Investigation of the Potential Targets behind the Promising and Highly Selective Antileishmanial Action of Synthetic Flavonoid Derivatives.

Leishmaniases are among the neglected tropical diseases that still cause devastating health, social, and economic consequences to more than 350 million people worldwide. Despite efforts to combat these vector-borne diseases, their incidence does not decrease. Meanwhile, current antileishmanial drugs are old and highly toxic, and safer presentations are unaffordable to the most severely affected human populations. In a previous study by our research group, we synthesized 17 flavonoid derivatives that demonstrated impressive inhibition capacity against rCPB2.8, rCPB3, and rH84Y. These cysteine proteases are highly expressed in the amastigote stage, the target form of the parasite. However, although these compounds have been already described in the literature, until now, the amastigote effect of any of these molecules has not been proven. In this work, we aimed to deeply analyze the antileishmanial action of this set of synthetic flavonoid derivatives by correlating their ability to inhibit cysteine proteases with the action against the parasite. Among all the synthesized flavonoid derivatives, 11 of them showed high activity against amastigotes of Leishmania amazonensis, also providing safety to mammalian host cells. Furthermore, the high production of nitric oxide by infected cells treated with the most active cysteine protease B (CPB) inhibitors confirms a potential immunomodulatory response of macrophages. Besides, considering flavonoids as multitarget drugs, we also investigated other potential antileishmanial mechanisms. The most active compounds were selected to investigate another potential biological pathway behind their antileishmanial action using flow cytometry analysis. The results confirmed an oxidative stress after 48 h of treatment. These data represent an important step toward the validation of CPB as an antileishmanial target, as well as aiding in new drug discovery studies based on this protease.

Metabolomics Approach Expands the Classification of Propolis Samples from Midwest Brazil.

Propolis samples collected from five areas in Mato Grosso do Sul state, Midwest Brazil, comprising portions of the Cerrado, Pantanal, and Atlantic Forest ecosystems, were investigated for metabolomic profiles and evaluated for antioxidant and antitumor potential. Chemical profiles were determined by HPLC-DAD-MS/MS data and evaluated using principal component analysis and hierarchical clustering analysis to discern chemical composition patterns. Based on phytogeographical origin and chemical composition, 20 potential markers were identified and five groups were distinguished: (I) Cerrado/Central, (II) Atlantic Forest/South, (III) Cerrado-Pantanal transition area/Northwest, (IV) Cerrado/North, and (V) Pantanal/West. Drawing on HPLC-DAD-MS/MS and NMR data, 47 compounds were successfully or tentatively identified, including prenylated phenylpropanoids, flavonoids, isoflavonoids, and di- and triterpenoids, among other constituents. Isoflavonoids, typically found in red propolis from Northeast Brazil, are being reported for the first time in a propolis sample from the Midwest. A new prenylated aromatic compound, (E)-3-[4-hydroxy-3-(2-hydroxy-3-methylbut-3-en-1-yl)-5-(3-methylbut-2-en-1-yl)phenyl]propenoic acid, was obtained. Samples in group II exhibited promising antitumor potential against prostate and breast carcinoma cells, as did samples in groups III and IV against the latter cell line. The sample in group I, despite containing the highest amount of total phenolic compounds and being the only sample to exhibit scavenging activity against DPPH, was not the most cytotoxic against the cell lines tested.

S-(4-Methoxyphenyl)-4-methoxybenzenesulfonothioate as a Promising Lead Compound for the Development of a Renal Carcinoma Agent.

Organosulfur compounds show cytotoxic potential towards many tumor cell lines. Disulfides and thiosulfonates act through apoptotic processes, inducing proteins associated with apoptosis, endoplasmic reticulum stress, and the unfolded protein response. Three p-substituted symmetric diaryl disulfides and three diaryl thiosulfonates were synthesized and analyzed for inhibition of tubulin polymerization and for human cancer cell cytotoxic activity against seven tumor cell lines and a non-tumor cell line. S-(4-methoxyphenyl)-4-methoxybenzenesulfonothioate (6) exhibited inhibition of tubulin polymerization and showed the best antiproliferative potential, especially against the 786-0 cell line, being six times more selective as compared with the non-tumor cell line. In addition, compound 6 was able to activate caspase-3 after 24 and 48 h treatments of the 786-0 cell line and induced cell-cycle arrest in the G2/M stage at the highest concentration evaluated at 24 and 48 h. Compound 6 was able to cause complete inhibition of proliferation, inducing the death of 786-0 cells, by increasing the number of cells at G2/M and greater activation of caspase-3.

Design, synthesis, antileishmanial, and antifungal biological evaluation of novel 3,5-disubstituted isoxazole compounds based on 5-nitrofuran scaffolds.

Nineteen 3,5-disubstituted-isoxazole analogs were synthesized based on nitrofuran scaffolds, by a [3 + 2] cycloaddition reaction between terminal acetylenes and 5-nitrofuran chloro-oxime. The compounds were obtained in moderate to very good yields (45-91%). The antileishmanial activity was assayed against the promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. Alkylchlorinated compounds 14p-r were active on both the promastigote and amastigote forms, with emphasis on compound 14p, which showed strong activity against the amastigote form (IC50 = 0.6 µM and selectivity index [SI] = 5.2). In the alkyl series, compound 14o stands out with an IC50 = 8.5 µM and SI = 8.0 on the amastigote form. In the aromatic series, the most active compounds were those containing electron-donor groups, such as trimethoxy isoxazole 14g (IC50 = 1.2 µM and SI = 20.2); compound 14h, with IC50 = 7.0 µM and SI = 6.1; and compound 14j containing the 4-SCH3 group, with IC50 = 5.7 µM and SI = 10.2. In addition, the antifungal activity of 19 nitrofuran isoxazoles was evaluated against five strains of Candida (C. albicans, C. parapsilosis, C. krusei, C. tropicalis, and C. glabrata). Eleven isoxazole derivatives were active against C. parapsilosis, and compound 14o was found to be the most active (minimal inhibitory concentration [MIC] = 3.4 µM) for this strain. Compound 14p was active against all the strains tested, with an MIC = 17.5 µM for C. glabrata, lower than that of the fluconazole used as the reference drug.

Cytotoxic and Apoptotic Activity of Majoranolide from Mezilaurus crassiramea on HL-60 Leukemia Cells.

Majoranolide, a butanolide isolated from the nonpolar fraction of an ethanol extract of Mezilaurus crassiramea (Lauraceae) fruits, is being reported for the first time in this genus and the third time in plants. Structurally identified from 1D and 2D NMR and HRESIMS data, majoranolide proved cytotoxic against cancer cells-MCF-7 and MDA-MB-231 (breast), HT-29 (colon), PC-3 (prostate), 786-0 (renal), and HL-60 (leukemia)-inhibiting growth in HL-60 cells (GI50 = 0.21 µM) and exhibiting higher selectivity for this line than for nonneoplastic NIH/3T3 murine fibroblasts. Effects on the cell cycle, caspase-3 activation, and plasma membrane integrity were evaluated by flow cytometry. Expression of genes related to apoptotic pathways (BAX, BCL2, BIRC5, and CASP8) was investigated using RT-qPCR. At 50 µM, majoranolide induced cell cycle arrest at G1 in 24 h increased the sub-G1 population in 48 h and increased caspase-3 activation in a time-dependent manner. The compound upregulated BAX and CASP8 transcription (proapoptotic genes) and downregulated BIRC5 (antiapoptotic). Loss of plasma membrane integrity in 30% of cells occurred at 48 h, but not at 24 h, characterizing gradual, programmed death. The results suggest that majoranolide cytotoxicity involves apoptosis induction in HL-60 cells, although other mechanisms may contribute to this cell death.

Design, synthesis and antitrypanosomatid activities of 3,5-diaryl-isoxazole analogues based on neolignans veraguensin, grandisin and machilin G.

Using bioisosterism as a medicinal chemistry tool, 16 3,5-diaryl-isoxazole analogues of the tetrahydrofuran neolignans veraguensin, grandisin and machilin G were synthesized via 1,3-dipolar cycloaddition reactions, with yields from 43% to 90%. Antitrypanosomatid activities were evaluated against Trypanosoma cruzi, Leishmania (L.) amazonensis and Leishmania (V.) braziliensis. All compounds were selective for the Leishmania genus and inactive against T. cruzi. Isoxazole analogues showed a standard activity on both promastigotes of L. amazonensis and L. braziliensis. The most active compounds were 15, 16 and 19 with IC50 values of 2.0, 3.3 and 9.5 µM against L. amazonensis and IC50 values of 1.2, 2.1 and 6.4 µM on L. braziliensis, respectively. All compounds were noncytotoxic, showing lower cytotoxicity (>250 µM) than pentamidine (78.9 µM). Regarding the structure-activity relationship (SAR), the methylenedioxy group was essential to antileishmanial activity against promastigotes. Replacement of the tetrahydrofuran nucleus by an isoxazole core improved the antileishmanial activity.

Antiproliferative Activity of Extracts of Campomanesia adamantium (Cambess.) O. Berg and Isolated Compound Dimethylchalcone Against B16-F10 Murine Melanoma.

Campomanesia adamantium, a native species of the Brazilian Cerrado, is characterized as a natural source of phenolic compounds and has known potential anticancer activities. This study aimed to evaluate the chemical profile of dichloromethane extracts of pulp (DEGPU) and peel (DEGPE) from the fruits of C. adamantium and to identify compounds with antiproliferative effects in vitro against melanoma cells by sulforhodamine B (SRB) assay, apoptosis induction assay, caspase-3 activation assay, nitric oxide (NO) release in coculture of B16-F10 cells and murine peritoneal macrophages. The chemical profiles of DEGPU and DEGPE were analyzed by high performance liquid chromatography coupled to diode array detector and mass spectrometer using the electrospray ionization interface (HPLC-DAD-ESI-MS/MS). Thirteen compounds were identified in both extracts and the chromatographic study of the most active extract in SRB assay DEGPU (GI50 of 16.17 µg/mL) resulted in the isolation of seven compounds. The isolated compound dimethylchalcone (DMC) had the highest antiproliferative activity against B16-F10 with a GI50 of 7.11 µg/mL. DEGPU extract activated caspase-3 in 29% of cells at 25 µg/mL and caused a 50% decrease in NO release in coculture. DEGPU can be characterized as a source of bioactive compounds such as DMC, as seen from its antiproliferative effect in vitro by inducing B16-F10 cells to undergo apoptosis, essential feature in the search for new anticancer drugs.

Rubiaceae-Type Cyclopeptides from Galianthe thalictroides.

Two Rubiaceae-type cyclopeptides, 6-O-methylbouvardin (1) and the new cyclopeptide 5ß-hydroxy-RA-III (2), were isolated from the roots of Galianthe thalictroides. Employing the sulforhodamine B assay, compounds 1 and 2 were tested in vitro against three cancer cell lines--786-0 (kidney carcinoma), PC-3 (prostate carcinoma), and HT-29 (colon carcinoma)--and showed GI50 values ranging from 0.06 to 1.80 µg mL(-1). This is the first report on the isolation of Rubiaceae-type cyclopeptides from a genus other than Rubia or Bouvardia.

Synthesis and evaluation of diaryl sulfides and diaryl selenide compounds for antitubulin and cytotoxic activity.

We have devised a procedure for the synthesis of analogs of combretastatin A-4 (CA-4) containing sulfur and selenium atoms as spacer groups between the aromatic rings. CA-4 is well known for its potent activity as an inhibitor of tubulin polymerization, and its prodrugs combretastatin A-4 phosphate (CA-4P) and combretastatin A-1 phosphate (CA-1P) are being investigated as antitumor agents that cause tumor vascular collapse in addition to their activity as cytotoxic compounds. Here we report the preparation of two sulfur analogs and one selenium analog of CA-4. All synthesized compounds, as well as several synthetic intermediates, were evaluated for inhibition of tubulin polymerization and for cytotoxic activity in human cancer cells. Compounds 3 and 4 were active at nM concentration against MCF-7 breast cancer cells. As inhibitors of tubulin polymerization, both 3 and 4 were more active than CA-4 itself. In addition, 4 was the most active of these agents against 786, HT-29 and PC-3 cancer cells. Molecular modeling binding studies are also reported for compounds 1, 3, 4 and CA-4 to tubulin within the colchicine site.

A new cytotoxic ß-carboline alkaloid from Galianthe thalictroides.

A cytotoxicity-guided fractionation of the roots of Galianthe thalictroides afforded a new ß-carboline alkaloid, 1-(hydroxymethyl)-3-(2-hydroxypropan-2-yl)-2-(5-methoxy-9H- ß-carbolin-1-yl)cyclopentanol, which exhibited strong cytotoxic activity against three human cancer cell lines (MCF-7, 786-0, and UACC62). Its structure was established on the basis of 1D- and 2D-NMR spectroscopic techniques supported by HRMS data.