Manuel Jesús Chan-Bacab

Design, synthesis and in vitro antiprotozoal activity of benzimidazole-pentamidine hybrids

A series of ten novel hybrids from benzimidazole and pentamidine were prepared using a short synthetic route. Each compound was tested in vitro against the protozoa Trichomonas vaginalis, Giardia lamblia, Entamoeba histolytica, Leishmania mexicana, and Plasmodium berghei, in comparison with pentamidine and metronidazole. Some analogues showed high bioactivity in the low micromolar range (IC(50)<1 microM) against the first four protozoa, which make them significantly more potent than either standard. 1,5-bis[4-(5-methoxy-1H-benzimidazole-2-yl)phenoxy]pentane (2) was 3- and 9-fold more potent againstG. lamblia than metronidazole and pentamidine, respectively. This compound was 23-, 108-, and 13-fold more active than pentamidine against T. vaginalis, E. histolytica and L. mexicana, respectively. Studying further structure-activity relationships through the use of bioisosteric substitution in these hybrids should provide new leads against protozoal diseases.

In vitro activity of Tridax procumbens against promastigotes of Leishmania mexicana.

ETHNOPHARMACOLOGICAL RELEVANCE Tridax procumbens is an active herb against leishmaniasis. AIM OF THE STUDY Leishmaniasis is a group of diseases caused by Leishmania protozoa. We investigated the antileishmanial activity of Tridax procumbens extracts and a pure compound against promastigotes of Leishmania mexicana, the causative agent of cutaneous leishmaniasis in the New World. MATERIALS AND METHODS Extracts and (3S)-16,17-didehydrofalcarinol (1) were obtained by chromatographic methods from Tridax procumbens, and the latter identified by spectroscopic analysis. The effect of these extracts and 1 on the growth inhibition of promastigotes of Leishmania mexicana was evaluated. In order to test the safety of extracts and 1, mammalian cells were treated with them, and cell viability was assessed using trypan blue and MTT. RESULTS We demonstrated that extracts of Tridax procumbens and 1 showed a pronounced activity against Leishmania mexicana. The methanol extract inhibited promastigotes growth of Leishmania mexicana with a 50% inhibitory concentration (IC(50)) of 3 microg/ml, while oxylipin 1 exhibited the highest inhibition at IC(50)=0.478 microg/ml. CONCLUSIONS In this study we report the biological activity of extracts and (3S)-16,17-didehydrofalcarinol (1), obtained from Tridax procumbens, on the promastigote form of Leishmania mexicana, with no effect upon mammalian cells.

Variation of leishmanicidal activity in four populations of Urechites andrieuxii

Urechites andrieuxii Muell.-Arg. (Apocynaceae) is widely used in the Yucatan Peninsula for the treatment of cutaneous leishmaniasis. The influence of the environment in the variability of the leishmanicidal activity of the plant was evaluated using crude methanol extracts of roots from individuals belonging to four natural populations growing in the Yucatan Peninsula. The results of the growth inhibition test using three Leishmania spp. promastigotes showed a stronger leishmanicidal activity in populations of U. andrieuxii growing in more humid environments. Further evaluation against four human cancer cell lines and in the brine shrimp bioassay of both extracts from various parts of the plant and from the most active methanol root extracts, suggested that while the leaf extract appears to have selective toxicity against Leishmania parasites, the strong leishmanicidal activity detected in the root extracts of the plant might be due to its cytotoxicity.

Synthesis of benzologues of Nitazoxanide and Tizoxanide: A comparative study of their in vitro broad-spectrum antiprotozoal activity

We have synthesized two new benzologues of Nitazoxanide (NIT) and Tizoxanide (TIZ), using a short synthetic route. Both compounds were tested in vitro against six protozoa (Giardia intestinalis, Trichomonas vaginalis, Entamoeba histolytica, Plasmodium berghei, Leishmania mexicana and Trypanosoma cruzi). Compound 1 (benzologue of NIT) showed broad antiprotozoal effect against all parasites tested, showing IC(50)s<5 μM. This compound was five-times more active than NIT, and 18-times more potent than metronidazole against G. intestinalis. It was 10-times more active than pentamidine against L. mexicana, and it was sevenfold more potent than benznidazole versus T. cruzi. This compound could be considered as a new broad spectrum antiprotozoal agent.

Valuable processes and products from marine intertidal microbial communities

Microbial communities are ubiquitous in marine intertidal environments. These communities, which grow preferentially as biofilms on natural and artificial surfaces, carry out key processes contributing to the functioning of coastal environments and providing valuable services to human society, including carbon cycling, primary productivity, trophic linkage, and transfer and removal of pollutants. In addition, their surface-associated life style greatly influences the integrity and performance of marine infrastructure and archaeological heritage materials. The fluctuating conditions of the intertidal zone make it an extreme environment to which intertidal biofilm organisms must adapt at varying levels. This requirement has probably favored the development and spread of specific microorganisms with particular physiological and metabolic processes. These organisms may have potential biotechnological utility, in that they may provide novel secondary metabolites, biopolymers, lipids, and enzymes and even processes for the production of energy in a sustainable manner.

Nitazoxanide, tizoxanide and a new analogue [4-nitro-N-(5-nitro-1,3-thiazol-2-yl)benzamide; NTB] inhibit the growth of kinetoplastid parasites (Trypanosoma cruzi and Leishmania mexicana) in vitro

Sir, Parasitic diseases such as leishmaniasis and trypanosomiasis, both caused by protozoan parasites of the Kinetoplastida order, represent a serious problem to the health and the economy of many developing countries. The Leishmania species cause a variety of diseases, from self-healing cutaneous lesions to lifethreatening visceral infections. Clinical manifestations depend on the species of the infecting parasites. There are an estimated annual 1.5–2.0 million new cases of leishmaniasis, of which approximately 500000 belong to the visceral form, which is potentially fatal. American trypanosomiasis, or Chagas’ disease, is still one of the major causes of morbidity and mortality due to cardiovascular diseases in Latin America. Common chemotherapeutic agents currently used against both diseases are often inadequate since they require long courses of parenteral administration, may have toxic side effects or become less effective due to the emergence of resistant strains. Therefore, new, effective and inexpensive drugs that can be used to treat these diseases are urgently required. Nitazoxanide [2-(5-nitrothiazol-2-ylcarbamoyl)phenyl acetate; Alinia] is a broad-spectrum antiparasitic compound belonging to a nitroheterocyclic class named thiazolides. In humans, nitazoxanide is rapidly metabolized to tizoxanide, which is a compound that is as effective as the parent drug (Figure 1). Detailed in vitro and in vivo studies have been conducted regarding the efficacy of nitazoxanide and other nitroheterocyclic drugs against helminths, extracellular anaerobic protozoa and bacteria, intracellular parasites and viruses. – 6 Due to the limited information regarding the efficacy of nitazoxanide against kinetoplastid parasites such as Trypanosoma cruzi and Leishmania mexicana, the goal of this work was to show the potential of nitazoxanide and its major metabolite, tizoxanide, as antileishmanial and trypanocidal drugs. In this work, we evaluated the in vitro activity of nitazoxanide, tizoxanide and the newly synthesized analogue 4-nitro-N-(5-nitro-1,3-thiazol-2-yl)benzamide (NTB) (Figure 1) against T. cruzi and L. mexicana. The activity was compared with the activities of benznidazole and pentamidine, well-known drugs that act against T. cruzi and L. mexicana, respectively. Nitaxozanide was synthesized in our laboratory starting from the acylation of 2-amino-5-nitrothiazole with acetylsalicyloyl chloride and triethylamine in methylene chloride as solvent. An alkaline hydrolysis of nitazoxanide produced tizoxanide in good yields. NTB was synthesized by means of the same procedure, using 4-nitrobenzoyl chloride instead of acetylsalicyloyl chloride. All compounds were characterized by spectroscopic and spectrometric techniques and all data agreed with reported literature values. The growth inhibition test was performed on promastigotes of L. mexicana (MHOM/MX/84/ISETGS; clinical strain originally isolated from a patient with diffuse cutaneous leishmaniasis) and epimastigotes of T. cruzi (MHOM/MX/1994/Ninoa; clinical strain originally isolated from a patient with Chagas’ disease in the acute phase). Parasites were cultivated at 268C in Schneider’s Drosophila medium, supplemented with 10% fetal bovine serum, penicillin (100 IU/mL) and streptomycin (100 mg/ mL). Biological assays were performed in 96-well plates and all compounds were evaluated in duplicate. Compounds were solubilized in dimethylsulphoxide and diluted in a liquid medium. Aliquots of 100 mL of compound solution and 100 mL of culture medium containing 10000 Leishmania promastigotes or 20000 T. cruzi epimastigotes were added to obtain concentrations of 10, 5, 2.5 and 1.25 mg/mL. Benznidazole (first-line antichagasic drug) and pentamidine (second-line antileishmanial drug) were used as positive controls. Cultures containing parasites without compound solution were also included. Plates were incubated at 268C for 72 h and the leishmanicidal activity of compounds was determined by direct count of parasites in a Neubauer chamber. The concentrations required to inhibit 50% of parasite growth (IC50) were calculated by probit analysis and are summarized in Table 1. It is clear from Table 1 that nitazoxanide and tizoxanide reduced the growth of the kinetoplastid parasites L. mexicana and T. cruzi in vitro with IC50s 2-fold lower than those of pentamidine and benznidazole, respectively. The newly synthesized analogue NTB was 2-fold more potent than nitazoxanide and tizoxanide and 6-fold more active

2-acylamino-5-nitro-1,3-thiazoles: preparation and in vitro bioevaluation against four neglected protozoan parasites.

The 2-acylamino-5-nitro-1,3-thiazole derivatives (1-14) were prepared using a one step reaction. All compounds were tested in vitro against four neglected protozoan parasites (Giardia intestinalis, Trichomonas vaginalis, Leishmania amazonensis and Trypanosoma cruzi). Acetamide (9), valeroylamide (10), benzamide (12), methylcarbamate (13) and ethyloxamate (14) derivatives were the most active compounds against G. intestinalis and T. vaginalis, showing nanomolar inhibition. Compound 13 (IC50=10nM), was 536-times more active than metronidazole, and 121-fold more effective than nitazoxanide against G. intestinalis. Compound 14 was 29-times more active than metronidazole and 6.5-fold more potent than nitazoxanide against T. vaginalis. Ureic derivatives 2, 3 and 5 showed moderate activity against L. amazonensis. None of them were active against T. cruzi. Ligand efficiency indexes analysis revealed higher intrinsic quality of the most active 2-acylamino derivatives than nitazoxanide and metronidazole. In silico toxicity profile was also computed for the most active compounds. A very low in vitro mammalian cytotoxicity was obtained for 13 and 14, showing selectivity indexes (SI) of 246,300 and 141,500, respectively. Nitazoxanide showed an excellent leishmanicidal and trypanocidal effect, repurposing this drug as potential new antikinetoplastid parasite compound.

Emulsifying Activity and Stability of a Non-Toxic Bioemulsifier Synthesized by Microbacterium sp. MC3B-10

A previously reported bacterial bioemulsifier, here termed microbactan, was further analyzed to characterize its lipid component, molecular weight, ionic character and toxicity, along with its bioemulsifying potential for hydrophobic substrates at a range of temperatures, salinities and pH values. Analyses showed that microbactan is a high molecular weight (700 kDa), non-ionic molecule. Gas chromatography of the lipid fraction revealed the presence of palmitic, stearic, and oleic acids; thus microbactan may be considered a glycolipoprotein. Microbactan emulsified aromatic hydrocarbons and oils to various extents; the highest emulsification index was recorded against motor oil (96%). The stability of the microbactan-motor oil emulsion model reached its highest level (94%) at 50 °C, pH 10 and 3.5% NaCl content. It was not toxic to Artemia salina nauplii. Microbactan is, therefore, a non-toxic and non-ionic bioemulsifier of high molecular weight with affinity for a range of oily substrates. Comparative phylogenetic assessment of the 16S rDNA gene of Microbacterium sp. MC3B-10 with genes derived from other marine Microbacterium species suggested that this genus is well represented in coastal zones. The chemical nature and stability of the bioemulsifier suggest its potential application in bioremediation of marine environments and in cosmetics.

Trinorsesquiterpenoids from the Root Extract of Pentalinon andrieuxii

Two unusual trinorsesquiterpenoids, urechitols A (1) and B (2), were isolated from the root extract of Pentalinon andrieuxii, a plant used commonly in Yucatecan traditional medicine to treat leishmaniasis. The structures of 1 and 2 were identified by interpretation of their spectroscopic data and chemical correlation reactions. The relative stereochemistry of 1 was confirmed through an X-ray crystallographic study.

Bioweathering Potential of Cultivable Fungi Associated with Semi-Arid Surface Microhabitats of Mayan Buildings

Soil and rock surfaces support microbial communities involved in mineral weathering processes. Using selective isolation, fungi were obtained from limestone surfaces of Mayan monuments in the semi-arid climate at Yucatan, Mexico. A total of 101 isolates representing 53 different taxa were studied. Common fungi such as Fusarium, Pestalotiopsis, Trichoderma, and Penicillium were associated with surfaces and were, probably derived from airborne spores. In contrast, unusual fungi such as Rosellinia, Annulohypoxylon, and Xylaria were predominantly identified from mycelium particles of biofilm biomass. Simulating oligotrophic conditions, agar amended with CaCO3 was inoculated with fungi to test for carbonate activity. A substantial proportion of fungi, in particular those isolated from mycelium (59%), were capable of solubilizing calcium by means of organic acid release, notably oxalic acid as evidenced by ion chromatography. Contrary to our hypothesis, nutrient level was not a variable influencing the CaCO3 solubilization ability among isolates. Particularly active fungi (Annulohypoxylon stygium, Penicillium oxalicum, and Rosellinia sp.) were selected as models for bioweathering experiments with limestone-containing mesocosms to identify if other mineral phases, in addition to oxalates, were linked to bioweathering processes. Fungal biofilms were seen heavily covering the stone surface, while a biomineralized front was also observed at the stone-biofilm interface, where network of hyphae and mycogenic crystals was observed. X-ray diffraction analysis (XRD) identified calcite as the main phase, along with whewellite and wedellite. In addition, lower levels of citrate were detected by Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Overall, our results suggest that a diverse fungal community is associated with limestone surfaces insemi-arid climates. A subset of this community is geochemically active, excreting organic acids under quasi-oligotrophic conditions, suggesting that the high metabolic cost of exuding organic acids beneficial under nutrient limitation. Oxalic acid release may deteriorate or stabilize limestone surfaces, depending on microclimatic dynamics.