Design, synthesis, electrochemistry and anti-trypanosomatid hit/lead identification of nitrofuranylazines.

Chagas disease and leishmaniasis are vector-borne infectious diseases affecting both humans and animals. These neglected tropical diseases can be fatal if not treated. Hundreds to thousands of new Chagas disease and leishmaniasis cases are being reported by the WHO every year, and currently available treatments are insufficient. Severe adverse effects, impractical administrations and increased pathogen resistance against current clinical treatments underscore a serious need for the development of new drugs to curb these ailments. In search for such drugs, we investigated a series of nitrofuran-based azine derivatives. Herein, we report the design, synthesis, electrochemistry, and biological activity of these derivatives against promastigotes and amastigotes of Leishmania major, and L. donovani strains, as well as epimastigotes and trypomastigotes of Trypanosoma cruzi. Two leishmanicidal early leads and one trypanosomacidal hit with submicromolar activity were uncovered and stand for further in vivo investigation in the search for new antitrypanosomatid drugs. Future objective will focus on the identification of involved biological targets with the parasites.

Diagnostic applications and limitations for the use of cell-free fetal DNA (cffDNA) in animal husbandry and wildlife management.

In animal breeding, a species sex can influence the value of the animal. For example, in the horse breeding industry, mares are preferred as polo horses, while in wildlife breeding males with larger horns are more valuable. Therefore, the economic advantages of knowing the unborn fetus' sex are important to successful animal management. Ultrasonography is used to determine the sex of unborn fetuses, but this method places additional stress on the animal and require specialized equipment and expertise. Conversely, molecular-based sexing techniques require less invasive sampling and can determine sex more reliably. Although in humans, various studies have evaluated the use of cell-free fetal DNA (cffDNA) for prenatal sexing, very few animal studies have been published in this field. Several factors can affect the sensitivity of cffDNA-based sex determination, for example the gestational age. These factors are often not optimized and validated when establishing a protocol for prenatal sexing. In this review, we summarize the current literature on cffDNA in animals. We discuss the diagnostic applications and limitations in the use thereof in animal husbandry and wildlife management. Lastly, the feasibility of implementing diagnostic tests is evaluated and solutions are given to the current drawbacks of the technology.

Exploration of ethylene glycol linked nitrofurantoin derivatives against Leishmania: Synthesis and in vitro activity.

Leishmaniasis is a neglected tropical disease that is caused by the Leishmania parasite. It is estimated that there are more than 350 million people at risk of infection annually. Current treatments that are in clinical use are expensive, have toxic side effects, and are facing parasitic resistance. Therefore, new drugs are urgently required. In the quest for new, safe, and cost-effective drugs, a series of novel ethylene glycol derivatives of nitrofurantoin was synthesised and the in vitro antileishmanial efficacy of the compounds tested against Leishmania donovani and Leishmania major strains. Arylated ethylene glycol derivatives were found to be the most potent, with submicromolar activity up to 294-fold greater than the parent compound nitrofurantoin. Analogues 2j and 2k had the best antipromastigote activities with submicromolar IC50 values against L. major IR-173 and antimonial-resistant L. donovani 9515 strains.

Synthesis and in vitro antileishmanial activity of alkylene-linked nitrofurantoin-triazole hybrids.

Leishmaniasis is a vector-borne parasitic disease that mostly affects populations in tropical and sub-tropical countries. There is currently no protective anti-leishmanial vaccine and only a paucity of clinical drugs is available to treat this disease albeit their toxicity. Leishmaniasis is curable but its eradication and elimination have been hampered by the emergence of multidrug resistant strains of the causative pathogens. This heightens the necessity for new and effective antileishmanial drugs. In search for such agents, nitrofurantoin, a clinical antibiotic, was appended to triazole scaffold through alkylene linkers of various length, and the resulting hybrids were evaluated for in vitro antileishmanial efficacy against Leishmania (L.) parasite of two strains. The hybrid 13, harboring a n-pentylene linker was uncovered as a leishmanicidal hit with micromolar activity against antimonial-resistant L. donovani, the causative of deadly visceral Leishmaniasis.

Exploring novel nitrofuranyl sulfonohydrazides as anti-Leishmania and anti-cancer agents: Synthesis, in vitro efficacy and hit identification.

Leishmaniasis and cancer are two deadly diseases that plague the human population. There are a limited number of drugs available for the treatment of these diseases; however, their overuse has resulted in pathogenic resistance. Recent studies have indicated the repurposing of nitro-containing compounds to be a new avenue into finding new treatments. In this study, new nitrofuranyl sulfonohydrazide derivatives were synthesized and evaluated for their in vitro antileishmanial and anticancer activities. The analogue 2h, featuring biphenyl moiety exhibited selective (SI > 10) submicromolar activity (IC50 0.97 µM) against acute promyelocytic leukemia cells hence was identified anticancer hit. This study revealed no antileishmanial hit. However, several promising analogues were uncovered and are worthy of further structural modifications to improve their toxicity and bioactivity profiles.

Probing O-substituted nifuroxazide analogues against Leishmania: Synthesis, in vitro efficacy, and hit/lead identification.

Leishmaniasis is a neglected tropical disease affecting millions of people worldwide, with 650 000 to 1.1 million new infections reported annually by the World Health Organization. Current antileishmanial treatments are unsatisfactory due to the development of parasitic resistance and the toxicity associated with the drugs used, and this highlights the need for the development of new antileishmanial drugs. In this study, a series of nifuroxazide analogues were synthesized in a single step reaction and investigated for their antileishmanial potential. The sulfonate 1l, bearing pyridine ring, was deemed an antileishmanial hit, targeting the amastigotes of Leishmania (L.) donovani and L. major, the pathogens of visceral and cutaneous leishmaniasis, respectively, with micromolar potencies. The benzyl analogues 2c and 2d were also confirmed as submicromolar active leads against amastigotes of L. major. These analogues stand as promising candidates for further investigation involving the evaluation of their in vivo activities and molecular targets.

Synthesis, in†vitro Antileishmanial Efficacy and Hit/Lead Identification of Nitrofurantoin-Triazole Hybrids.

Leishmaniasis is a vector-borne neglected parasitic infection affecting thousands of individuals, mostly among populations in low- to moderate-income developing countries. In the absence of protective vaccines, the management of the disease banks solely on chemotherapy. However, the clinical usefulness of current antileishmanial drugs is threatened by their toxicity and the emergence of multidrug-resistant strains of the causative pathogens. This emphasizes the imperative for the development of new and effective antileishmanial agents. In this regard, we synthesized and evaluated in vitro the antileishmanial activity and cytotoxicity profile of a series of nitrofurantoin-triazole hybrids. The nitrofurantoin derivative 1 featuring propargyl moiety was distinctively the most active of all, was nontoxic to human cells and possessed submicromolar cellular activity selectively directed towards the pathogens of the life threatening visceral leishmaniasis. Hence it was identified as potential antileishmanial lead for further investigation into its prospective to act as alternative to therapies.

In vitro antileishmanial efficacy of antiplasmodial active aminoquinoline-chalcone hybrids.

Significant overlaps in the geographical distribution of malaria and leishmaniasis increase the risk for comorbidity, which can affect treatment efficacy, cotreatment compatibility and disease progression. These concerns are also exacerbated by the existing shortcomings of malaria and leishmaniasis treatments. There is, therefore, a pressing need for new anti-infective drugs for both individual diseases and coinfections. The in vitro antileishmanial activity of previously synthesized antiplasmodial aminoquinoline-chalcone hybrids was evaluated. Hybrid 6, featuring a N-methyl-1,3-propylene diamine linker between pharmacophores, was 11-fold more potent in anti-amastigote activity against Leishmania major, responsible for cutaneous leishmaniasis, the most common form of the disease, in comparison to chloroquine. Hybrid 7, with a 2,2-(ethylenedioxy)bis(ethylamine) linker, was nearly 7-fold more active in anti-amastigote activity against Leishmania donovani, responsible for visceral leishmaniasis, the most lethal form of the infection. Although these two hybrids were less potent than the clinically used antileishmanial, amphotericin B, they still qualify as hits against both Plasmodium and Leishmania strains. Accordingly, this may lend them as potential agents against Leishmania-Plasmodium coinfections, which will require further investigation using in vitro co-cultures and subsequent in vivo testing for confirmation.

Chalcone-inspired rA1 /A2A adenosine receptor ligands: Ring closure as an alternative to a reactive substructure.

Over the past few years, great progress has been made in the development of high-affinity adenosine A1 and/or A2A receptor antagonists-promising agents for the potential treatment of Parkinson's disease. Unfortunately, many of these compounds raise structure-related concerns. The present study investigated the effect of ring closures on the rA1 /A2A affinity of compounds containing a highly reactive α,ß-unsaturated carbonyl system, hence providing insight into the potential of heterocycles to address these concerns. A total of 12 heterocyclic compounds were synthesised and evaluated in silico and in vitro. The test compounds performed well upon qualitative assessment of drug-likeness and were generally found to be free from potentially problematic fragments. Most also showed low/weak cytotoxicity. Results from radioligand binding experiments confirm that heterocycles (particularly 2-substituted 3-cyanopyridines) can replace the promiscuous α,ß-unsaturated ketone functional group without compromising A1 /A2A affinity. Structure-activity relationships highlighted the importance of hydrogen bonds in binding to the receptors of interest. Compounds 3c (rA1 Ki  = 16 nM; rA2A Ki  = 65 nM) and 8a (rA1 Ki  = 102 nM; rA2A Ki  = 37 nM), which both act as A1 antagonists, showed significant dual A1 /A2A affinity and may, therefore, inspire further investigation into heterocycles as potentially safe and potent adenosine receptor antagonists.

Synthesis and comparison of in vitro dual anti-infective activities of novel naphthoquinone hybrids and atovaquone.

A principal factor that contributes towards the failure to eradicate leishmaniasis and tuberculosis infections is the reduced efficacy of existing chemotherapies, owing to a continuous increase in multidrug-resistant strains of the causative pathogens. This accentuates the dire need to develop new and effective drugs against both plights. A series of naphthoquinone-triazole hybrids was synthesized and evaluated in vitro against Leishmania (L.) and Mycobacterium tuberculosis (Mtb) strains. Their cytotoxicities were also evaluated, using the human embryonic kidney cell line (HEK-293). The hybrids were found to be non-toxic towards human cells and had demonstrated micromolar cellular antileishmanial and antimycobacterial potencies. Hybrid 13, i.e. 2-{[1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl]methoxy}naphthalene-1,4-dione was the most active of all. It was found with MIC90 0.5 µM potency against Mtb in a protein free medium, and with half-maxima inhibitory concentrations (IC50) of 0.81 µM and 1.48 µM against the infective promastigote parasites of L. donavani and L. major, respectively, with good selectivity towards these pathogens (SI 22 - 65). Comparatively, the clinical naphthoquinone, atovaquone, although less cytotoxic, was found to be two-fold less antimycobacterial potent, and six- to twelve-fold less active against leishmania. Hybrid 13 may therefore stand as a potential anti-infective hit for further development in the search for new antitubercular and antileishmanial drugs. Elucidation of its exact mechanism of action and molecular targets will constitute future endeavour.

Synthesis and in vitro antileishmanial efficacy of novel benzothiadiazine-1,1-dioxide derivatives.

Leishmaniasis is a major vector-borne parasitic disease that affects thousands of people in tropical and subtropical developing countries. In 2019 alone, it killed 26,000-65,000 individuals. Leishmaniasis is curable, yet its eradication and elimination are hampered by major hurdles, such as the availability of only a handful of clinical toxic drugs and the emergence of pathogenic resistance against them. This underscores the imperative need for new and effective antileishmanial drugs. In search for such agents, we synthesized and evaluated the in vitro antileishmanial potential of a small library of benzothiadiazine derivatives by assessing their activity against the promastigotes of three strains of Leishmania and toxicity in healthy cells. The derivatives were found to have no toxicity to the mammalian cells and were, in general, active against all parasites. The benzothiadiazine derivative 1e, 3-methyl-2-[3-(trifluoromethyl)benzyl]-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide, was found to be the most active (IC50 , 0.2 µM) against Leishmania major, responsible for the most prevalent disease form, cutaneous leishmaniasis. Conversely, benzothiadiazine 2c, 2-(4-bromobenzyl)-3-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide, was the most potent (IC50 , 6.5 µM) against Leishmania donovani, a causative strain of the lethal visceral leishmaniasis. Both compounds stand as antipromastigote hits for further lead investigation into their potential to act as new antileishmanial agents.

Synthesis and in vitro antileishmanial efficacy of novel quinazolinone derivatives.

Currently available drugs being used to treat leishmaniasis have several shortcomings, including high toxicity, drug administration that requires hospitalization, and the emergence of parasite resistance against clinically used drugs. As a result, there is a dire need for the development of new antileishmanial drugs that are safe, affordable, and efficient. In this study, two new series of synthesized quinazolinone derivatives were investigated as potential future antileishmanial agents, by assessing their activities against the Leishmania (L.) donovani and L. major species. The cytotoxicity profiles of these derivatives were assessed in vitro on Vero cells. The compounds were found to be safer and without any toxic activities against mammalian cells, compared to the reference drug, halofuginone, a clinical derivative of febrifugine. However, they had demonstrated poor antileishmanial growth inhibition efficacies. The two compounds that had been found the most active were the mono quinazolinone 2d and the bisquinazolinone 5b with growth inhibitory efficacies of 35% and 29% for the L. major and L. donovani 9515 promastigotes, respectively. These outcomes had suggested structural redesign, inter alia the inclusion of polar groups on the quinazolinone ring, to potentially generate novel quinazolinone derivatives, endowed with effective antileishmanial potential.

Synthesis and in vitro antileishmanial efficacy of benzyl analogues of nifuroxazide.

Leishmaniasis is a vector-borne parasitic disease that mostly affects populations in tropical and subtropical countries. There is currently no vaccine to protect against and only a handful of drugs are available to treat this disease. Leishmaniasis is curable, but its eradication and elimination are hindered by the emergence of multidrug resistant strains of the causative pathogens, accentuating the need for new and effective antileishmanial drugs. In search for such agents, nifuroxazide, a clinical antibiotic, was evaluated through investigation of its benzyl analogues for in vitro antileishmanial efficacy against promastigotes of various Leishmania (L.) strains. The monobenzylated analogues 1 and 2 were the most potent of all, possessing nanomolar activities up to 10-fold higher than the parent drug nifuroxazide against all three tested Leishmania strains. Both analogues stand as antipromastigote hits for further lead investigation into their potential to act as new antileishmanial agents.

In vitro efficacy of synthesized artemisinin derivatives against Leishmania promastigotes.

Leishmaniasis is a neglected tropical disease affecting thousands worldwide, especially in developing countries where it co-exists with malaria. Only a handful of drugs are clinically available to treat the disease, but significant limitations threaten their very use. New, safe and effective drugs, including those against malaria-leishmaniasis co-infections, are thus imperative. We assessed the in vitro anti-infective potential of previously synthesized, potent antimalarial artemisinin derivatives. Analogue esters featuring 1,1'-biphenyl and thiophenyl moieties were as much as 30-fold more potent than clinical artemisinins against L. donovani parasites, qualifying them as antipromastigote hits for further investigation in the search for malaria-leishmaniasis co-infection therapies.

Single-step synthesis and in vitro anti-mycobacterial activity of novel nitrofurantoin analogues.

The emergence of drug-resistant tuberculosis (DR-TB) as well as the requirement for long, expensive and toxic drug regimens impede efforts to control and eliminate TB. Therefore, there's a need for effective and affordable anti-mycobacterial agents which can shorten the duration of therapy and are active against Mycobacterium tuberculosis (Mtb) in both active and latent phases. Nitrofurantoin (NFT) is a hypoxic agent with activity against a myriad of anaerobic pathogens and, like the first-line TB drug, rifampicin (RIF), kills non-replicating bacilli. However, the poor ability of NFT to cross host cell membranes and penetrate tissue means that it does not reach therapeutic concentrations. To improve TB efficacy of NFT, a series of NFT analogues was synthesized and evaluated in vitro for anti-mycobacterial activity against the laboratory strain, Mtb H37Rv, and for potential cytotoxicity using human embryonic kidney (HEK-293) and Chinese hamster ovarian (CHO) cells. The NFT analogues showed good safety profiles, enhanced anti-mycobacterial potency, improved lipophilicity, as well as reduced protein binding affinity. Analogue 9 which contains an eight carbon aliphatic chain was the most active, equipotent to isoniazid (INH), a major front-line agent, with MIC90 = 0.5 µM, 30-fold more potency than the parent drug, nitrofurantoin (MIC90 = 15 µM), and 100-fold more selective towards mycobacteria. Therefore, 9 was identified as a validated hit for further investigation in the urgent search for new, safe and affordable TB drugs.

Synthesis and evaluation of methoxy substituted 2-benzoyl-1-benzofuran derivatives as lead compounds for the development adenosine A1 and/or A2A receptor antagonists.

A series of fourteen methoxy substituted 2-benzoyl-1-benzofuran derivatives were synthesised and their affinities determined for adenosine A1 and A2A receptors via radioligand binding assays to establish the structure activity relationships pertinent for A1 and A2A affinity. Compound 3j (6,7-dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone exhibited A1 affinity (A1Ki (rat) = 6.880 µM) as well as A2A affinity (A2AKi (rat) = 0.5161 µM). Compounds 3a-b &3i-k exhibited selective affinity towards A1 with Ki values below 10 µM. The results indicate that C6,7-diOCH3 substitution on ring A in combination with meta (C3')-OCH3 substitution on ring B is beneficial for A1 and A2A affinity and activity. Compounds 3a-b &3j-k showed low cytotoxicity. Upon in vitro and in silico evaluation, compound 3j may be considered lead-like (i.e. a molecular entity suitable for optimization) and, thus, of value in the design of novel, potent and selective adenosine A1 and A2A receptor antagonists.

An update on derivatisation and repurposing of clinical nitrofuran drugs.

5-nitrofurans (NFs) have been in clinical use for over 60 years. These affordable drugs are used for the treatment of a broad spectrum of diseases ranging from urinary tract infections to cancer. The anti-pathogenic effect of clinical NFs occurs following a step-wise process involving activation by azoreduction, followed by nitroreduction catalysed by azoreductases and nitroreductases (NTRs), respectively. Azoreduction yields stable metabolites that have the ability to covalently bind to cellular proteins. Nitroreduction, on the other hand, occurs by type I or II reduction of the nitro group in the presence of parasitic NADPH-cytochrome P450 reductases. Type I NTRs catalyse, under anaerobic conditions, the reduction of NFs to produce anti-pathogenic hydroxylamine. Under aerobic conditions, nitroreduction catalysed by type II NTRs produces reactive oxygen and nitrogen species (ROS/RNS), causing oxidative stress to pathogens and ultimate death. This multi-activity nature of NFs thus allows the repurposing of these drugs from agricultural chemicals and basic antibiotics to efficient therapies against human life-threatening diseases. Cases of NF resistance in pathogens are also limited likely due to this multi-activity, as well as effectivity under both aerobic and anaerobic conditions. However, multi-activity of these drugs can also infer toxicity. Molecular derivatisation is an effective strategy to improve efficacy, lower toxicity, diversify activity and address pathogen resistance associated with the use of these drugs.

Design, synthesis, and antimycobacterial activity of novel ciprofloxacin derivatives.

Tuberculosis is the deadliest infectious disease affecting humankind with a death toll of approximately 1.7 million people in 2016. The increasing prevalence of multidrug-resistant strains of the causative pathogen, Mycobacterium tuberculosis (Mtb) which results in reduced effectiveness of the current therapies, underscores the urgent need for the development of new antitubercular drugs. In the search for such drugs, we investigated two series of ciprofloxacin (CPX) derivatives (analogues and hybrids). We herein report the design, synthesis, and biological activity of these series against the human virulent Mtb H37Rv strain in vitro. The small propionyl analogue 11 (MIC90 1.6 µM; SI > 61) and the large cholesteryl hybrid 32 (MIC90 2.0 µM; SI > 6) were the most active derivatives, comparable to CPX (MIC90 1.8 µM). However, the slightly less active but non-cytotoxic para-fluorobenzyl hybrid 28 (MIC90 3.7 µM; SI 27) was more selective toward bacteria than 32. Thus, the CPX derivatives 11 and 28 were identified as preferred antitubercular hits for further investigation including distribution, metabolism and pharmacokinetic parameters determination and in vivo activity assessment in animal models.

Sequence analysis of cell-free DNA derived from cultured human bone osteosarcoma (143B) cells.

The true importance of cell-free DNA in human biology, together with the potential scale of its clinical utility, is tarnished by a lack of understanding of its composition and origin. In investigating the cell-free DNA present in the growth medium of cultured 143B cells, we previously demonstrated that the majority of cell-free DNA is neither a product of apoptosis nor necrosis. In the present study, we investigated the composition and origin of this cell-free DNA population using next-generation sequencing. We found that the cell-free DNA comprises mainly of repetitive DNA, including α-satellite DNA, mini satellites, and transposons that are currently active or exhibit the capacity to become reactivated. A significant portion of these cell-free DNA fragments originates from specific chromosomes, especially chromosomes 1 and 9. In healthy adult somatic cells, the centromeric and pericentromeric regions of these chromosomes are normally densely methylated. However, in many cancer types, these regions are preferentially hypomethylated. This can lead to double-stranded DNA breaks or it can directly impair the formation of proper kinetochore structures. This type of chromosomal instability is a precursor to the formation of nuclear anomalies, including lagging chromosomes and anaphase bridges. DNA fragments derived from these structures can recruit their own nuclear envelope and form secondary nuclear structures known as micronuclei, which can localize to the nuclear periphery and bud out from the membrane. We postulate that the majority of cell-free DNA present in the growth medium of cultured 143B cells originates from these micronuclei.

The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature.

Since the detection of cell-free DNA (cfDNA) in human plasma in 1948, it has been investigated as a non-invasive screening tool for many diseases, especially solid tumours and foetal genetic abnormalities. However, to date our lack of knowledge regarding the origin and purpose of cfDNA in a physiological environment has limited its use to more obvious diagnostics, neglecting, for example, its potential utility in the identification of predisposition to disease, earlier detection of cancers, and lifestyle-induced epigenetic changes. Moreover, the concept or mechanism of cfDNA could also have potential therapeutic uses such as in immuno- or gene therapy. This review presents an extensive compilation of the putative origins of cfDNA and then contrasts the contributions of cellular breakdown processes with active mechanisms for the release of cfDNA into the extracellular environment. The involvement of cfDNA derived from both cellular breakdown and active release in lateral information transfer is also discussed. We hope to encourage researchers to adopt a more holistic view of cfDNA research, taking into account all the biological pathways in which cfDNA is involved, and to give serious consideration to the integration of in vitro and in vivo research. We also wish to encourage researchers not to limit their focus to the apoptotic or necrotic fraction of cfDNA, but to investigate the intercellular messaging capabilities of the actively released fraction of cfDNA and to study the role of cfDNA in pathogenesis.