no significant difference was observed in ADME profile between active and non-active compounds.

Synthesis and Biological Evaluation of 1,3-Dideazapurine-Like 7-Amino-5-Hydroxymethyl-Benzimidazole Ribonucleoside Analogues as Aminoacyl-tRNA Synthetase Inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these “base-flipped” analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

Design and Synthesis of New Substituted Pyrazolopyridines with Potent Antiproliferative Activity.

Purine isosteres are often endowed with interesting pharmacological properties, due to their involvement in cellular processes replacing the natural purines. Among these compounds, pyrazolopyridines are under active investigation for potential anticancer properties. Based on previously discovered substituted pyrazolopyridines with promising antiproliferative activity, we designed and synthesized new, suitably substituted analogues aiming to investigate their potential activity and contribute to SAR studies of this class of bioactive compounds. The new compounds were synthesized using suitably substituted 2-amino-4-picolines, which upon ring-closure provided substituted pyrazolo[3,4-c] pyridine-5-carbonitriles that served as key intermediates for the preparation of the target 3,5,7 trisubstituted derivatives. The antiproliferative activity of 31 new target derivatives was evaluated against three cancer cell lines (MIA PaCa-2, PC-3 and SCOV3), whereas cell-cycle perturbations of exponentially growing PC-3 cells, using three selected derivatives were also performed. Eight compounds displayed IC50 values in the low μM range, allowing the extraction of interesting SAR's. Two of the most potent compounds against all cell lines share a common pattern, by accumulating cells at the G0/G1 phase. From this project, a new carboxamidine-substituted hit has emerged. Among the new compounds, those possessing the 3-phenylpyrazolo[3,4-c]pyridine scaffold, proved to be worth investigating and the majority of them showed strong cytotoxic activity against all cell lines, with IC50 values ranging from 0.87-4.3 µM. A carboxamidine analogue that resulted from the synthetic procedure, proved to be highly active against the cancer cells and could be considered as a useful lead for further optimization.

A spectroscopic and computational intervention of interaction of lysozyme with 6-mercaptopurine.

In the present work, biophysical insight into the binding interactions of the protein, hen egg white (HEW) lysozyme (Lyz) with an anticancer drug, 6-mercaptopurine (6-MP)' was investigated by using a combination of spectroscopic and computational tools. 6-MP, a synthetic analog of natural purines, is a well-known anticancer drug and antiviral agent that inhibits the synthesis of RNA, DNA, and proteins. Lysozyme is a single-chain protein that can combine with endogenous and exogenous substances to exert its antiviral, antibacterial, and antitumor effects. The intrinsic fluorescence of lysozyme was quenched with the increased addition of 6-MP. The quenching mechanism was found to be static in nature as shown by the fluorescence lifetime and excitation spectrum measurements. The conformational changes of Lyz in the presence of 6-MP were monitored both at the ensemble and single-molecule level by using synchronous fluorescence spectroscopy, circular dichroism (CD), and fluorescence correlation spectroscopy (FCS). Molecular docking results predicted the probable binding sites for 6-MP on Lyz. The experimental findings are in good agreement with the results obtained by the molecular dynamics (MD) simulation study. Graphical abstract.

CpRu Complexes Containing Water Soluble Phosphane PTA and Natural Purines Adenine, Guanine and Theophylline: Synthesis, Characterization, and Antiproliferative Properties

Complexes [RuCp(Adeninate‐κN9)(PTA)2] (1), [RuCp(Guaninate‐κN9)(PTA)2] (2), [RuCp(Theophyllinate‐κN7)(PTA)2] (3), [RuCp(Adeninate‐κN9)(PPh3)(PTA)] (4), [RuCp(Guaninate‐κN9)(PPh3)(PTA)] (5) and [RuCp(Theophyllinate‐κN7)(PPh3)(PTA)] (6) were synthesized and characterized by NMR spectroscopy, elemental analysis and FT‐IR (PTA = 1,3,5‐triaza‐7‐phosphaadamantane). Crystal structures of 1·H2O·3EtOH, 3·CH3COCH3 and 4·H2O were also determined by single‐crystal X‐ray diffraction. The antiproliferative activities of the complexes against cisplatin‐sensitive T2 and cisplatin‐resistant SKOV3 cell lines have also been evaluated. Theoretical studies were preformed to elucidate how the adeninate ligand is coordinated to the metal.

THE DIRHENIUM(III) COMPLEX COMPOUNDS WITH IMIDAZOLE AND BENZIMIDAZOLE

Imidazole and it’s derivatives are structural fragments of certain enzymes, aminoacids, alkaloids and drugs. To date, the participation of imidazole in human metabolism, especially in the biosynthesis of natural purine bases of RNA and DNA, has been proven. Benzimidazole is one of the most used compound in medical chemistry. The various derivatives of substances, that exhibit analgesic, antidiabetic, anti-inflammatory, antitumor and antiviral activities were synthesized based on benzimidazole. This fact paves the way for the synthesis of antimetabolites, which can delay biosynthesis during biotransformation and inhibit the growth of malignant tumors. Thus, in the present work, the interaction of complex compounds with a single cluster fragment of Re 2 6+ (NBu 4 ) 2 Re 2 Cl 8 and trans-tetrachlorodi-μ-alkylcarboxylates of dirhenium(III) with representatives of the azole class – imidazole and benzimidazole was studied under various conditions. As a result of the experiments, methods for the synthesis of cis-[Re 2 (X) 4 Cl 4 (CH 3 CN) 2 ]Cl 2 (X is imidazole, benzimidazole) in an electron-donating organic solvent – CH3CN under heating in an inert atmosphere were developed. The starting materials were (NBu4)2Re2Cl8 and the selected azoles at a molar ratio of 1:10. The yield of the target compounds was 67-75%. We also investigated the reaction between trans-tetrachloridi-μ-alkylcarboxylates of dirhenium(III) and indicated azoles in a non-donor solvent (1,2-dichloroethane) in inert atmosphere at a molar ratio of reactants of 1:20. As a result of the interaction, complex compounds of the general formula (XH) 2 Re 2 Cl 8 (X – imidazole, benzimidazole) are formed, in which azoles are cations. The yield of reaction products was 83-87%. All target substances are synthesized, isolated in individual state, their spectral properties are investigated. The composition and structure of the obtained complex compounds were confirmed by IR- and NMR- spectroscopy on 1H and 13C, conductometry, and electronic absorption spectroscopy.

Metal-Modified Nucleic Acids: Metal-Mediated Base Pairs, Triples, and Tetrads.

The incorporation of metal ions into nucleic acids by means of metal-mediated base pairs represents a promising and prominent strategy for the site-specific decoration of these self-assembling supramolecules with metal-based functionality. Over the past 20 years, numerous nucleoside surrogates have been introduced in this respect, broadening the metal scope by providing perfectly tailored metal-binding sites. More recently, artificial nucleosides derived from natural purine or pyrimidine bases have moved into the focus of AgI -mediated base pairing, due to their expected compatibility with regular Watson-Crick base pairs. This minireview summarizes these advances in metal-mediated base pairing but also includes further recent progress in the field. Moreover, it addresses other aspects of metal-modified nucleic acids, highlighting an expansion of the concept to metal-mediated base triples (in triple helices and three-way junctions) and metal-mediated base tetrads (in quadruplexes). For all types of metal-modified nucleic acids, proposed or accomplished applications are briefly mentioned, too.

A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition

Natural purines like ATP, ADP and adenosine have crucial roles in platelet physiology. This knowledge has been significant in drug development and today ADP receptor antagonists are widely used for prevention of thrombotic events following myocardial infarction and ischaemic stroke.Recent studies have shown that a purine analogue bearing nitrate ester group (denoted MK128) has anti-inflammatory effects probably due to its ability to donate nitric oxide (NO). However, other pharmacological mechanisms may contribute to the observed effect. The aim of the present study was to establish the anti-platelet activity and elucidate the underlying molecular mechanism(s) of the purine analogue MK128.We found that MK128 reduced aggregation and secretion induced by the thrombin receptor agonist SFLLRN and nearly abolished aggregation and secretion induced by thromboxane A2 (TxA2) and collagen receptor agonists. The inhibition took place despite blockage of the NO/cGMP signalling system. Furthermore, interaction between MK128 and platelet purinergic receptors did not explain the observed inhibition. Instead, we found that MK128 concentration-dependently inhibited Rho-associated kinase (ROCK), which led to decreased ROCK-dependent myosin phosphatase target subunit (MYPT)-1 phosphorylation and suppression of platelet functional responses.

Fused Heterocyclic Systems with an s-Triazine Ring. 34. Development of a Practical Approach for the Synthesis of 5-Aza-isoguanines.

Purine isosteres present excellent opportunities in drug design and development. Using isosteres of natural purines as scaffolds for the construction of new therapeutic agents has been a valid strategy of medicinal chemistry. Inspired by the similarity to isoguanine, we attempted to develop a practical method for the preparation of 5-aza-isoguanines. Several synthetic approaches were explored to establish a robust general protocol for the preparation of these compounds. The significant difference in the reactivity of the C-5 and C-7 electrophilic centers of 1,2,4-triazolo[1,5-a][1,3,5]triazines (5-azapurines) towards nucleophiles was demonstrated. The most practical and general method for the preparation of 5-aza-isoguanines involved a regioselective reaction of ethoxycarbonyl isothiocyanate with a 5-aminotriazole. The intramolecular ring closure of the resulted product followed by the S-methylation afforded 7-methylthio-2-phenyl-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-one, which could be effectively aminated with various amines. The resulted 5-aza-isoguanines resemble a known purine nucleoside phosphorylase inhibitor and could be interesting for further investigations as potential anticancer agents.

Trypanosoma brucei bloodstream forms express highly specific and separate transporters for adenine and hypoxanthine; evidence for a new protozoan purine transporter family?

The transport of nucleobases and nucleosides in protozoan parasites is known to be performed by Equilibrative Nucleoside Transporter (ENT) family members, including the extensively studied P1 and P2 nucleoside transporters of T. brucei bloodstream forms. Studies with P2 knockout parasites suggested the existence of as yet uncharacterised purine transport mechanisms in these cells. Here, we deleted several ENT genes, in addition to P2, including an array comprising three genes encoding for high-affinity broad-selectivity nucleobase transporters − the longest multi-gene locus deletion in T. brucei to date. It was verified that none of them appreciably contributed to the transport of hypoxanthine in bloodstream forms grown axenically in HMI-9 medium, which was mainly performed by a previously not described hypoxanthine-specific transporter (HXT1) with a Km of 22 ± 1.7 μM and Vmax of 0.49 ± 0.06 pmol(107 cells)−1 s−1. The uptake of adenine was also assessed in the knockout cells and was performed by a highly specific adenine transporter (ADET1) with a Km of 573 ± 62 nM and Vmax of 0.23 ± 0.06 pmol(107 cells)−1 s−1. Neither HXT1 nor ADET1 displayed any affinity for other natural purines or pyrimidines and could not be completely inhibited by hypoxanthine or adenine analogues. These carriers may be the final pieces in the substantial transporter array trypanosomes can employ to fine-tune the uptake of purines from diverse environments during their life cycles, and may be encoded by genes other than those of the ENT family.