Synthesis of a dehydrodieugenol B derivative as a lead compound for visceral leishmaniasis-mechanism of action and in vivo pharmacokinetic studies.

Leishmaniasis is a parasitic neglected tropical disease, affecting 12 million people. Available treatments present several limitations, with an increasing number of resistance cases. In the search for new chemotherapies, the natural product dehydrodieugenol B was used as a scaffold for the synthesis of a series of derivatives, resulting in the discovery of the promising analog [4-(4-(5-allyl-3-methoxy-2-((4-methoxybenzyl)oxy)phenoxy)-3-methoxybenzyl)morpholine, 1]. In this work, we investigated the effect of compound 1 on cell signaling in Leishmania (L.) infantum, culminating in cell death, as well as its immunomodulatory effect in the host cell. Additionally, we performed a pharmacokinetic profile study in an animal model. After treatment, compound 1 induced the alkalinization of acidocalcisomes and concomitant Ca2+ release in the parasite. These events may induce depolarization of the mitochondrial potential, with successive collapse of the bioenergetic system, leading to a reduction of ATP and reactive oxygen species (ROS) levels. The analysis of total proteins and protein profile by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) demonstrated that compound 1 also altered the parasite proteins after treatment. Transmission electron microscopy studies revealed ultrastructural damage to mitochondria; together, these data suggest that compound 1 may promote autophagic cell death. Additionally, compound 1 also induced an immunomodulatory effect in host cells, with a reduction of Th1 and Th2 cytokine response, characterizing an anti-inflammatory compound. The obtained pharmacokinetic profile in rats enhances the potential of the compound, with a mean plasma half-life (T1/2) of 21 h. These data reinforce the potential of compound 1 as a new lead for future efficacy studies.

Exploring the antileishmanial activity of dicentrine from Ocotea puberula (Lauraceae) using biomembrane models.

This study aimed to assess the antiprotozoal efficacy of dicentrine, an aporphine alkaloid isolated from Ocotea puberula, against amastigote forms of Leishmania (L.) infantum. Our findings reveal that dicentrine demonstrated a notable EC50 value of 10.3 µM, comparable to the positive control miltefosine (EC50 of 10.4 µM), while maintaining moderate toxicity to macrophages (CC50 of 51.9 µM). Utilizing an in silico methodology, dicentrine exhibited commendable adherence to various parameters, encompassing lipophilicity, water solubility, molecule size, polarity, and flexibility. Subsequently, we conducted additional investigations to unravel the mechanism of action, employing Langmuir monolayers as models for protozoan cell membranes. Tensiometry analyses unveiled that dicentrine disrupts the thermodynamic and mechanical properties of the monolayer by expanding it to higher areas and increasing the fluidity of the film. The molecular disorder was further corroborated through dilatational rheology and infrared spectroscopy. These results contribute insights into the role of dicentrine as a potential antiprotozoal drug in its interactions with cellular membranes. Beyond elucidating the mechanism of action at the plasma membrane's external surface, our study sheds light on drug-lipid interface interactions, offering implications for drug delivery and other pharmaceutical applications.

Lignans Isolated from Piper truncatum Act as New Potent Antitrypanosomal Compounds.

The hexane extract from twigs of Piper truncatum Vell (Piperaceae) displayed activity against Trypanosoma cruzi and was subjected to chromatographic steps to afford six dibenzylbutyrolactolic lignans, being four knowns: cubebin (1), (-)-9α-O-methylcubebin (2), (+)-9ß-O-methylcubebinin (3) and 3,4-dimethoxy-3,4-demethylenedioxycubebin (4) as well as two new, named truncatin A (5) and B (6). Initially, in vitro activity against trypomastigotes was evaluated and compounds 1, 4 and 6 exhibited EC50 values of 41.6, 21.0 and 39.6 µM, respectively. However, when tested against amastigotes, the relevant clinical form in the chronic phase of Chagas disease, compounds 1-6 displayed activities with EC50 values ranging from 1.6 to 13.7 µM. In addition, the mammalian cytotoxicity of compounds 1-6 was evaluated against murine fibroblasts (NCTC). Compounds 2, 3 and 4 exhibited reduced toxicity against NCTC cells (CC50>200 µM), resulting in SI values of>21.9,>14.5 and>121.9, respectively. Compound 4 showed the highest potency with an SI value twice superior to that determined by the standard drug benznidazole (SI>54.6) against the intracellular amastigotes. These data suggest that lignan 4 can be considered a possible scaffold for designing a new drug candidate for Chagas disease.

Selective Activity Against Amastigote Forms of Trypanosoma cruzi and Leishmania infantum of Diastereomeric Dicentrine N-oxides.

As part of our continuous research for the discovery of bioactive compounds against Trypanosoma cruzi and Leishmania infantum, the alkaloid (6aS)-dicentrine (1) was oxidized to afford (6aS,6S)- (2) and (6aS,6R)- (3) dicentrine-N-oxides. Evaluation of the cytotoxicity against NCTC cells indicated that 2 and 3 are non-toxic (CC50>200 µM) whereas 1 demonstrated CC50 of 52.0 µM. Concerning T. cruzi activity against amastigotes, derivatives 2 and 3 exhibited EC50 values of 9.9 µM (SI>20.2) and 27.5 µM (SI>7.3), respectively, but 1 is inactive (EC50>100 µM). Otherwise, when tested against L. infantum amastigotes, 1 and 3 exhibited EC50 values of 10.3 µM (SI=5.0) and 12.7 µM (SI>15.7), respectively, being 2 inactive (EC50>100 µM). Comparing the effects of positive controls benznidazol (EC50=6.5 µM and SI>30.7) and miltefosine (EC50=10.2 µM and SI=15.2), it was observed a selective antiparasitic activity to diastereomers 2 and 3 against T. cruzi and L. infantum. Considering stereochemical aspects, it was suggested that the configuration of the new stereocenter formed after oxidation of 1 played an important role in the bioactivity against amastigotes of both tested parasites.

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.

Evaluation of Anti-Trypanosoma cruzi Activity of Chemical Constituents from Baccharis sphenophylla Isolated Using High-Performance Countercurrent Chromatography.

Endemic in 21 countries, Chagas disease, also known as American Trypanosomiasis, is a neglected tropical disease (NTD) caused by the protozoan parasite Trypanosoma cruzi. The available drugs for the treatment of this disease, benznidazole and nifurtimox, are outdated and display severe side effects. Thus, the discovery of new drugs is crucial. Based on our continuous studies aiming towards the discovery of natural products with anti-T. cruzi potential, the MeOH extract from aerial parts of Baccharis sphenophylla Dusén ex. Malme (Asteraceae) displayed activity against this parasite and was subjected to high-performance countercurrent chromatography (HPCCC), to obtain one unreported syn-labdane diterpene - sphenophyllol (1) - as well as the known compounds gaudichaudol C (2), ent-kaurenoic acid (3), hispidulin (4), eupafolin (5), and one mixture of di-O-caffeoylquinic acids (6-8). Compounds 1-8 were characterized by analysis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. When tested against trypomastigote forms, isolated labdane diterpenes 1 and 2 displayed potent activity, with EC50 values of 20.1 µM and 2.9 µM, respectively. The mixture of chlorogenic acids 6-8, as well as the isolated flavones 4 and 5, showed significant activity against the clinically relevant amastigotes, with EC50 values of 24.9, 12.8, and 2.7 µM, respectively. Nonetheless, tested compounds 1-8 displayed no cytotoxicity against mammalian cells (CC50 > 200 µM). These results demonstrate the application of HPCCC as an important tool to isolate bioactive compounds from natural sources, including the antitrypanosomal extract from B. sphenophylla, allowing for the development of novel strategic molecular prototypes against tropical neglected diseases.

Piperazine amides with desirable solubility, physicochemical and drug-like properties: Synthesis and evaluation of the anti-Trypanosoma cruzi activity.

The absence of effective chronic treatment, expansion to non-endemic countries and the significant burden in public health have stimulated the search for novel therapeutic options to treat Chagas disease, a protozoan disease caused by Trypanosoma cruzi. Despite current efforts, no new drug candidates were approved in clinical trials in the past five decades. Considering this, our group has focused on the expansion of a series (LINS03) with low micromolar activity against amastigotes, considering the optimization of pharmacokinetic properties through increasing drug-likeness and solubility. In this work, we report a new set of 13 compounds with modifications in both the arylpiperazine and the aromatic region linked by an amide group. Five analogues showed activity against intracellular amastigotes (IC50 17.8 to 35.9 µM) and no relevant cytotoxicity to mammalian cells (CC50 > 200 µM). Principal component analysis (PCA) was performed to identify structural features associated to improved activity. The data revealed that polarity, hydrogen bonding ability and flexibility were key properties that influenced the antiparasitic activity. In silico drug-likeness assessments indicated that compounds with the 4-methoxycinammyl (especially compound 2b) had the most prominent balance between properties and activity in the series, as confirmed by SAR analysis.

Feature-Based Molecular Networking Discovery of Bromopyrrole Alkaloids from the Marine Sponge Agelas dispar.

Investigation of the marine sponge Agelas dispar MeOH fractions using feature-based molecular networking, dereplication, and isolation led to the discovery of new bromopyrrole-derived metabolites. An in-house library of bromopyrrole alkaloids previously isolated from A. dispar and Dictyonella sp. was utilized, along with the investigation of an MS/MS fragmentation of these compounds. Our strategy led to the isolation and identification of the disparamides A-C (1-3), with a novel carbon skeleton. Additionally, new dispyrins B-F (4-8) and nagelamides H2 and H3 (9 and 10) and known nagelamide H (11), citrinamine B (12), ageliferin (13), bromoageliferin (14), and dibromoageliferin (15) were also isolated and identified by analysis of spectroscopic data. Analysis of MS/MS fragmentation data and molecular networking analysis indicated the presence of hymenidin (16), oroidin (17), dispacamide (18), monobromodispacamide (19), keramadine (20), longamide B (21), methyl ester of longamide B (22), hanishin (23), methyl ester of 3-debromolongamide B (24), and 3-debromohanishin (25). Antibacterial activity of ageliferin (13), bromoageliferin (14), and dibromoageliferin (15) was evaluated against susceptible and multi-drug-resistant ESKAPE pathogenic bacteria Klabsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterococcus faecalis. Dibromoageliferin (15) displayed the most potent antimicrobial activity against all tested susceptible and MDR strains. Compounds 13-15 presented no significant hemolytic activity up to 100 µM.

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.

Marine alkaloids as bioactive agents against protozoal neglected tropical diseases and malaria.

Covering: 2000 up to 2021Natural products are an important resource in drug discovery, directly or indirectly delivering numerous small molecules for potential development as human medicines. Among the many classes of natural products, alkaloids have a rich history of therapeutic applications. The extensive chemodiversity of alkaloids found in the marine environment has attracted considerable attention for such uses, while the scarcity of these natural materials has stimulated efforts towards their total synthesis. This review focuses on the biological activity of marine alkaloids (covering 2000 to up to 2021) towards Neglected Tropical Diseases (NTDs) caused by protozoan parasites, and malaria. Chemotherapy represents the only form of treatment for Chagas disease, human African trypanosomiasis, leishmaniasis and malaria, but there is currently a restricted arsenal of drugs, which often elicit severe adverse effects, show variable efficacy or resistance, or are costly. Natural product scaffolds have re-emerged as a focus of academic drug discovery programmes, offering a different resource to discover new chemical entities with new modes of action. In this review, the potential of a range of marine alkaloids is analyzed, accompanied by coverage of synthetic efforts that enable further studies of key antiprotozoal natural product scaffolds.

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.

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.

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.

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.

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.

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.