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The alkaloid physostigmine is an approved anticholinergic drug and an important lead structure for the development of novel therapeutics. Using a complementary approach that merged chemical synthesis with pathway refactoring, we produced a series of physostigmine analogues with altered specificity and toxicity profiles in the heterologous host Myxococcus xanthus. The compounds that were generated by applying a simple feeding strategy include the promising drug candidate phenserine, which was previously accessible only by total synthesis.

We now describe the physicochemical profiling, in vitro ADME, and antiparasitic activity of eight N,N′-diarylureas to assess their potential as a broad-spectrum antiprotozoal chemotype. Chromatographic LogD(7.4) values ranged from 2.5 to 4.5; kinetic aq. solubilities were ≤6.3 μg/mL, and plasma protein binding ranged from 95 to 99%. All of the compounds had low intrinsic clearance values in human, but not mouse, liver microsomes. Although no N,N′-diarylurea had submicromolar potency against Trypanosoma cruzi, two had submicromolar potencies against Toxoplasma gondii and Trypanosoma brucei rhodesiense, and five had submicromolar potencies against Leishmania donovani. Plasmodium falciparum appeared to be the most susceptible to growth inhibition by this compound series. Most of the N,N′-diarylureas had antiprotozoal selectivities ≥10. One N,N′-diarylurea had demonstrable activity in mouse models of malaria and toxoplasmosis.

Spirombandakamine A3 (7) is only the third known naphthylisoquinoline dimer with a spiro-fused novel molecular framework and the first such representative to possess a relative trans-configuration at the two chiral centers in both tetrahydroisoquinoline subunits. It was found in the leaves of a botanically as yet unidentified Congolese Ancistrocladus plant, which is morphologically closely related to the Central African taxon Ancistrocladus ealaensis. Likewise isolated were the new cyclombandakamines A8 (8) and A9 (9), which belong to another most recently discovered type of unusual oxygen-bridged naphthylisoquinoline dimers and two previously described "open-chain" analogues, mbandakamines C (10) and D (11). The full absolute stereostructures of these compounds were assigned by combining spectroscopic, chemical, and chiroptical methods. Preliminary biomimetic investigations indicated that both spirombandakamine- and cyclombandakamine-type dimers result from the oxidation of their open-chain mbandakamine-type congeners. The new dimeric alkaloids 7-9 displayed potent growth-inhibitory activity against Plasmodium falciparum, the protozoal pathogen causing malaria, and moderate effects on Trypanosoma brucei rhodesiense, the parasite responsible for African sleeping sickness.

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei the proteins that make up the kinetochore are highly divergent, with the inner kinetochore comprising at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2 and KKT3. We performed a phenotypic screen of T. brucei bloodstream forms with a Novartis kinase-focused inhibitor library, which identified a number of selective inhibitors with potent pan-kinetoplastid activity. Deconvolution of an amidobenzimidazole series using a selection of 37 T. brucei mutants that over-express known essential protein kinases identified CLK1 as the primary target. Biochemical studies show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with C215 in the ATP binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. We show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 to be essential for KKT2 function and for kinetochore assembly. We propose that CLK1 is part of a novel signalling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. This work highlights a novel drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

PURPOSE: Zhumeria majdae, a unique species of the Zhumeria genus, is an endemic Iranian plant in the Lamiaceae family. Phytochemical investigation and biological activity of this plant are rarely reported. The current study aimed to find new antiprotozoal compounds from the roots of Z. majdae and to determine the absolute configuration of isolated compounds by circular dichroism. METHODS: The extraction process from roots and aerial parts of Z. majdae was carried out by hexane, ethyl acetate and methanol followed by testing their antiprotozoal effects against Leishmania donovani, Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum, respectively. Structure elucidation was done using 1D and 2D NMR spectroscopy and HREIMS spectrometry. In addition, experimental and theoretical circular dichroism spectroscopy was used to establish absolute configuration. RESULTS: In comparison with aerial parts, the hexane extract from roots showed superior activity against T. b. rhodesiense, L. donovani and P. falciparum with IC(50) values of 5.4, 1.6 and 2.1 μg/ml, respectively. From eight abietane-type diterpenoids identified in roots, six were reported for the first time in the genus Zhumeria. 11,14-dihydroxy-8,11,13-abietatrien-7-one (6) exhibited a promising biological activity against P. falciparum (IC(50) 8.65 μM), with a selectivity index (SI) of 4.6, and lanugon Q (8) showed an IC(50) value of 0.13 μM and SI of 15.4 against T. b. rhodesiense. CONCLUSION: Altogether, according to the results, of 8 isolated compounds, dihydroxy-8,11,13-abietatrien-7-one (6) and lanugon Q (8) exhibited a promising activity against T. b. rhodesiense and P. falciparum. In conclusion, these compounds could be potential candidates for further analysis and may serve as lead compounds for the synthesis of antiprotozoal agents. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s40199-020-00345-w) contains supplementary material, which is available to authorized users.

As part of our ongoing search for antiprotozoal natural products from plants, we examined different resins from the Burseraceae family. The dichloromethane extract obtained from myrrh, the oleo-gum-resin of Commiphora species, showed promising in vitro activity against Plasmodium falciparum with an IC50 value of 1 µg/mL. Bioactivity-guided fractionation led to the isolation and characterization of 18 sesquiterpenoids, namely, β-elemene (1), elemyl acetate (2), curzerenone (3), 8-hydroxyisogermafurenolide (4), 2-methoxyisogermafurenolide (5), 8-epi-2-methoxyisogermafurenolide (6), furanodienone (7), 1(10)Z,4Z-furanodien-6-one (8), rel-2R-methyl-5S-acetoxy-4R-furanogermacr-1(10)Z-en-6-one (9), (1(10)E)-2-methoxy-8,12-epoxygermacra-1(10),7,11-trien-6-one (10), 2R-methoxyfuranodiene (11), 2-acetyloxyglechomanolide (12), 8-epi-2-acetyloxyglechomanolide (13), (1R,2R,4S)-1,2-epoxyfuranogermacr-10(15)-en-6-one (14), hydroxylindestrenolide (15), isohydroxylindestrenolide (16), myrrhone (17), and myrrhterpenoid O (18). Moreover, nine (nor-)triterpenoids were isolated: mansumbinol (19), mansumbinol epoxide (20), mansumbinone (21), mansumbin-13(17)-en-3,16-dione (22), 3,4-seco-mansumbinoic acid (23), rel-20S-hydroxy-dammar-24-en-3,16-dione (24), rel-(16S,20S)-dihydroxydammar-24-en-3-one (25), cycloart-24-en-1α,2α,3β-triol (26), and 3β-isovaleroyloxycycloart-24-en-1α,2α-diol (27). All compounds were identified by MS and NMR spectroscopic analyses. To the best of our knowledge, compounds 5, 6, 12, 13, 16, 18, and 20 are reported for the first time. All isolated compounds were tested in vitro for activity against P. falciparum and cytotoxicity. The sesquiterpene 7 and the triterpene 25 were the most active compounds found in this study with IC50 values of 7.4 and 2.8 µM, respectively.

Janadolide is a cyclic depsipeptide natural product isolated from the marine cyanobacterium Okeania sp. Herein, we describe the total synthesis of janadolide, along with eight simplified analogues, via an efficient solid-phase strategy. Crucial to the synthesis of the natural product was the construction of a key polyketide fragment via an enantioselective (-)-B-chlorodiisopinocampheylborane-mediated reduction and a B-alkyl Suzuki reaction. Janadolide and the simplified analogues exhibited antitrypanosomal activity against pathogenic Trypanosoma brucei rhodesiense and Trypanosoma cruzi parasites.

A new cyclic lipopeptide, phototemtide A (1), was isolated from Escherichia coli expressing the biosynthetic gene cluster pttABC from Photorhabdus temperata Meg1. The structure of 1 was elucidated by HR‐ESI‐MS and NMR experiments. The absolute configurations of amino acids and 3‐hydroxyoctanoic acid in 1 were determined by using the advanced Marfey's method and comparison after total synthesis of 1, respectively. Additionally, three new minor derivatives, phototemtides B–D (2–4), were identified by detailed HPLC–MS analysis. Phototemtide A (1) showed weak antiprotozoal activity against Plasmodium falciparum, with an IC50 value of 9.8 μm. The biosynthesis of phototemtides A–D (1–4) was also proposed., Cyclic lipopeptides (CLPs) are important pharmaceutical lead compounds. We report a new antimalarial CLP phototemtide A with three minor derivatives, phototemtides B–D, which are generated after the silent NRPS‐encoding gene cluster pttABC from entomopathogenic P. temperata Meg1 was activated by heterologous expression in E. coli. The structures of these new CLPs were unequivocally identified by combining biological and chemical methods.

African sleeping sickness is a potentially fatal neglected disease affecting sub-Saharan Africa. High-throughput screening identified the thiazolyl–benzothiophenamide 1 to be active against the causative parasite, Trypanosoma brucei. This work establishes structure–activity relationships of 1, guiding the design of second generation derivatives. After screening against the clinically relevant species T. b. rhodesiense, the derivative 16 was identified as a suitable candidate for further investigation.

Chemical investigation of the alkaloid extract of the aerial parts of Schizanthus tricolor led to the targeted isolation of 26 hygroline derivatives of which 20 were fully characterized. They have not yet been described in the literature and their structures were established by 1D and 2D NMR, UV and IR spectroscopy, and HRESIMS. The configuration was determined by Gauge-Independent Atomic Orbital NMR chemical shift calculations supported by the advanced statistical method DP4 plus, vibrational circular dichroism, and measurement of optical rotation. Their anti-trypanosomatid, antiplasmodial and cytotoxic activities were measured. Several compounds exhibited low micromolar activity against Plasmodium falciparum. None of the identified molecules was cytotoxic.

Chagas disease is a zoonosis caused by Trypanosoma cruzi and transmitted by triatomine bugs. Autochthonous to Latin America, Chagas disease has spread globally through travel and migration. New drugs are needed urgently, in particular drugs that cure the chronic stage. This is where high-content imaging makes a key contribution: assays with fluorescent parasites in cell culture allow to determine pharmacodynamic parameters and to better assess the antichagasic potential of new molecules.

The 2-phenoxybenzamide 1 from the Medicines for Malaria Venture Malaria Box Project has shown promising multi-stage activity against different strains of P. falciparum. It was successfully synthesized via a retrosynthetic approach. Subsequently, twenty-one new derivatives were prepared and tested for their in vitro activity against blood stages of the NF54 strain of P. falciparum. Several insights into structure-activity relationships were revealed. The antiplasmodial activity and cytotoxicity of compounds strongly depended on the substitution pattern of the anilino partial structure as well as on the size of substituents. The diaryl ether partial structure had further impacts on the activity. Additionally, several physicochemical and pharmacokinetic parameters were calculated (log P, log D7.4 and ligand efficiency) or determined experimentally (passive permeability and CYP3A4 inhibition). The tert-butyl-4-{4-[2-(4-fluorophenoxy)-3-(trifluoromethyl)benzamido]phenyl}piperazine-1-carboxylate possesses high antiplasmodial activity against P. falciparum NF54 (PfNF54 IC50 = 0.2690 µM) and very low cytotoxicity (L-6 cells IC50 = 124.0 µM) resulting in an excellent selectivity index of 460. Compared to the lead structure 1 the antiplasmodial activity was improved as well as the physicochemical and some pharmacokinetic parameters.

A new cyclic peptide photoditritide (1), containing two rare amino acid d-homoarginine residues, was isolated from Photorhabdus temperata Meg1 after the nonribosomal peptide synthetase encoding gene pdtS was activated via promoter exchange. The structure of 1 was elucidated by HR-MS and NMR experiments. The absolute configurations of amino acids were determined according to the advanced Marfey's method after hydrolysis of 1. Bioactivity testing of 1 revealed potent antimicrobial activity against Micrococcus luteus with an MIC value of 3.0 μM and weak antiprotozoal activity against Trypanosoma brucei rhodesiense with an IC50 value of 13 μM. Additionally, the biosynthetic pathway of 1 was also proposed.

Protozoal infections are still a global health problem, threatening the lives of millions of people around the world, mainly in impoverished tropical and sub‐tropical regions. Thus, in view of the lack of efficient therapies and increasing resistances against existing drugs, this study describes the antiprotozoal potential of synthetic cinnamate ester analogues and their structure‐activity relationships. In general, Leishmania donovani and Trypanosoma brucei were quite susceptible to the compounds in a structure‐dependent manner. Detailed analysis revealed a key role of the substitution pattern on the aromatic ring and a marked effect of the side chain on the activity against these two parasites. The high antileishmanial potency and remarkable selectivity of the nitro‐aromatic derivatives suggested them as promising candidates for further studies. On the other hand, the high in vitro potency of catechol‐type compounds against T. brucei could not be extrapolated to an in vivo mouse model., Fighting parasitic diseases: A classical phenotypic approach combined with a comprehensive SAR study led to establishment of potent and selective cinnamate ester analogues acting in vitro against Leishmania donovani and Trypanosoma brucei rhodesiense. Key structural requirements for activity were determined resulting in interesting candidates for further development.

Three new naphthylisoquinoline dimers, jozibrevines A-C (1a-c), were isolated from the West African shrub Ancistrocladus abbreviatus, along with the known dimer jozimine A2 (1d). The two molecular moieties of 1a-d are coupled via the sterically constrained 3',3″-positions of their two naphthalene units, so that the central biaryl linkage is rotationally hindered. With the two outer axes also being chiral, 1a-d possess three consecutive stereogenic axes. The four isolated dimers all have the same constitutions and identical absolute configurations at the four stereogenic centers, but differ by their axial chirality. They belong to the extremely small class of Dioncophyllaceae-type naphthylisoquinoline dimers, i.e., being devoid of oxygen functions at C-6 and bearing the R-configuration at C-3 in their isoquinoline portions. Besides these dimers, the plant produces predominantly typical Ancistrocladaceae-type monomeric compounds, i.e., with the S-configuration at C-3 and an oxygen function at C-6, such as the new ancistrobrevines K (5) and L (6). Furthermore, a new hybrid-type (i.e., mixed Ancistrocladaceae/Dioncophyllaceae-type) alkaloid was identified, named ancistrobrevine M (7), which is 3R-configured and 6-oxygenated. Remarkable was the discovery of its "inverse hybrid-type" counterpart, dioncoline A (8). It is the as yet only known 3S-configured naphthylisoquinoline lacking an O-functionality at C-6. The new jozibrevines A-C (1a-c) exhibited pronounced antiplasmodial activities in the submicromolar range, with 1a being the most potent compound (IC50, 0.012 μM). Furthermore, jozimine A2 (1d) showed cytotoxicity against human colon carcinoma (HT-29), fibrosarcoma (HT1080), and multiple myeloma (MM.1S) cancer cells, displaying IC50 values of 12.0, 9.0, and 5.0 μM, respectively, whereas jozibrevines A (1a) and B (1b) were nontoxic in this concentration range.

New derivatives of 3-azabicyclo[3.2.2]nonanes were prepared and characterized using FT-IR spectroscopy, HRMS, and NMR spectroscopy. The new compounds were investigated in vitro for their antiplasmodial activities against the sensitive NF54 strain and the multiresistant K1 strain of Plasmodium falciparum, and for their antitrypanosomal activity against Trypanosoma brucei rhodesiense. The N-methyl-6,9-diphenyl-N-[(pyridin-4-yl)methyl]-3-azabicyclo[3.2.2]nonan-1-amine possessed high antiplasmodial in vitro activity against both strains of P. falciparum (NF54: IC50 = 0.848 nm; K1: IC50 = 2 nm). The most promising ones were further investigated in a mouse model for their in vivo activity against Plasmodium berghei.

We have designed and synthesized a series of new imidazole-based compounds structurally related to an antiprotozoal agent with nanomolar activity which we identified recently. The new analogues possess micromolar activities against Trypanosoma brucei rhodesiense and Leishmania donovani and nanomolar potency against Plasmodium falciparum. Most of the analogues displayed IC 50 within the low nanomolar range against Trypanosoma cruzi, with very high selectivity toward the parasite. Discussion of structure-activity relationships and in vitro biological data for the new compounds are provided against a number of different protozoa. The mechanism of action for the most potent derivatives (5i, 6a-c, and 8b) was assessed by a target-based assay using recombinant T. cruzi CYP51. Bioavailability and efficacy of selected hits were assessed in a T. cruzi mouse model, where 6a and 6b reduced parasitemia in animals >99% following intraperitoneal administration of 25 mg/kg/day dose for 4 consecutive days.

Redox-active drugs are the mainstay of parasite chemotherapy. To assess their repurposing potential for eumycetoma, we have tested a set of nitroheterocycles and peroxides in vitro against two isolates of Madurella mycetomatis, the main causative agent of eumycetoma in Sudan. All the tested compounds were inactive except for niclosamide, which had minimal inhibitory concentrations of around 1 µg/mL. Further tests with niclosamide and niclosamide ethanolamine demonstrated in vitro activity not only against M. mycetomatis but also against Actinomadura spp., causative agents of actinomycetoma, with minimal inhibitory concentrations below 1 µg/mL. The experimental compound MMV665807, a related salicylanilide without a nitro group, was as active as niclosamide, indicating that the antimycetomal action of niclosamide is independent of its redox chemistry (which is in agreement with the complete lack of activity in all other nitroheterocyclic drugs tested). Based on these results, we propose to further evaluate the salicylanilides, niclosamidein particular, as drug repurposing candidates for mycetoma.

In continuation of our search for leads from medicinal plants against protozoal pathogens, we detected antileishmanial activity in polar fractions of a dichloromethane extract from Boswellia serrata resin. 11-keto-β-boswellic acid (KBA) could be isolated from these fractions and was tested in vitro against Leishmania donovani axenic amastigotes along with five further boswellic acid derivatives. 3-O-acetyl-11-keto-β-boswellic acid (AKBA) showed the strongest activity with an IC50 value of 0.88 µM against axenic amastigotes but was inactive against intracellular amastigotes in murine macrophages

As part of our studies on antiprotozoal activity of approved herbal medicinal products, we previously found that a commercial tincture from Salvia officinalis L. (common Sage, Lamiaceae) possesses high activity against Trypanosoma brucei rhodesiense (Tbr), causative agent of East African Human Trypanosomiasis. We have now investigated in detail the antitrypanosomal constituents of this preparation. A variety of fractions were tested for antitrypanosomal activity and analyzed by UHPLC/+ESI QqTOF MS. The resulting data were used to generate a partial least squares (PLS) regression model that highlighted eight particular constituents that were likely to account for the major part of the bioactivity. These compounds were then purified and identified and their activity against the pathogen tested. All identified compounds (one flavonoid and eight diterpenes) displayed significant activity against Tbr, in some cases higher than that of the total tincture. From the overall results, it can be concluded that the antitrypanosomal activity of S. officinalis L. is, for the major part, caused by abietane-type diterpenes of the rosmanol/rosmaquinone group.

Azoles such as posaconazole (Posa) are highly potent against Trypanosoma cruzi. However, when tested in chronic Chagas disease patients, a high rate of relapse after Posa treatment was observed. It appears that inhibition of T. cruzi cytochrome CYP51, the target of azoles, does not deliver sterile cure in monotherapy. Looking for suitable combination partners of azoles, we have selected a set of inhibitors of sterol and sphingolipid biosynthetic enzymes. A small-scale phenotypic screening was conducted in vitro against the proliferative forms of T. cruzi, extracellular epimastigotes and intracellular amastigotes. Against the intracellular, clinically relevant forms, four out of 15 tested compounds presented higher or equal activity as benznidazole (Bz), with EC50 values ≤2.2 μM. Ro48-8071, an inhibitor of lanosterol synthase (ERG7), and the steroidal alkaloid tomatidine (TH), an inhibitor of C-24 sterol methyltransferase (ERG6), exhibited the highest potency and selectivity indices (SI = 12 and 115, respectively). Both were directed to combinatory assays using fixed-ratio protocols with Posa, Bz, and fexinidazole. The combination of TH with Posa displayed a synergistic profile against amastigotes, with a mean ΣFICI value of 0.2. In vivo assays using an acute mouse model of T. cruzi infection demonstrated lack of antiparasitic activity of TH alone in doses ranging from 0.5 to 5 mg/kg. As observed in vitro, the best combo proportion in vivo was the ratio 3 TH:1 Posa. The combination of Posa at 1.25 mpk plus TH at 3.75 mpk displayed suppression of peak parasitemia of 80% and a survival rate of 60% in the acute infection model, as compared to 20% survival for Posa at 1.25 mpk alone and 40% for Posa at 10 mpk alone. These initial results indicate a potential for the combination of posaconazole with tomatidine against T. cruzi.

Three new compounds (1–3) with unusual skeletons were isolated from the n-hexane extract of the air-dried aerial parts of Hypericum scabrum. Compound 1 represents the first example of an esterified polycyclic polyprenylated acylphloroglucinol that features a unique tricyclo-[4.3.1.11,4]-undecane skeleton. Compound 2 is a fairly simple MPAP, but with an unexpected cycloheptane ring decorated with prenyl substituents, and compound 3 has an unusual 5,5-spiroketal lactone core. Their structures were determined by extensive spectroscopic and spectrometric techniques (1D and 2D NMR, HRESI-TOFMS). Absolute configurations were established by ECD calculations, and the absolute structure of 2 was confirmed by a single crystal determination. Plausible biogenetic pathways of compounds 1–3 were also proposed. The in vitro antiprotozoal activity of the compounds against Trypanosoma brucei rhodesiense and Plasmodium falciparum and cytotoxicity against rat myoblast (L6) cells were determined. Compound 1 showed a moderate activity against T. brucei and P. falciparum, with IC50 values of 3.07 and 2.25 μM, respectively.

The antiprotozoal activity of 1-benzyltetrahydropyridin-4-yliden iminium salts is reported. This paper describes the preparation of a series of analogs from dihydropyridines or dihydrothiopyrans as educts. The new compounds were investigated for their activity against Plasmodium falciparum NF54, a causative organism of Malaria tropica and Trypanosoma brucei rhodesiense, the causative organism of Human African Trypanosomiasis (sleeping sickness). Several structure–activity relationships were detected. Both the substituents in ring positions 1 and 4 of the tetrahydropyridinium moiety had a strong impact on the antiprotozoal activities as well as on the cytotoxicity of compounds against L-6 cells (rat skeletal myoblasts). All new compounds were characterized using FT-IR spectroscopy, HRMS, and NMR spectroscopy. Graphic abstract

In the course of our studies on antiprotozoal natural products and following our recent discovery that certain aminosteroids and aminocycloartanoid compounds from Holarrhena africana A. DC. (Apocynaceae) and Buxus sempervirens L. (Buxaceae), respectively, are strong and selective antitrypanosomal agents, we have extended these studies to another plant, related to the latter&mdash, namely, Pachysandra terminalis Sieb. and Zucc. (Buxaceae). This species is known to contain aminosteroids similar to those of Holarrhena and structurally related to the aminocycloartanoids of Buxus. The dicholoromethane extract obtained from aerial parts of P. terminalis and, in particular, its alkaloid fraction obtained by acid&ndash, base partitioning showed prominent activity against Trypanosoma brucei rhodesiense (Tbr). Activity-guided fractionation along with extended UHPLC-(+)ESI QTOF MS analyses coupled with partial least squares (PLS) regression modelling relating the analytical profiles of various fractions with their bioactivity against Tbr highlighted eighteen constituents likely responsible for the antitrypanosomal activity. Detailed analysis of their (+)ESI mass spectral fragmentation allowed identification of four known constituents of P. terminalis as well as structural characterization of ten further amino-/amidosteroids not previously reported from this plant.

The polyamine synthesis inhibitor eflornithine is a recommended treatment for the neglected tropical disease Gambian human African trypanosomiasis in late stage. This parasitic disease, transmitted by the tsetse fly, is lethal unless treated. Eflornithine is administered by repeated intravenous infusions as a racemic mixture of L-eflornithine and D-eflornithine. The study compared the in vitro antitrypanosomal activity of the two enantiomers with the racemic mixture against three Trypanosoma brucei gambiense strains. Antitrypanosomal in vitro activity at varying drug concentrations was analysed by non-linear mixed effects modelling. For all three strains, L-eflornithine was more potent than D-eflornithine. Estimated 50% inhibitory concentrations of the three strains combined were 9.1 μM (95% confidence interval [8.1; 10]), 5.5 μM [4.5; 6.6], and 50 μM [42; 57] for racemic eflornithine, L-eflornithine and D-eflornithine, respectively. The higher in vitro potency of L-eflornithine warrants further studies to assess its potential for improving the treatment of late-stage Gambian human African trypanosomiasis., Author summary The neglected tropical disease human African trypanosomiasis is lethal unless treated. One of the treatments for the late stage–i.e. when parasites have invaded the central nervous system–of Gambian human African trypanosomiasis is the drug eflornithine, which is dosed as 50:50 racemic mixture of the two enantiomers L-eflornithine and D-eflornithine. This study showed that L-eflornithine was better than D-eflornithine at inhibiting the growth of parasites in vitro. The 50% inhibitory concentration for L-eflornithine was 5.5 μM in comparison to 50 μM for D-eflornithine. This higher in vitro potency for L-eflornithine warrants further studies to assess its potential as an improved treatment for late-stage Gambian human African trypanosomiasis.

Leucinostatin A is one of the most potent antiprotozoal compounds ever described, but little was known on structure–activity relationships (SAR). We used Trypanosoma brucei as a protozoal model organism to test synthetically modified derivatives, resulting in simplified but equally active compounds 2 (ZHAWOC6025) and 4 (ZHAWOC6027), which were subsequently modified in all regions of the molecule to gain an in‐depth SAR understanding. The antiprotozoal SAR matched SAR in phospholipid liposomes, where membrane integrity, leaking, and dynamics were studied. The mode of action is discussed based on a structure–activity analysis of derivatives in efficacy, ultrastructural studies in T. brucei, and artificial membrane models, mimicking membrane stability and membrane potential. The main site of antiprotozoal action of natural and synthetic leucinostatins lies in the destabilization of the inner mitochondrial membrane, as demonstrated by ultrastructural analysis, electron microscopy and mitochondrial staining. Long‐time sublethal exposure of T. brucei (200 passages) and siRNA screening of 12′000 mutants showed no signs of resistance development to the synthetic derivatives., A structure–activity relationship of the natural product Leucinostatin A was performed to result in two highly potent compounds against the protozoa Trypanosoma brucei with decreased toxic effects in vitro and in vivo. Those compounds did not form any resistance by long‐time sublethal exposure and their mode of action lies in the destabilization of the inner mitochondrial membrane.

A new series of compounds was prepared from 6-methoxyquinolin-8-amine or its N-(2-aminoethyl) analogue via Ugi-azide reaction. Their linkers between the quinoline and the tert-butyltetrazole moieties differ in chain length, basicity and substitution. Compounds were tested for their antiplasmodial activity against Plasmodium falciparum NF54 as well as their cytotoxicity against L-6-cells. The activity and the cytotoxicity were strongly influenced by the linker and its substitution. The most active compounds showed good activity and promising selectivity.

The glidobactin‐like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881–1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy., The silent treatment: Glidobactin‐like proteasome inhibitors are regarded as promising candidates for anticancer drug development. We report the largest subset of glidobactin‐like proteasome inhibitors, which are produced and identified after the silent biosynthetic gene cluster plu1881–1877 is activated in the native host P. laumondii.

New assay designs are needed to improve the predictive value of the Trypanosoma cruzi in vitro tests used as part of the Chagas' disease drug development pipeline. Here, we employed a green fluorescent protein (eGFP)-expressing parasite line and live high-content imaging to monitor the growth of T. cruzi amastigotes in mouse embryonic fibroblasts. A novel assay design allowed us to follow parasite numbers over 6 days, in four-hour intervals, while occupying the microscope for only 24 hours per biological replicate. Dose-response curves were calculated for each time point after addition of test compounds, revealing how EC50 values first decreased over the time of drug exposure, and then leveled off. However, we observed that parasite numbers could vary, even in the untreated controls, and at different sites in the same well, which caused variability in the EC50 values. To overcome this, we established that fold change in parasite number per hour is a more robust and informative measure of drug activity. This was calculated based on an exponential growth model for every biological sample. The net fold change per hour is the result of parasite replication, differentiation, and death. The calculation of this fold change enabled us to determine the tipping point of drug action, i.e. the time point when the death rate of the parasites exceeded the growth rate and the fold change dropped below 1, depending on the drug concentration and exposure time. This revealed specific pharmacodynamic profiles of the benchmark drugs benznidazole and posaconazole., Author summary Chagas' disease, caused by Trypanosoma cruzi, is a chronic debilitating infection occurring mostly in Latin America. There is an urgent need for new, well tolerated drugs. However, the latest therapeutic candidates have yielded disappointing outcomes in clinical trials, despite promising preclinical results. This demands new and more predictive in vitro assays. To address this, we have developed an assay design that enables the growth of T. cruzi intracellular forms to be monitored in real time, under drug pressure, for 6 days post-infection. This allowed us to establish the tipping point of drug action, when the death rate of the parasites exceeded the growth rate. The resulting pharmacodynamics profiles can provide robust and informative details on anti-chagasic candidates, as demonstrated for the benchmark drugs benznidazole and posaconazole.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. It is endemic in South and Central America and recently has been found in other parts of the world, due to migration of chronically infected patients. The current treatment for Chagas disease is not satisfactory, and there is a need for new treatments. In this work, we describe the optimization of a hit compound resulting from the phenotypic screen of a library of compounds against T. cruzi. The compound series was optimized to the level where it had satisfactory pharmacokinetics to allow an efficacy study in a mouse model of Chagas disease. We were able to demonstrate efficacy in this model, although further work is required to improve the potency and selectivity of this series.

Twelve new pyridine-4(1H)-one derivatives, namely, 8-demethoxywaltherione F (1), waltheriones R-V (2, 6, 7, 10, and 11), 1-methoxywaltherione O (3), (S)-15-hydroxywaltherione G (4), (8R)-8-hydroxywaltherione M (5), (9S,13S)-2-hydroxymethylwaltherione C (8), (9S,10S,13S)-10-hydroxywaltherione C (9), and (S)-13-methoxywaltherione V (12), as well as melovinone (13) and 5'-methoxywaltherione A (14) were isolated from the CH2Cl2 extract of the aerial parts of Waltheria indica. Their chemical structures were determined by means of a comprehensive analysis including 1H NMR, DEPTQ, HSQC, HMBC, 1H-1H COSY, ROESY, and HRESIMS data. The absolute configurations were assigned via comparison of the experimental and calculated ECD data. In addition, the isolated constituents as well as the known waltheriones M-Q were evaluated for their in vitro antitrypanosomal activity. Compounds 2, 5, and 7 as well as waltheriones M, P, and Q showed potent growth inhibition toward Trypanosoma cruzi with IC50 values of 2.1, 0.8, 2.1, 1.3, 0.5, and 0.1 μM, respectively, and selectivity indices of >12, >33, >13, 5, 25, and 14. These findings further demonstrate that the waltheriones are a promising class of antichagasic compounds worthy of further investigations.

Abstract Novel 2-aminopyrimidine derivatives were prepared from acyclic starting materials, benzylidene acetones and ammonium thiocyanates, via 5 steps, including ring closure, aromatization, S-methylation, oxidation to methylsulfonyl compounds, and formation of guanidines with suitable amines. The prepared compounds differ from each other by the substitutions of their amino group and of their phenyl ring. The 2-aminopyrimidines were tested by use of microplate assays for their in vitro activities against a causative organism of sleeping sickness, Trypanosoma brucei rhodesiense, as well as against a causative organism of malaria, Plasmodium falciparum NF54. Their cytotoxic properties were determined with L-6 cells (rat skeletal myoblasts). Some of the compounds exhibited quite good antitrypanosomal activity, and others showed excellent antiplasmodial activity. The influence of the structural modifications on these activities is discussed. Graphic abstract

The recent endorsement of fexinidazole by the European Medicines Agency for the treatment of human African trypanosomiasis has demonstrated the high predictive value of cell-based assays for parasite chemotherapy. Here we describe three in vitro drug susceptibility tests with Trypanosoma brucei that have served as the basis for the identification of fexinidazole as a promising lead: (1) a standard assay with end-point measurement to determine drug efficacy; (2) a wash-out assay to test for reversibility and speed of drug action; (3) isothermal microcalorimetry for real-time measurement of onset of drug action and time to kill. Together, these assays allow to estimate pharmacodynamic parameters in vitro and to devise appropriate treatment regimens for subsequent in vivo experiments.

Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are ...

Fractionation of the n-hexane extract of Salvia hydrangea afforded seven isoprenoids including six new compounds (1–6) and salvadione A (7). Their structures were established by comprehensive spectroscopic and spectrometric data analysis (1D and 2D NMR, HRMS). The absolute configuration of salvadione A (7) was established by single-crystal X-ray diffraction analysis with Cu/Kα radiation. In addition, the absolute configuration of all compounds was determined by electronic circular dichroism spectroscopy. A biosynthetic pathway for the formation of the scaffold of 1 is proposed. The antiprotozoal activity of the compounds against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum was determined, and cytotoxicity was assessed in rat myoblast L6 cells. Perovskone C (2) exhibited good activity against P. falciparum (IC50 0.6 μM) and a selectivity index of 62.2.

From the roots of the West African liana Ancistrocladus abbreviatus (Ancistrocladaceae), ten new naphthylisoquinoline alkaloids (7a, 7b, 8a, 8b, and 9-14), displaying three different coupling types (5,1', 5,8', and 7,8'), were isolated, among them a series of five 5,1'-linked representatives and four metabolites belonging to the rare group of 7,8'-coupled alkaloids. Two of the alkaloids, the ancistrobrevines I (13) and J (14), are only the fourth and fifth examples of 7,8'-linked naphthyldihydroisoquinolines ever found in nature. The stereostructures of the new plant metabolites were determined by spectroscopic, chemical (oxidative degradation), and chiroptical (electronic circular dichroism) methods. For the assignment of the axial configuration of 13 and 14 relative to the stereocenter at C-3, which is too far away for significant NOE long-range interactions, these 7,8'-coupled naphthyldihydroisoquinolines were stereoselectively converted into the respective cis-configured tetrahydroisoquinoline analogs. The newly generated 'auxiliary' stereocenter at C-1 permitted decisive NOE interactions between the isoquinoline and the naphthalene parts, and thus a reliable attribution of the axial configuration of 13 and 14. In addition, five known compounds (3, 5, 16, 17, and 20), previously discovered in related African and Asian Ancistrocladus species, have now for the first time been identified in A. abbreviatus. All of these alkaloids are S-configured at C-3 and bear an oxygen function at C-6, and are, thus, typical Ancistrocladaceae-type compounds. Some of the alkaloids of A. abbreviatus exhibited promising activities against the malaria parasite Plasmodium falciparum and PANC-1 human pancreatic cancer cells.

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k2nd (M−1s−1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC50 values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values against Leishmania donovanii.

Seven new rhabdopeptide/xenortide-like peptides (RXPs) (1-7) with putrescine or ammonia as the C-terminal amines were isolated from Xenorhabdus innexi DSM 16336. Their chemical structures were elucidated by high-resoultion mass spectroscopy (HR-MS) and one-dimensional (1D) and two-dimensional (2D) NMR. They were evaluated for their activities against protozoan parasites and cytotoxicity against rat skeletal myoblasts (L6 cells). All tested compounds exhibited strong effects against Trypanosoma brucei rhodesiense and Plasmodium falciparum, with IC50 values of 0.07-6.25 and 0.091-3.16 μM, respectively, making them the most active RXP derivatives known to date.

Here, we describe an efficient and diversity-oriented entry to both (-)-artemisinin (1) and its natural antipode (+)-artemisinin, starting from commercially and readily available S-(+)- and R-(-)-citronellene, respectively. Subsequently, we answered the still open question regarding the specificity of artemisinins action. By using a drug-sensitive Plasmodium falciparum NF54 strain, we showed that the antimalarial activity of artemisinin is not stereospecific. Our straightforward and biomimetic approach to this natural endoperoxide enables the synthesis of artemisinin derivatives that are not accessible through applying current methods and may help to address the problem of emerging resistance of Plasmodium falciparum towards artemisinin.

Phytochemical investigation of the lipophilic extract of the roots of Salvia leriifolia resulted in the isolation of the new rearranged abietane diterpenoids leriifoliol (1) and leriifolione (2), together with 10 known diterpenoids. Structure elucidations were performed via extensive NMR and HRESIMS data, and the absolute configurations of compounds 1 and 3–5 were established by evaluation of experimental and calculated ECD spectra. The antiplasmodial activity of the new isolates was assayed against Trypanosoma brucei rhodesiense, T. cruzi, Plasmodium falciparum, and Leishmania donovani and also toxicity against rat myoblast (L6) cells. Compound 1 displayed antimalarial and low cytotoxic activity with IC50 values of 0.4 and 33.6 μM, respectively, and a selectivity index of 84. Compound 2 displayed activity against T. brucei, T. cruzi, and L. donovani, with IC50 values of 1.0, 4.6, and 1.0 μM, respectively. Putative biosynthetic pathways toward the formation of 1, 2, and 3 are proposed. Leriifoliol (1) is ...

A series of natural products-based phenyl sulfone derivative and their property-based analogues were investigated as potential growth inhibitors of Trypanosoma brucei. Trypanosoma brucei is a kinetoplastid protozoan parasite that causes trypanosomiasis. In this work, we found that nopol- and quinoline-based phenyl sulfone derivative were the most active and selective for T. brucei, and they were not reactive towards the active thiol of T. brucei’s cysteine protease rhodesain. A thiol reactive variant of the quinoline-based phenyl sulfone was subsequently investigated and found to be a moderate inhibitor of rhodesain. The quinoline-based compound that is not reactive towards rhodesain can serve a template for phenotypic-based lead discovery while its thiol-active congener can serve as template for structure-based investigation of new antitrypanosomal agents.

Rhabdopeptides are a large class of nonribosomal peptides from the bacteria Xenorhabdus and Photorhabdus with low micromolar activity against different protozoa, which are the causative agents of several tropical diseases. The development of a facile and flexible synthesis combining backbone amide linking with on-resin peralkylation for the synthesis of permethylated rhabdopeptides is described. This strategy allows the fast generation of permethylated naturally occurring and artificial rhabdopeptides for a structure-activity study. Furthermore, in vitro experiments revealed their superior properties regarding their stability and passive membrane diffusion.

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure-based design. The best inhibitors were freely soluble and showed competitive inhibition constants (K; i; ) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half-maximal inhibitory concentration, IC; 50; ) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X-ray co-crystal structures confirmed the binding of the ligands to the hydrophobic wall of the "mepacrine binding site" with the new, solubility-providing vectors oriented toward the surface of the large active site.

A series of monocationic new guanidinobenzimidazole derivatives were prepared in a four step process starting from 2-nitro-1,4-phenylendiamine. Their antiparasitic activity against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani were evaluated in vitro. Two out of 20 tested monocationic compounds (7, 14) showed close activity with reference drug chloroquine against P. Falciparum. To understand the interactions between DNA minor groove and in vitro active compounds (7, 14) molecular docking studies were carried out. Stability and binding energies of DNA-ligand complexes formed by DNA with compounds 7 and 14 were measured by molecular dynamics simulations throughout 200 ns time. Root mean square deviation (RMSD) values of the ligands remained stable below 0.25 mm and root mean square fluctuation (RMSF) values of the active site residues with which it interacted decreased compared to the apo form. All compounds exhibited theoretical absorption, distribution, metabolism and excretion (ADME) profiles conforming to Lipinski's and Ghose's restrictive rules. (C) 2021 Elsevier Masson SAS. All rights reserved.

In continuation of a search for new antiprotozoal agents from plants of the family Asteraceae, Tarchonanthus camphoratus and Schkuhria pinnata have been investigated. By following the promising in vitro activity of the dichloromethane extracts from their aerial parts, bioassay-guided chromatographic isolation yielded two known sesquiterpene lactones (1 and 2) from T. camphoratus and 20 known compounds of this type from S. pinnata. From the latter, a new eudesmanolide, (1R*,5S*,6R*,7R*,8R*,10R*)-1-hydroxy-8-[5″-hydroxy-4'-(2″-hydroxyisovaleroyloxy)tigloyloxy]-3-oxoeudesma-11(13)-en-6,12-olide (3), and two new germacranolides, 3β-(2″-hydroxyisovaleroyloxy)-8β-(3-furoyloxy)costunolide (14) and 1(10)-epoxy-3β-hydroxy-8β-[5'-hydroxy-4'-(2″-hydroxyisovaleroyloxy)tigloyloxy]costunolide (16), were obtained. Additionally, the flavonoid pectolinarigenin (24) and 3-hydroxy-4,5-dimethoxybenzenepropanol (25) were also isolated from S. pinnata. The compounds were characterized by analysis of 1D and 2D NMR spectroscopic and HR/MS data. In vitro antitrypanosomal activity and cytotoxicity against mammalian cells (L6 cell line) were evaluated for all the compounds. Santhemoidin A (13) and 3β-(2″-hydroxyisovaleroyloxy)-8β-(3-furoyloxy)costunolide (14) were the most active compounds found in this study, with IC50 values of 0.10 and 0.13 μM against Trypanosoma brucei rhodesiense trypomastigotes and selectivity indices of 20.5 and 29.7, respectively.

Some new piperazine derivatives of antiprotozoal active 3-azabicyclo[3.2.2]nonanes were prepared. Verification of the structures of the new compounds was done by one- and two-dimensional NMR measurements. All new compounds were investigated for their antiplasmodial and antitrypanosomal activities against Plasmodium falciparum NF54 and Trypanosoma brucei rhodesiense. Their cytotoxicity was assessed against L6 cells. Some of the new compounds showed in vitro very good antiplasmodial and excellent antitrypanosomal activities. The most antitrypanosomal active compound was screened in vivo in a mouse model against T. b. rhodesiense.

Xenorhabdus doucetiae, the bacterial symbiont of the entomopathogenic nematode Steinernema diaprepesi produces several different fatty acid amides. Their biosynthesis has been studied using a combination of analysis of gene deletions and promoter exchanges in X. doucetiae and heterologous expression of candidate genes in E. coli. While a decarboxylase is required for the formation of all observed phenylethylamides and tryptamides, the acyltransferase XrdE encoded in the xenorhabdin biosynthesis gene cluster is responsible for the formation of short chain acyl amides. Additionally, new, long-chain and cytotoxic acyl amides were identified in X. doucetiae infected insects and when X. doucetiae was grown in Galleria Instant Broth (GIB). When the bioactivity of selected amides was tested, a quorum sensing modulating activity was observed for the short chain acyl amides against the two different quorum sensing systems from Chromobacterium and Janthinobacterium.

International audience; Marine meroterpenoids, thiaplidiaquinones A and B and their respective non-natural dioxothiazine regioisomers have been shown to inhibit mammalian and protozoal farnesyltransferase (FTase) with the regioisomers exhibiting activity in the nanomolar range. In order to explore the structure-activity relationship (SAR) of this class of marine natural products, analogues of thiaplidiaquinones A and B and their regioisomers were synthesised, with variation in the number of isoprene units present in their side chains to afford prenyl and farnesyl analogues. The previously reported geranyl series of compounds were found to be the most potent FTase inhibitors closely followed by the novel farnesyl series. The pre-nyl series exhibited the most potent anti-plasmodial activity but the series was also the most cytotoxic. Overall, the farnesyl series exhibited moderate anti-plasmodial activity with one analogue, 14 also exhibiting low cytotoxicity, identifying it as a scaffold worthy of further exploration.

In the course of our ongoing search for new natural products as leads against protozoal diseases, the dichloromethane extract of Indian frankincense, the oleo-gum-resin obtained from Boswellia serrata, showed in vitro activity against Plasmodium falciparum. Bioactivity-guided fractionation led to the isolation of eight diterpenes: (1S,3E,7E,11R)-verticilla-3,7,12(18)-triene (1), cembrene A (2), serratol (3), 1S,3E,7R,8R,11E-7,8-epoxy-cembra-3,11-dien-1-ol (4), incensole oxide (5), rel (1S,3R,7E,11S,12R)-1,12-epoxy-4-methylenecembr-7-ene-3, 11-diol (6), isoincensole oxide (7), and isodecaryiol (8). Furthermore, 10 triterpenes, namely, oleanolic acid (9), 11-keto-β-boswellic acid (10), 3-epi-neoilexonol (11), uvaol (12), β-boswellic aldehyde (13), 5α-tirucalla-8,24-dien-3α-ol (14), isoflindissone lactone (15), isoflindissol lactone (16), rel (8R,9S,20R)-tirucall-24-ene-3β,20-diol (17), and rel (3α,8R, 9S,20R,24S)-20,24-epoxytirucalla-3,25-diol (18) as well as the sesquiterpene β-bourbonene (19), the monoterpene carvacrol (20) and the phenyl propanoids methyleugenol (21), and p-methoxycinnamaldehyde (22) were isolated. All compounds were identified by mass spectrometry and nuclear magnetic resonance spectroscopic measurements. Compounds 6, 11, and 16–18 are described for the first time. Compounds 13 – 15 are isolated as natural products for the first time, compound 8 for the first time from a plant. Antiplasmodial IC50 values and cytotoxicity against L6 rat skeletal myoblasts were determined. Isoflindissone lactone (15) was the most active compound with an IC50 of 2.2 µM against P. falciparum and a selectivity index of 18.

A new bibenzyl derivative (4), together with two glycosylated flavonoids (1 and 2), batatasin III (3) and the phenanthrene isohircinol (5) were isolated from the aerial parts of Gavilea lutea. Their structures were elucidated on the basis of spectroscopic studies including 1D and 2D NMR, UV, IR and HRESIMS. All isolated compounds were evaluated for their antifungal activity towards Candida albicans. The new compound 4 showed inhibitory activity with a MIQ of 50 μg. In addition, compound 4 exhibited a selective activity (IC50 = 2.3 μg/mL) against Leishmania donovani.