Structure-activity relationship study of antitrypanosomal analogues of gibbilimbol B using multivariate analysis and computation-aided drug design.

Gibbilimbol B and analogues were isolated from the Brazilian plant Piper malacophyllum and displayed activity against trypomastigote forms of Trypanosoma cruzi as well as reduced toxicity against NCTC cells. These results stimulated the preparation of a series of 24 chemically related analogues to study the potential of these compounds against T. cruzi trypomastigotes and explore structure-activity relationships. Initially, 12 compounds were planned, maintaining the same extension of the linear side chain of gibbilimbol B and unsaturation on the C-4 position but changing the functional groups - ester and amide - and variating the substituent at the p-position in the aromatic ring. Other 12 compounds were prepared using a branched side chain containing an ethyl group at the C-2 position. Overall, these structurally-related analogues demonstrated promising activity against trypomastigote forms (EC50 < 20 µM) and no mammalian cytotoxicity to fibroblasts (CC50 > 200 µM). Using multivariate statistics and machine learning analysis, aspects associated with structure/activity were related to their three-dimensional structure and, mainly, to the substituents on the aromatic ring. Obtained results suggested that the presence of t-butyl or nitro groups at p-position with appropriate side chains causes an alteration in the electron topological state, Van der Waals volumes, surface areas, and polarizabilities of tested compounds which seem to be essential for biological activity against T. cruzi parasites.

Essential Oils from Different Myrtaceae Species from Brazilian Atlantic Forest Biome - Chemical Dereplication and Evaluation of Antitrypanosomal Activity.

Chagas Disease (CD), caused by flagellate protozoan Trypanosoma cruzi, is a Neglected Tropical Diseases (NTD) that affect approximately seven million people worldwide with a restrict therapeutical arsenal. In the present study, the essential oils from 18 Myrtaceae species were extracted, chemically dereplicated, and evaluated in vitro against T. cruzi. From these, eight essential oils were considered promising (IC50 <10 µg/mL and SI>10) against the protozoan: Eugenia florida, E. acutata, E. widgrenii, Calyptranthes brasilienses, C. widgreniana, Plinia cauliflora, Campomanesia xanthocarpa, and Psidium guajava. Multivariate data analysis pointed out (E)-caryophyllene, α-humulene, limonene, caryophyllene oxide, and α-copaene playing an important role in the anti-T. cruzi activity. The obtained results demonstrated the potential of essential oils of Myrtaceae species as valuable sources of bioactive compounds against T. cruzi.

Antileishmanial Effects of Acetylene Acetogenins from Seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae) and Semisynthetic Derivatives.

As part of our continuous studies involving the prospection of natural products from Brazilian flora aiming at the discovery of prototypes for the development of new antiparasitic drugs, the present study describes the isolation of two natural acetylene acetogenins, (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11'-yn-19'-enyl)butanolide (1) and (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11'-ynyl)butanolide (2), from the seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae). Using an ex-vivo assay, compound 1 showed an IC50 value of 29.9 µM against the intracellular amastigote forms of Leishmania (L.) infantum, whereas compound 2 was inactive. These results suggested that the terminal double bond plays an important role in the activity. This effect was also observed for the semisynthetic acetylated (1a and 2a) and eliminated (1b and 2b) derivatives, since only compounds containing a double bond at C-19 displayed activity, resulting in IC50 values of 43.3 µM (1a) and 23.1 µM (1b). In order to evaluate the effect of the triple bond in the antileishmanial potential, the mixture of compounds 1 + 2 was subjected to catalytic hydrogenation to afford a compound 3 containing a saturated side chain. The antiparasitic assays performed with compound 3, acetylated (3a), and eliminated (3b) derivatives confirmed the lack of activity. Furthermore, an in-silico study using the SwissADME online platform was performed to bioactive compounds 1, 1a, and 1b in order to investigate their physicochemical parameters, pharmacokinetics, and drug-likeness. Despite the reduced effect against amastigote forms of the parasite to the purified compounds, different mixtures of compounds 1 + 2, 1a + 2a, and 1b + 2b were prepared and exhibited IC50 values ranging from 7.9 to 38.4 µM, with no toxicity for NCTC mammalian cells (CC50 > 200 µM). Selectivity indexes to these mixtures ranged from >5.2 to >25.3. The obtained results indicate that seeds of Porcelia macrocarpa are a promising source of interesting prototypes for further modifications aiming at the discovery of new antileishmanial drugs.

Antitrypanosomal Lactones from Nectandra barbellata.

Twigs of Nectandra barbellata were extracted using a solution of the ionic liquid 1-butyl-3-methylimidazolium bromide (BMImBr) in H2O, assisted by microwave (MAE). After successive chromatographic steps, one sesquiterpene, costic acid, and three new related lactones, (R)-3(7)-Z-3-hexadec-21-enylidene-5-(hydroxymethyl)tetrahydrofuran-2-one (1), (R)-3(7)-Z-3-hexadecylidene-5-(hydroxymethyl)tetrahydrofuran-2-one (2), and (R)-3(7)-Z-3-docosylidene-5-(hydroxymethyl)tetrahydrofuran-2-one (3), were isolated. After structural elucidation using IR, UV, HRESIMS, NMR, ECD, and VCD, compounds 1-3 were tested against trypomastigote forms of Trypanosoma cruzi. The mechanism of action of bioactive isolated compounds was studied using different fluorescent-based approaches to investigate alterations of the plasma membrane, permeability/electric potential (ΔΨp), reactive oxygen species levels, mitochondria (electric membrane potential, ΔΨm/ATP levels), Ca2+ levels, and pH of the acidocalcisomes. In addition, in silico studies predicted no resemblance to pan assay interference compounds (PAINS).

Chemical Constituents from Aerial Parts of Baccharis sphenophylla and Effects against Intracellular Forms of Trypanosoma cruzi.

The hexane extract from aerial parts Baccharis sphenophylla Dusén ex Malme (Asteraceae) displayed activity against amastigote forms of Trypanossoma cruzi and was subjected to chromatographic steps to afford one unreported - 7α-hydroxy-ent-abieta-8(14),13(15)-dien-16,12ß-olide (1) and three known diterpenes - ent-kaur-16-en-19-oic acid, (2), grandifloric acid (3), and 15ß-tiglinoyloxy-ent-kaur-16-en-19-oic acid (4), two sesquiterpenes - spathulenol (5) and oplopanone (6) - as well as hexacosyl p-coumarate (7). Isolated compounds were characterized by NMR and ESI-HR-MS spectra and were evaluated in vitro for activity against amastigote forms of the parasite T. cruzi - the relevant clinical form in the chronic phase of Chagas disease. In addition, the activity of compounds 1-7 against NCTC cells was evaluated. Compounds 1 and 7 showed effectiveness with EC50 values of 21.3 and 16.9 µM, respectively. Both compounds also exhibited reduced toxicity against NCTC cells (CC50 >200 µM) with SI values higher than 9.4 and 11.9. Obtained results suggest that the new ent-abietane diterpene 1 and alkyl coumarate 7 could be used as prototypes for the development of novel and selective semisynthetic derivatives against intracellular forms of T. cruzi.

Simplified Derivatives of Dibenzylbutyrolactone Lignans from Hydrocotyle bonariensis as Antitrypanosomal Candidates.

The search for the pharmacophore of a bioactive compound, crucial for drug discovery studies, involves the adequate arrangement of different atoms in the molecule. As part of a continuous work aiming discovery of new drug candidates against the protozoan parasite Trypanosoma cruzi, the hexane extract of Hydrocotyle bonariensis was subjected to a bioactivity-guided fractionation to afford two chemically related dibenzylbutyrolactone lignans - hinokinin (1) and hibalactone (2). Compounds 1 and 2 showed activity against trypomastigote with EC50 values of 17.0 and 69.4 µM, respectively. Compound 1 was also active against the clinically relevant form of the parasite, amastigotes, displaying an EC50 value of 34.4 µM. The structure-activity relationship (SAR) indicated that the absence of the double bond at C-7 is a crucial feature for the increment of the antiparasitic activity. The lethal action of the most potent compound 1 was investigated in the trypomastigotes. The fluorescent-based assay with SYTOX Green demonstrated a significant alteration of the plasma membrane permeability of the parasite. Additionally, compound 1 demonstrated no significant hemolytic activity in mice erythrocytes at 200 µM. To search the pharmacophore, three different simplified compounds - 3,4-methylenedioxydihydrocinnamic acid (3), 3,4-methylenedioxydihydrocinnamic alcohol (4) and 3,4-methylenedioxycinnamic acid (5) - were prepared and tested against T. cruzi. These derivatives displayed EC50 values of 37.2 (3), 25.8 (4) and 73.5 (5) µM against trypomastigotes, and 41.3 (3) and 48.2 (4) µM against amastigotes, whereas compound 5 was inactive. Except for compound 2, which resulted in a CC50 value of 114.5 µM, all compounds showed no mammalian cytotoxicity at 200 µM. An in silico ADMET study was performed and predicted values demonstrated an acceptable drug-likeness profile for compounds 1-5. Despite the minor reduction in the potency, the simplified derivatives retained the antitrypanosomal activity against the intracellular amastigotes, even with 95 % reduction of their molecular weight. Additionally, in silico studies suggested them as more soluble compounds, making these simplified structures promising scaffolds for optimization studies in Chagas disease.

Aporphine Alkaloids from Ocotea puberula with Anti-Trypanosoma Cruzi Potential - Activity of Dicentrine-ß-N-Oxide in the Plasma Membrane Electric Potentials.

One new aporphine, dicentrine-ß-N-oxide (1), together with five related known alkaloids dehydrodicentrine (2), predicentrine (3), N-methyllaurotetanine (4), cassythicine (5), and dicentrine (6) were isolated from the leaves of Ocotea puberula (Lauraceae). Antiprotozoal activity of the isolated compounds was evaluated in vitro against trypomastigote forms of Trypanosoma cruzi. Among the tested compounds, alkaloid 1 exhibited higher potential with EC50 value of 18.2 µM and reduced toxicity against NCTC cells (CC50 >200 µM - SI>11.0), similar to positive control benznidazole (EC50 of 17.7 µM and SI=10.7). Considering the promising results of dicentrine-ß-N-oxide (1) against trypomastigotes, the mechanism of parasite death caused by this alkaloid was investigated. As observed, this compound reached the plasma membrane electric potential directly after 2 h of incubation and triggered mitochondrial depolarization, which probably leads to trypomastigote death. Therefore, dicentrine-ß-N-oxide (1), reported for the first time in this work, can contribute to future works for the development of new trypanocidal agents.

γ-Lactones from Persea americana and Persea fulva - in Vitro and in Silico Evaluation of Trypanosoma cruzi Activity.

In the present study, five known γ-lactones (majoranolide B - 1, majorenolide - 2, majorynolide - 3, lincomolide D - 4, and isolinderanolide E - 5), as well as a new one (perseanolide - 6), were isolated from Persea fulva and P. americana. All isolated compounds exhibited potential activity against trypomastigote forms of Trypanosoma cruzi, whereas compounds 2 (EC50 of 4.8 µM) and 6 (EC50 of 3.6 µM) displayed superior activity than the positive control benznidazole (EC50 of 16.4 µM), with selectivity index (SI) values of 17.8 and >55.6, respectively (benznidazole, SI>12.2). Molecular docking studies were performed for 1-6 against six T. cruzi molecular targets. Using this approach, we observed that, even though perseanolide (6) showed favorable docking to several studied targets, the results were especially promising for hypoxanthine phosphoribosyl transferase (PDB 1TC1). As PDB 1TC1 is associated to the transference of a monophosphorylated ribose from phosphoribosylpyrophosphate (PRPP) in the ribonucleotide synthesis pathway, this interaction may affect the survival of T. cruzi in mammalian cells. The data herein also indicate that possible intermolecular interactions between 6 and PDB 1TC1 derive from (i) hydrogen bonds in the α,ß-unsaturated-γ-lactone unity and (ii) hydrophobic interactions in the long-chain alkyl group. Based on our results, perseanolide (6), reported for the first time in this work, can auspiciously contribute to future works regarding new trypanocidal agents.

Discovery of New Hits as Antitrypanosomal Agents by In Silico and In Vitro Assays Using Neolignan-Inspired Natural Products from Nectandra leucantha.

In the present study, the phytochemical study of the n-hexane extract from flowers of Nectandra leucantha (Lauraceae) afforded six known neolignans (1-6) as well as one new metabolite (7), which were characterized by analysis of NMR, IR, UV, and ESI-HRMS data. The new compound 7 exhibited potent activity against the clinically relevant intracellular forms of T. cruzi (amastigotes), with an IC50 value of 4.3 µM and no observed mammalian cytotoxicity in fibroblasts (CC50 > 200 µM). Based on the results obtained and our previous antitrypanosomal data of 50 natural and semi-synthetic related neolignans, 2D and 3D molecular modeling techniques were employed to help the design of new neolignan-based compounds with higher activity. The results obtained from the models were important to understand the main structural features related to the biological response of the neolignans and to aid in the design of new neolignan-based compounds with better biological activity. Therefore, the results acquired from phytochemical, biological, and in silico studies showed that the integration of experimental and computational techniques consists of a powerful tool for the discovery of new prototypes for development of new drugs to treat CD.

Interaction of dicentrinone, an antitrypanosomal aporphine alkaloid isolated from Ocotea puberula (Lauraceae), in cell membrane models at the air-water interface.

In the present work, the oxoaporphine alkaloid dicentrinone was isolated, for the first time, from leaves of Ocotea puberula (Lauraceae). This alkaloid exhibited antiparasitic activity against trypomastigote forms of Trypanosoma cruzi (IC50 of 16.4 ± 1.7 µM), similar to the positive control benznidazole (IC50 of 18.7 ± 4.1 µM), reduced mammalian cytotoxicity (CC50 > 200 µM), and a selectivity index (SI) higher than 12. These results were correlated with the effects observed using cellular membrane models, represented by 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), in Langmuir monolayers. Dicentrinone was incorporated in the films, submitted to lateral compression, and characterized by tensiometry. As observed in compression-decompression and time-stability curves, dicentrinone expanded the lipid monolayers, decreased the compressional modulus of the film, and reduced the stability of the monolayer. Brewster Angle Microscopy and interfacial Infrared Spectroscopy showed that dicentrinone causes the monolayers to be segregated in phases, and to increase the number of gauche/trans conformers ratio for the lipid acyl methylene groups, indicating configurational disorder. As a result, dicentrinone caused a disturbance in the cell membrane models, altering the physicochemical properties of the lipid surface such as thermodynamic, rheological, morphological, and structural aspects. These results can be useful to understand the interactions between dicentrinone and lipid biological surfaces at the molecular level.

Anti-Trypanosoma cruzi activity of costic acid isolated from Nectandra barbellata (Lauraceae) is associated with alterations in plasma membrane electric and mitochondrial membrane potentials.

As part of our continuous studies on prospecting metabolites from Brazilian plant species with pharmacologic activity against Trypanosoma cruzi, the n-hexane extract from twigs of Nectandra barbellata (Lauraceae) was subjected to a bioactivity-guided fractionation to afford the sesquiterpene costic acid. As results, costic acid induced a trypanocidal effect with IC50 of 37.8 and 7.9 µM to trypomastigotes and intracellular amastigotes, respectively. When tested in L929 cells, no cytotoxicity was detected in the highest tested concentration (CC50 > 200 µM), resulting in SI values >5 and >25 to trypomastigotes and amastigotes, respectively. Based on these promising results against T. cruzi, a mechanistic study of the parasite death was investigated. The flow cytometry analysis of costic acid-treated parasites showed depolarization of the plasma membrane electric potential. Spectrofluorimetrical analysis and transmission electron microscopy showed no evidence of plasma membrane permeability alteration of trypomastigotes, but strong ultrastructural damage, evidenced by large vacuoles. Although Ca2+ and reactive oxygen species (ROS) levels were unaltered after short time incubation with costic acid, it rapidly affected the mitochondria, leading to a depolarized potential of the membrane, reducing the ATP levels. In silico studies of costic acid showed good predictions for drug-likeness, with adherence to Lipinskís rules of five (RO5), good ADMET properties and no alerts for Pan-Assay Interference Compounds (PAINS). Therefore, costic acid demonstrated promising activity against T. cruzi parasites, with high selectivity to intracellular amastigotes. Considering the lethal action of costic acid in affecting a vital and unique organelle as the mitochondria, it could be considered a new hit compound for future drug design studies for Chagas disease.

Differential lethal action of C17:2 and C17:0 anacardic acid derivatives in Trypanosoma cruzi - A mechanistic study.

The n-hexane extract from leaves of Schinus terebinthifolius (Anacardiaceae) induced 100% of death of trypomastigote forms of T. cruzi at 300 µg/mL and was subjected to a bioactivity-guided fractionation to afford a C17:2 derivative of anacardic acid [6-(8'Z,11'Z)-heptadecadienyl-salicylic acid, 1]. Additionally, compound 1 was subjected to hydrogenation procedures to afford a C17:0 derivative (6-heptadecanyl-salicylic acid, 1a). Compounds 1 and 1a were effective in killing trypomastigote forms of T. cruzi with IC50 values of 8.3 and 9.0 µM, respectively, while a related compound, salicylic acid, was inactive. Furthermore, no cytotoxicity was observed for the highest tested concentration (CC50 > 200 µM) for all evaluated compounds. Due to the promising results, the mechanism of parasite death was investigated for compounds 1 and 1a using flow cytometry and spectrofluorimetry. The cell membrane permeability assay with SYTOX Green indicated that compound 1 significantly altered this parameter after 40 min of incubation, while compound 1a caused no alteration. Considering that the hydrogenation rendered a differential cellular target in parasites, additional assays were performed with 1a. Despite no permeabilization of the plasma membrane, compound 1a induced depolarization of the electric potential after two hours of incubation. The mitochondria of the parasite were also affected by compound 1a, with depolarization of the mitochondrial membrane potential, and reduction of reactive oxygen species (ROS) levels. The Ca2+ levels were not affected during the time of incubation. Considering that the mitochondrion is a single organelle in Trypanosoma cruzi for ATP generation, compounds affecting the bioenergetic system are of interest for drug discovery against Trypanosomatids.

Structure-activity relationship study of antitrypanosomal chalcone derivatives using multivariate analysis.

Chagas disease represents one of several neglected diseases with a reduced number of chemotherapeutical drugs including the highly toxic compounds benznidazole and nifurtimox. In this sense, natural products represent an import scaffold for the discovery of new biologically active compounds, in which chalcones are promising representatives due to their antitrypanosomal potential. In this work, a series of 36 chalcone derivatives were synthesized and tested against trypomastigotes of Trypanosoma cruzi. In addition, a detailed investigation on their molecular features was performed. The obtained results suggest that certain molecular features are fundamental for an efficient antitrypanosomal potential of chalcones, such as allylic groups, α,ß-unsaturated carbonyl system, and aromatic hydroxyl groups. These results were obtained based on the interpretation of machine-learning and multivariate statistical methods, which revealed the essential characteristics of chalcone prototypes against trypomastigotes of T. cruzi.

Antitrypanosomal Activity of Acetogenins Isolated from the Seeds of Porcelia macrocarpa Is Associated with Alterations in Both Plasma Membrane Electric Potential and Mitochondrial Membrane Potential.

As part of a drug discovery program aimed at the identification of anti- Trypanosoma cruzi metabolites from Brazilian flora, four acetogenins (1-4) were isolated from the seeds of Porcelia macrocarpa and were identified by NMR spectroscopy and HRESIMS. The new compounds 1 and 2 displayed activity against the trypomastigote (IC50 = 0.4 and 3.6 µM) and amastigote (IC50 = 23.0 and 27.7 µM) forms. The structurally related known compound 3 showed less potency to the amastigotes, with an IC50 value of 58 µM, while the known compound 4 was inactive. To evaluate the potential mechanisms for parasite death, parameters were evaluated by fluorometric assays: (i) plasma membrane permeability, (ii) plasma membrane electric potential (ΔΨp), (iii) reactive oxygen species production, and (iv) mitochondrial membrane potential (ΔΨm). The results obtained indicated that compounds 1 and 2 depolarize plasma membranes, affecting ΔΨp and ΔΨm and contributing to the observed cellular damage and disturbing the bioenergetic system. In silico studies of pharmacokinetics and toxicity (ADMET) properties predicted that all compounds were nonmutagenic, noncarcinogenic, nongenotoxic, and weak hERG blockers. Additionally, none of the isolated acetogenins 1-4 were predicted as pan-assay interference compounds.

Evaluation of the antitrypanosoma activity and SAR study of novel LINS03 derivatives.

Chagas' disease is a parasitic infection caused by Trypanosoma cruzi that is still treated by old and toxic drugs. In the search for novel alternatives, natural sources are an important source for new drug prototypes against T. cruzi to further structural exploitation. A set of natural-based compounds (LINS03) was designed, showing promising antitrypanosoma activity and low cytotoxicity to host cells. In this paper, nine novel LINS03 derivatives were evaluated against T. cruzi trypomastigotes and amastigotes. The selectivity was assessed through cytotoxicity assays using NCTC mammalian cells and calculating the CC50/IC50 ratio. The results showed that compounds 2d and 4c are noteworthy, due their high activity against amastigotes (IC50 13.9 and 5.8 µM) and low cytotoxicity (CC50 107.7 µM and >200 µM, respectively). These compounds did not showed alteration on plasma membrane permeability in a Sytox green model. SAR analysis suggested an ideal balance between hydrosolubility and lipophilicity is necessary to improve the activity, and that insertion of a meta-substituent is detrimental to the activity of the amine derivatives but not to the neutral derivatives, suggesting different mechanisms of actions. The results presented herein are valuable for designing novel compounds with improved activity and selectivity to be applied in future studies.

Antitrypanosomal activity and effect in plasma membrane permeability of (-)-bornyl p-coumarate isolated from Piper cernuum (Piperaceae).

This work describes the isolation of six metabolites from leaves and branches of Piper cernuum (Piperaceae): (-)-cubebin (1), (-)-hinokinin (2), (-)-kusunokinin (3), trans-dehydroagarofuran (4), 11-hydroxi-4,5-secoeudesmane-4,5-dione (5), and (-)-bornyl p-coumarate (6). Antitrypanosomal activity and toxicity of purified compounds were performed in vitro against trypomastigote forms of Trypanosoma cruzi and NCTC cells, respectively. Compounds 2, 3 and 5 showed moderate activities with IC50 values of 33.1, 31.8 and 45.9 µM, respectively, while compounds 1 and 4 were inactive (IC50 > 100 µM). On the other hand, compound 6 displayed an IC50 value of 2.1 µM, a selectivity index (SI) of 18 and induced a considerable interference in the plasma membrane permeability (87%) in trypomastigotes of T. cruzi. Additionally, the lethal effect of compound 6 in T. cruzi could be associated to the plasma membrane permeability. Finally, experiments using scanning electron microscopy (SEM) confirmed the obtained results in which was possible to observe total alteration parasites topography after treatment with compound 6 in comparison to untreated parasites. These data indicated that the lethal action of compound 6 is directly related to structural disruption of the membrane.

Acetylenic fatty acids from Porcelia macrocarpa (Annonaceae) against trypomastigotes of Trypanosoma cruzi: Effect of octadec-9-ynoic acid in plasma membrane electric potential.

Porcelia macrocarpa (Warm.) R. E. Fries (Annonaceae) is an endemic plant in Brazil where its tasty pulp has been eaten fresh. The hexane extract from its flowers was subjected to chromatographic procedures to afford four acetylene derivatives identified as octadec-9-ynoic (stearolic acid - 1), (11E)-octadec-11-en-9-ynoic (santalbic acid - 2), 8-hydroxyoctadec-9,11-diynoic (3) and 8-hydroxyoctadec-17-en-9,11-diynoic (isanolic acid - 4) acids by NMR and HRESIMS. Among tested compounds against trypomastigote forms of T. cruzi, octadec-9-ynoic acid (1) displayed higher potential with IC50 = 27.6 µM and a selectivity index (SI) higher than 7. Compounds 2 and 3 showed IC50 of approximately 60 µM while compound 4 was inactive. The lethal action of the compound 1 was investigated using spectrofluorometric techniques to detect ROS content, plasma membrane permeability and plasma membrane potential by flow cytometry. Compound 1 showed no alteration in the production of ROS of treated trypomastigotes and no alteration of the plasma membrane permeability was observed as detected by the fluorescent probe SYTOX-green after 120 min of incubation. However, by using the potential-sensitive fluorescent probe DiSBAC2(3), compound 1 caused depolarization of the plasma membrane potential when compared to untreated parasites. Our results demonstrated the anti-T. cruzi effects of compounds 1-3 isolated from flowers of P. macrocarpa and indicated that the lethal effect of compound 1 in T. cruzi could be associated to the plasma membrane disturbance of the parasite.

Investigation of the Anti-Leishmania (Leishmania) infantum Activity of Some Natural Sesquiterpene Lactones.

Leishmaniases are neglected infectious diseases caused by parasites of the 'protozoan' genus Leishmania. Depending on the parasite species, different clinical forms are known as cutaneous, muco-cutaneous, and the visceral leishmaniasis (VL). VL is particularly fatal and the therapy presents limitations. In the search for new anti-leishmanial hit compounds, seven natural sesquiterpene lactones were evaluated against promastigotes and intracellular amastigotes of Leishmania (Leishmania) infantum, a pathogen causing VL. The pseudoguaianolides mexicanin I and helenalin acetate demonstrated the highest selectivity and potency against intracellular amastigotes. In addition, promastigotes treated with helenalin acetate were subject to an ultrastructural and biochemical investigation. The lethal action of the compound was investigated by fluorescence-activated cell sorting and related techniques to detect alterations in reactive oxygen species (ROS) content, plasma membrane permeability, and mitochondrial membrane potential. Helenalin acetate significantly reduced the mitochondrial membrane potential and the mitochondrial structural damage was also confirmed by transmission electron microscopy, displaying an intense organelle swelling. No alteration of plasma membrane permeability or ROS content could be detected. Additionally, helenalin acetate significantly increased the production of nitric oxide in peritoneal macrophages, probably potentiating the activity against the intracellular amastigotes. Helenalin acetate could hence be a useful anti-leishmanial scaffold for further optimization studies.

Analogues of Marine Guanidine Alkaloids Are in Vitro Effective against Trypanosoma cruzi and Selectively Eliminate Leishmania (L.) infantum Intracellular Amastigotes.

Synthetic analogues of marine sponge guanidine alkaloids showed in vitro antiparasitic activity against Leishmania (L.) infantum and Trypanosoma cruzi. Guanidines 10 and 11 presented the highest selectivity index when tested against Leishmania. The antiparasitic activity of 10 and 11 was investigated in host cells and in parasites. Both compounds induced depolarization of mitochondrial membrane potential, upregulation of reactive oxygen species levels, and increased plasma membrane permeability in Leishmania parasites. Immunomodulatory assays suggested an NO-independent effect of guanidines 10 and 11 on macrophages. The same compounds also promoted anti-inflammatory activity in L. (L.) infantum-infected macrophages cocultived with splenocytes, reducing the production of cytokines MCP-1 and IFN-γ. Guanidines 10 and 11 affect the bioenergetic metabolism of Leishmania, with selective elimination of parasites via a host-independent mechanism.

Anti-parasitic Guanidine and Pyrimidine Alkaloids from the Marine Sponge Monanchora arbuscula.

HPLC-UV-ELSD-MS-guided fractionation of the anti-parasitic extract obtained from the marine sponge Monanchora arbuscula, collected off the southeastern coast of Brazil, led to the isolation of a series of guanidine and pyrimidine alkaloids. The pyrimidines monalidine A (1) and arbusculidine A (7), as well as the guanidine alkaloids batzellamide A (8) and hemibatzelladines 9-11, represent new minor constituents that were identified by analysis of spectroscopic data. The total synthesis of monalidine A confirmed its structure. Arbusculidine A (7), related to the ptilocaulin/mirabilin/netamine family of tricyclic guanidine alkaloids, is the first in this family to possess a benzene ring. Batzellamide A (8) and hemibatzelladines 9-11 represent new carbon skeletons that are related to the batzelladines. Evaluation of the anti-parasitic activity of the major known metabolites, batzelladines D (12), F (13), L (14), and nor-L (15), as well as of synthetic monalidine A (1), against Trypanosoma cruzi and Leishmania infantum is also reported, along with a detailed investigation of parasite cell-death pathways promoted by batzelladine L (14) and norbatzelladine L (15).