Ealamines A-H, a Series of Naphthylisoquinolines with the Rare 7,8'-Coupling Site, from the Congolese Liana Ancistrocladus ealaensis, Targeting Pancreatic Cancer Cells.

From the twigs and leaves of the Central African liana Ancistrocladus ealaensis (Ancistrocladaceae), a series of ten 7,8'-coupled naphthylisoquinoline alkaloids were isolated, comprising eight new compounds, named ealamines A-H (4a, 4b, 5-10), and two known ones, 6-O-demethylancistrobrevine A (11) and yaoundamine A (12), which had previously been found in related African Ancistrocladus species. Only one of the new compounds within this series, ealamine H (10), is a typical Ancistrocladaceae-type alkaloid, with 3S-configuration at C-3 and an oxygen function at C-6, whereas seven of the new alkaloids are the first 7,8'-linked "hybrid-type" naphthylisoquinoline alkaloids, i.e., 3R-configured and 6-oxygenated in the tetrahydroisoquinoline part. The discovery of such a broad series of 7,8'-coupled naphthyltetrahydroisoquinolines is unprecedented, because representatives of this subclass of alkaloids are normally found in Nature quite rarely. The stereostructures of the new ealamines were assigned by HRESIMS, 1D and 2D NMR, oxidative degradation, and experimental and quantum-chemical ECD investigations, and-in the case of ealamine A (4a)-also confirmed by X-ray diffraction analysis. Ealamines A-D exhibited distinct-and specific-antiplasmodial activities, and they displayed pronounced preferential cytotoxic effects toward PANC-1 human pancreatic cancer cells in nutrient-deprived medium, without causing toxicity under normal, nutrient-rich conditions, with ealamine C (5) as the most potent agent.

A Near-Complete Series of Four Atropisomeric Jozimine A2-Type Naphthylisoquinoline Dimers with Antiplasmodial and Cytotoxic Activities and Related Alkaloids from Ancistrocladus abbreviatus.

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.

Ancistrobrevines E-J and related naphthylisoquinoline alkaloids from the West African liana Ancistrocladus abbreviatus with inhibitory activities against Plasmodium falciparum and PANC-1 human pancreatic cancer cells.

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.

Mbandakamine-Type Naphthylisoquinoline Dimers and Related Alkaloids from the Central African Liana Ancistrocladus ealaensis with Antiparasitic and Antileukemic Activities.

Four new dimeric naphthylisoquinoline alkaloids, michellamine A5 (2) and mbandakamines C-E (4-6), were isolated from the Congolese plant Ancistrocladus ealaensis, along with the known dimer mbandakamine A (3). They represent constitutionally unsymmetric dimers, each consisting of two 5,8'-coupled naphthylisoquinoline monomers. While the molecular halves of michellamine A5 (2) are linked via C-6' of both of the naphthalene moieties, i.e., via the least-hindered positions, so that the central biaryl axis is configurationally unstable and not an additional element of chirality, the mbandakamines 3-6 possess three consecutive stereogenic axes. Their monomeric units are linked through an unprecedented 6',1″-coupling in the binaphthalene core, leading to a high steric load, since the central axis is located in one of the peri-positions, neighboring one of the outer axes. In addition, four new 5,8'-coupled monomeric naphthylisoquinolines, viz., ancistroealaines C-F (7-10), were identified, along with four "naphthalene-devoid" tetra- and dihydroisoquinolines, named ealaines A-D (11-14). The new mbandakamines C (4) and D (5) showed pronounced activities against the malaria parasite Plasmodium falciparum, and they were likewise found to display strong cytotoxic activities against human leukemia (CCRF-CEM) and multi-drug-resistant tumor cells (CEM/ADR5000).

2 H-1,2,3-Triazole-Based Dipeptidyl Nitriles: Potent, Selective, and Trypanocidal Rhodesain Inhibitors by Structure-Based Design.

Macrocyclic inhibitors of rhodesain (RD), a parasitic cysteine protease and drug target for the treatment of human African trypanosomiasis, have shown low metabolic stability at the macrocyclic ether bridge. A series of acyclic dipeptidyl nitriles was developed using structure-based design (PDB ID: 6EX8 ). The selectivity against the closely related cysteine protease human cathepsin L (hCatL) was substantially improved, up to 507-fold. In the S2 pocket, 3,4-dichlorophenylalanine residues provided high trypanocidal activities. In the S3 pocket, aromatic residues provided enhanced selectivity against hCatL. RD inhibition ( Ki values) and in vitro cell-growth of Trypanosoma brucei rhodesiense (IC50 values) were measured in the nanomolar range. Triazole-based ligands, obtained by a safe, gram-scale flow production of ethyl 1 H-1,2,3-triazole-4-carboxylate, showed excellent metabolic stability in human liver microsomes and in vivo half-lives of up to 1.53 h in mice. When orally administered to infected mice, parasitaemia was reduced but without complete removal of the parasites.

Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors.

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei ( T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors ( Ki < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC50 < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys-SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival.

Development of a Focused Library of Triazole-Linked Privileged-Structure-Based Conjugates Leading to the Discovery of Novel Phenotypic Hits against Protozoan Parasitic Infections.

Protozoan infections caused by Plasmodium, Leishmania, and Trypanosoma spp. contribute significantly to the burden of infectious diseases worldwide, causing severe morbidity and mortality. The inadequacy of available treatments calls for cost- and time-effective drug discovery endeavors. To this end, we envisaged the triazole linkage of privileged structures as an effective drug design strategy to generate a focused library of high-quality compounds. The versatility of this approach was combined with the feasibility of a phenotypic assay, integrated with early ADME-tox profiling. Thus, an 18-membered library was efficiently assembled via Huisgen cycloaddition of phenothiazine, biphenyl, and phenylpiperazine scaffolds. The resulting 18 compounds were then tested against seven parasite strains, and counter-screened for selectivity against two mammalian cell lines. In parallel, hERG and cytochrome P450 (CYP) inhibition, and mitochondrial toxicity were assessed. Remarkably, 10-((1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-5-yl)methyl)-10H-phenothiazine (7) and 10-(3-(1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-4-yl)propyl)-10H-phenothiazine (12) showed respective IC50 values of 1.8 and 1.9 µg mL-1 against T. cruzi, together with optimal selectivity. In particular, compound 7 showed a promising ADME-tox profile. Thus, hit 7 might be progressed as an antichagasic lead.

Michellamines A6 and A7, and further mono- and dimeric naphthylisoquinoline alkaloids from a Congolese Ancistrocladus liana and their antiausterity activities against pancreatic cancer cells.

Michellamines A6 (1) and A7 (2) are the first dimers of 5,8'-coupled naphthylisoquinoline alkaloids with cis-configured stereocenters in both tetrahydroisoquinoline subunits. They were isolated from the leaves of a recently discovered, yet unidentified Congolese Ancistrocladus liana that shares some morphological characteristics with Ancistrocladus likoko. Two further new dimeric analogs, michellamines B4 (3) and B5 (4), were obtained, along with two previously likewise unknown monomers, ancistrobonsolines A1 (5) and A2 (6), which, besides one single known other example, are the only naphthyldihydroisoquinolines with an M-configured biaryl axis and R-configuration at C-3. Moreover, five compounds earlier reported from other Ancistrocladus species were identified, ancistroealaine C (7), korupensamines A (8a) and B (8b), and michellamines A2 (9) and E (10). Their complete structural elucidation succeeded due to the fruitful interplay of spectroscopic, chemical, and chiroptical methods. Chemotaxonomically, the stereostructures of the metabolites clearly delineate this Congolese Ancistrocladus liana from all known related species, showing that it might be a new taxon. Ancistrobonsolines A1 (5) and A2 (6) exhibited strong preferential cytotoxicities against human PANC-1 pancreatic cancer cells under nutrient-deprived conditions, without displaying toxicity in normal, nutrient-rich medium. Against cervical HeLa cancer cells, the dimeric alkaloids michellamines A6 (1) and E (10) displayed the highest cytotoxic activities, comparable to that of the standard agent, 5-fluorouracil. Furthermore, ancistrobonsolines A1 (5) and A2 (6) showed weak-to-moderate antiprotozoal activities.

Jozilebomines A and B, Naphthylisoquinoline Dimers from the Congolese Liana Ancistrocladus ileboensis, with Antiausterity Activities against the PANC-1 Human Pancreatic Cancer Cell Line.

Two new naphthylisoquinoline dimers, jozilebomines A (1a) and B (1b), were isolated from the roots of the Congolese plant Ancistrocladus ileboensis, along with the known dimer jozimine A2 (2). These compounds are Dioncophyllaceae-type metabolites, i.e., lacking oxygen functions at C-6 and with an R-configuration at C-3 in their tetrahydroisoquinoline moieties. The dimers 1a and 1b consist of two 7,1'-coupled naphthylisoquinoline monomers linked through an unprecedented 3',6″-coupling in the binaphthalene core and not, as in 2, via the C-3-positions of the two naphthalene units. Thus, different from the C2-symmetric jozimine A2 (2), the new jozilebomines are constitutionally unsymmetric. The central biaryl axis of each of the three dimers is rotationally hindered, so that 1a, 1b, and 2 possess three consecutive chiral axes. The two jozilebomines have identical constitutions and the same absolute configurations at all four stereogenic centers, but differ from each other in their axial chirality. Their structural elucidation was achieved by HRESIMS, 1D and 2D NMR, oxidative degradation, and experimental and calculated ECD data. They exhibited distinct and specific antiplasmodial activities. All dimers showed potent cytotoxicity against HeLa human cervical cancer cells and preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions. Furthermore, these dimers significantly inhibited the colony formation of PANC-1 cells, even when exposed to noncytotoxic concentration for a short time. Jozilebomines A (1a) and B (1b) and jozimine A2 (2) represent novel potential candidates for future drug development against pancreatic cancer.

Terpenoids from the Oleo-Gum-Resin of Boswellia serrata and Their Antiplasmodial Effects In Vitro.

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-ß-boswellic acid (10), 3-epi-neoilexonol (11), uvaol (12), ß-boswellic aldehyde (13), 5α-tirucalla-8,24-dien-3α-ol (14), isoflindissone lactone (15), isoflindissol lactone (16), rel (8R,9S,20R)-tirucall-24-ene-3ß,20-diol (17), and rel (3α,8R, 9S,20R,24S)-20,24-epoxytirucalla-3,25-diol (18) as well as the sesquiterpene ß-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.

Antileukemic ancistrobenomine B and related 5,1'-coupled naphthylisoquinoline alkaloids from the Chinese liana Ancistrocladus tectorius.

A striking feature of the metabolite pattern of the Southeast Asian liana Ancistrocladus tectorius (Ancistrocladaceae) is the predominance of 5,1'-coupled naphthylisoquinoline alkaloids. About 20 alkaloids of this coupling type have so far been discovered in this plant species. Here, we report on the isolation of four new 5,1'-linked naphthylisoquinolines from the twigs and stems of A. tectorius. Two of them, the ancistrobenomines B (5) and C (6), belong to the very rare group of alkaloids with a fully dehydrogenated isoquinoline portion. Likewise unusual for naphthylisoquinoline alkaloids is the presence of a hydroxymethylene group at C-3. Within the large class of meanwhile ca. 180 such natural products, this structural peculiarity had so far been known only from two other representatives isolated from the Malaysian species A. benomensis, and from one single naphthalene-devoid 3-hydroxymethyleneisoquinoline from A. tectorius. Seven further 5,1'-linked alkaloids, previously isolated from related Asian and African Ancistrocladus species, have now been identified for the first time in A. tectorius. Their structural elucidation was achieved by spectroscopic analysis including HRESIMS, 1D and 2D NMR, and by chemical (oxidative degradation) and chiroptical (electronic circular dichroism) methods. Ancistrobenomine B (5) exhibited moderate effects against Plasmodium falciparum and Trypanosoma brucei rhodesiense in vitro, and it was found to display strong cytotoxic activities against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ADR5000.

Synthesis and activity of nucleoside-based antiprotozoan compounds.

Parasitic protozoa employ a salvage pathway to synthesize purines and generate essential active nucleotides, whereas mammals are capable of their de novo biosynthesis. This difference provides opportunity for the design of potential new antiprotozoan compounds. A series of 47 adenosine analogues was prepared with modifications at the 2-, 6- and 5'-positions, based on the hypothesis that such compounds would serve as substrates for protozoan nucleoside salvage enzymes, while remaining refractory in mammalian cells. The nucleosides were designed to produce toxic metabolites upon cleavage to the corresponding purine base by the parasite. Three 7-deazaguanosine derivatives were prepared with similar objectives. All of these compounds were tested in vitro against T. brucei (African sleeping sickness), T. cruzi (Chagas' disease), L. donovani (leishmaniasis) and P. falciparum (malaria). In order to determine the therapeutic selectivity indices (SI) of the antiprotozoan nucleosides, their cytotoxicities toward a rat myoblast cell line were also determined. One adenosine derivative proved highly effective against P. falciparum (IC50=110nM and SI=1010, while a modified guanosine displayed potent activities against L. donovani (IC50=60nM, SI=2720) and T. brucei (IC50=130nM, SI=1250), as well as moderate activity against T. cruzi (IC50=3.4µM, SI=48). These results provide proof of concept for the nucleoside-based antiprotozoan strategy, as well as potential lead compounds for further optimization and validation.

Dioncophyllines C2, D2, and F and Related Naphthylisoquinoline Alkaloids from the Congolese Liana Ancistrocladus ileboensis with Potent Activities against Plasmodium falciparum and against Multiple Myeloma and Leukemia Cell Lines.

Dioncophylline F (1), the first 5,8'-coupled dioncophyllaceous alkaloid (i.e., lacking an oxygen function at C-6 and possessing an R-configuration at C-3), was isolated from the recently described Congolese liana Ancistrocladus ileboensis. Two further, likewise Dioncophyllaceae-type, alkaloids, the dioncophyllines C2 (2) and D2 (3), were identified, along with the Ancistrocladaceae-type compound ancistrocladisine B (4), which is oxygenated at C-6 and S-configured at C-3. The structures of the new compounds were determined by spectroscopic, chemical, and chiroptical methods. The stereostructure of 1 was further confirmed by total synthesis. As a consequence of the lack of a methyl group ortho to their biaryl axes, both dioncophylline F (1) and the 7,8'-coupled dioncophylline D2 (3) occur as pairs of configurationally semistable and, thus, slowly interconverting atropo-diastereomers, whereas dioncophylline C2 (2), with its 5,1'-linkage, is configurationally stable at the axis. Eight further known naphthylisoquinolines were isolated from A. ileboensis, among them dioncophylline A (P-10), its 4'-O-demethyl analogue P-11, and 5'-O-methyldioncophylline D (7), which were found to display strong cytotoxic activities against multiple myeloma INA-6 cells (P-10 even stronger than the standard drug melphalan) and against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells and their multidrug-resistant subline, CEM/ADR5000. Moreover, the dioncophyllines 1, 3, and 7 showed high-and specific-activities against the malaria parasite Plasmodium falciparum.

Ancistectorine D, a naphthylisoquinoline alkaloid with antiprotozoal and antileukemic activities, and further 5,8'- and 7,1'-linked metabolites from the Chinese liana Ancistrocladus tectorius.

From the twigs and stems of the Chinese liana Ancistrocladus tectorius (Ancistrocladaceae), two new 5,8'-coupled naphthylisoquinolines, ancistectorine D (5) and its 6-O-demethyl derivative 6, were isolated, along with two new 7,1'-linked alkaloids, 6-O-methylancistectorine B1 (7) and ancistectorine B2 (8). Two further compounds, ancistroealaine A (4) and 6-O-demethyl-8-O-methyl-7-epi-ancistrobrevine D (10), already known from related Asian and African Ancistrocladus species, were discovered for the first time in A. tectorius. The structure elucidation was achieved by spectroscopic analysis including HRESIMS, 1D and 2D NMR, and by chemical (oxidative degradation) and chiroptical (circular dichroism) methods. Chemotaxonomically remarkable, 5,8'-coupled naphthylisoquinolines have as yet been found quite rarely in Asian Ancistrocladus species, where only two examples have so far been detected, while alkaloids with this coupling type represent the by far the largest group of constituents in African taxa. Ancistectorine D (5) shows promising activities against the protozoan parasites Trypanosoma cruzi and Leishmania donovani, and it was likewise found to display strong cytotoxic activities against human leukemia (CCRF-CEM) and multidrug-resistant tumor cells (CEM/ADR5000).

Novel nucleoside-based antimalarial compounds.

The malaria-causing parasite Plasmodium falciparum employs a salvage pathway for the biosynthesis of nucleotides, in contrast to de novo biosynthesis that is utilized by the human host. A series of twenty-two 2-, 6- and 5'-modified adenosine ribonucleosides was synthesized, with the expectation that these compounds would generate toxic metabolites instead of active nucleotides by the pathogen, while remaining inert in host cells. Bioassays with P. falciparum (K1 strain) indicated IC50 values as low as 110nM and a selectivity index with respect to cytotoxicity toward an L6 rat myoblast cell line of >1000 for the most potent analogue.

Abietane-Type Diterpenoid Amides with Highly Potent and Selective Activity against Leishmania donovani and Trypanosoma cruzi.

Dehydroabietylamine (1) was used as a starting material to synthesize a small library of dehydroabietyl amides by simple and facile methods, and their activities against two disease-causing trypanosomatids, namely, Leishmania donovani and Trypanosoma cruzi, were assayed. The most potent compound, 10, an amide of dehydroabietylamine and acrylic acid, was found to be highly potent against these parasites, displaying an IC50 value of 0.37 µM against L. donovani axenic amastigotes and an outstanding selectivity index of 63. Moreover, compound 10 fully inhibited the growth of intracellular amastigotes in Leishmania donovani-infected human macrophages with a low IC50 value of 0.06 µM. This compound was also highly effective against T. cruzi amastigotes residing in L6 cells with an IC50 value of 0.6 µM and high selectivity index of 58, being 3.5 times more potent than the reference compound benznidazole. The potent activity of this compound and its relatively low cytotoxicity make it attractive for further development in pursuit of better drugs for patients suffering from leishmaniasis and Chagas disease.

Targeting the parasite's DNA with methyltriazenyl purine analogs is a safe, selective, and efficacious antitrypanosomal strategy.

The human and veterinary disease complex known as African trypanosomiasis continues to inflict significant global morbidity, mortality, and economic hardship. Drug resistance and toxic side effects of old drugs call for novel and unorthodox strategies for new and safe treatment options. We designed methyltriazenyl purine prodrugs to be rapidly and selectively internalized by the parasite, after which they disintegrate into a nontoxic and naturally occurring purine nucleobase, a simple triazene-stabilizing group, and the active toxin: a methyldiazonium cation capable of damaging DNA by alkylation. We identified 2-(3-acetyl-3-methyltriazen-1-yl)-6-hydroxypurine (compound 1) as a new lead compound, which showed submicromolar potency against Trypanosoma brucei, with a selectivity index of >500, and it demonstrated a curative effect in animal models of acute trypanosomiasis. We investigated the mechanism of action of this lead compound and showed that this molecule has significantly higher affinity for parasites over mammalian nucleobase transporters, and it does not show cross-resistance with current first-line drugs. Once selectively accumulated inside the parasite, the prodrug releases a DNA-damaging methyldiazonium cation. We propose that ensuing futile cycles of attempted mismatch repair then lead to G2/M phase arrest and eventually cell death, as evidenced by the reduced efficacy of this purine analog against a mismatch repair-deficient (MSH2(-/-)) trypanosome cell line. The observed absence of genotoxicity, hepatotoxicity, and cytotoxicity against mammalian cells revitalizes the idea of pursuing parasite-selective DNA alkylators as a safe chemotherapeutic option for the treatment of human and animal trypanosomiasis.

2-Phenoxy-1,4-naphthoquinones: From a Multitarget Antitrypanosomal to a Potential Antitumor Profile.

A small library of 2-phenoxy-1,4-naphthoquinone and 2-phenoxy-1,4-anthraquinone derivatives was initially developed to optimize the antitrypanosomatid profile of the multitarget hit compound B6 (1). The whole series was evaluated against the three most important human trypanosomatid pathogens (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani), and two compounds (14 and 21) showed good activity, despite a concomitant mammalian cytotoxicity. Furthermore, a subset also inhibited the glycolytic TbGAPDH enzyme in vitro. In light of these results and aware of the antitumor properties of quinones, the anticancer potential of some selected derivatives was investigated. Intriguingly, the tested compounds displayed antitumor activity, while being less toxic against noncancerous cells. The observed cytotoxic potency was ascribed to a multitarget mechanism of action accounting for hGAPDH inhibition and mitochondrial toxicity. Overall, the development of further derivatives, able to finely modulate multiple pathways of cancer or parasite cell metabolism, might lead to more effective treatments against these devastating diseases.

Antiprotozoal Effect of Artemisia indica Extracts and Essential Oil.

Diverse solvent extracts of Artemisia indica leaves originating from the West Bengal region (India) were assessed for the content of artemisinin and characteristic Artemisia polymethoxyflavonoids, namely eupatin (1), casticin (2), chrysoplenetin (3), cirsilineol (4), chrysophenol-D (5), and artemetin (6). HPLC-DAD and HPLC-MS were used to investigate the extracts macerated by solvents of increasing polarity, i.e., petroleum ether, n-hexane, dichloromethane, acetone, MeOH, or EtOH (either 96, 80, or 60 % v/v), and hot water. Artemisinin was absent in all extracts. The acetone and EtOH extracts comprised the highest levels of polymethoxyflavonoids, whereas no flavonoid could be detected in the infusion. None of the remaining extracts contained chryosphenol-D (5) or artemetin (6), while chrysoplenetin (3) was found in all extracts. The essential oil of the plant was also obtained by hydrodistillation and analysed by gas chromatography and gas chromatography-mass spectrometry simultaneously. Of the 92 compounds detected in the oil, camphor (13.0 %) and caryophyllene oxide (10.87 %) were the major components. All solvent extracts and the volatile oil showed in vitro antimalarial activity, plus a potential malaria prophylactic effect by inhibiting at least two recombinant plasmodial fatty acid biosynthesis (PfFAS-II) enzymes. Except for the infusion, all extracts were also active against other parasitic protozoa and displayed low cytotoxicity against mammalian cells. This is the first detailed study investigating both artemisinin and polymethoxyflavonoid content as well as in vitro malaria prophylactic and detailed antiprotozoal potential of A. indica extracts against a panel of protozoan parasites. This is also the first report of antiparasitic activity of the essential oil of the plant.