Emanuel Hernández-Núñez

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.

Synthesis and in vitro trichomonicidal, giardicidal and amebicidal activity of N-acetamide(sulfonamide)-2-methyl-4-nitro-1H-imidazoles

Two new series of imidazole derivatives (acetamides: 1-8 and sulfonamides: 9-15) were synthesized using a short synthetic route. Compound 1 as well as the intermediate 16g were characterized by X-ray crystallography. Imidazole derivatives 1-15 were tested in vitro against three unicellular parasites (Giardia intestinalis, Trichomonas vaginalis and Entamoeba histolytica) in comparison with benznidazole (Bzn) and metronidazole. Compound 1 [N-benzyl-2-(2-methyl-4-nitro-1H-imidazol-1-yl)acetamide] was 2 times more active than Bzn against T. vaginalis and G. intestinalis and it was as active as Bzn against E. histolytica. Sulfonamides showed selective toxicity against E. histolytica over the other parasites. Toxicity assay showed that all compounds are non-cytotoxic against MDCK cell line. The results revealed that compounds 1-15 have antiparasitic bioactivity in the micromolar range against the parasites tested, and could be considered as benznidazole bioisosteres.

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.

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.

Discovery of 2-(3,4-dichlorophenoxy)-N-(2-morpholin-4-ylethyl)acetamide: A selective σ1 receptor ligand with antinociceptive effect

Compound 2-(3,4-dichlorophenoxy)-N-(2-morpholin-4-ylethyl)acetamide (1) was designed, prepared and the in vitro binding evaluation against σ1 and σ2 receptors was measured. Compound 1 showed high σ1 receptor affinity (Ki=42 nM) and it was 36-times more selective for σ1 than σ2 receptor. Also, it was performed a molecular docking of compound 1 into the ligand binding pocket homology model of σ1 receptor, showing a salt bridge between the ionized morpholine ring and Asp126, as well as important short contacts with residues Tyr120, His154 and Trp164. Ligand efficiency indexes and predicted toxicity analysis revealed an excellent intrinsic quality of 1. The antinociceptive effect of compound 1 was determined using the formalin test. The ipsilateral local peripheral (10-300 μg/paw) and intrathecal (100 μg/rat) administration of 1 produced a reduction in formalin-induced nociception. The in vivo results indicated that 1 may be effective in treating inflammatory pain.

Design, Synthesis and in Combo Antidiabetic Bioevaluation of Multitarget Phenylpropanoic Acids

We have synthesized a small series of five 3-[4-arylmethoxy)phenyl]propanoic acids employing an easy and short synthetic pathway. The compounds were tested in vitro against a set of four protein targets identified as key elements in diabetes: G protein-coupled receptor 40 (GPR40), aldose reductase (AKR1B1), peroxisome proliferator-activated receptor gama (PPARγ) and solute carrier family 2 (facilitated glucose transporter), member 4 (GLUT-4). Compound 1 displayed an EC50 value of 0.075 μM against GPR40 and was an AKR1B1 inhibitor, showing IC50 = 7.4 μM. Compounds 2 and 3 act as slightly AKR1B1 inhibitors, potent GPR40 agonists and showed an increase of 2 to 4-times in the mRNA expression of PPARγ, as well as the GLUT-4 levels. Docking studies were conducted in order to explain the polypharmacological mode of action and the interaction binding mode of the most active molecules on these targets, showing several coincidences with co-crystal ligands. Compounds 1–3 were tested in vivo at an explorative 100 mg/kg dose, being 2 and 3 orally actives, reducing glucose levels in a non-insulin-dependent diabetes mice model. Compounds 2 and 3 displayed robust in vitro potency and in vivo efficacy, and could be considered as promising multitarget antidiabetic candidates. This is the first report of a single molecule with these four polypharmacological target action.

Synthesis, Screening and in silico Simulations of Anti-Parasitic Propamidine/Benzimidazole Derivatives

BACKGROUND We designed hybrid molecules between propamidine and benzimidazole in order to retain the antiprotozoal action, but decreasing the toxic effect of the molecule. OBJECTIVE Design and prepare 12 hybrids for testing their antiparasitic effect over three protozoa: Giardia intestinalis, Trichomonas vaginalis and Leishmania mexicana, as well as conduct several in silico simulations such as toxicological profile, molecular docking and molecular dynamics in order to understand their potential mode of action. METHODS Hybrids 1-3, 6-9 and 12 were obtained using a chemical pathway previously reported. Compounds 4, 5, 10 and 11 were prepared using a one-pot reduction-cyclization reaction. The in vitro antiparasitic and cytotoxic activities of these compounds were conducted. It was calculated several properties such as toxicity, PK behavior, as well as docking studies and molecular dynamics of the most active compound performed in a DNA sequence dodecamer in comparison with propamidine. RESULTS Compound 2 was 183, 127 and 202 times more active against G. intestinalis than metronidazole, pentamidine and propamidine. It was eleven times more active than pentamidine against L. mexicana. This compound showed low in vitro mammalian cytotoxicity. Molecular simulations showed a stable complex 2-DNA that occurred in the minor groove, analogous to propamidine-DNA complex. CONCLUSION Compound 2, exhibited the higher bioactivity, especially towards G. intestinalis and L. mexicana. This study demonstrated that the replacement of benzimidazole scaffold instead of toxic amidine group in propamidine, results in an enhancement of antiprotozoal bioactivity. The preliminary molecular dynamics simulation suggests that the ligand-DNA complex is stable.

Synthesis of syn-gamma-Amino-beta-hydroxyphosphonates by Reduction of beta-Ketophosphonates Derived from L-Proline and L-Serine

The reduction of γ-N-benzylamino-β-ketophosphonates 6 and 10, readily available from L-proline and L-serine, respectively, can be carried out in high diastereoselectivity with catecholborane (CB) in THF at -78 °C to produce the syn-γ-N-benzylamino-β-hydroxyphosphonates 11 and 13 as a single detectable diastereoisomer, under non-chelation or Felkin-Anh model control.

Synthesis, in vitro and in vivo giardicidal activity of nitrothiazole-NSAID chimeras displaying broad antiprotozoal spectrum ☆

We designed and synthesized five new 5-nitrothiazole-NSAID chimeras as analogues of nitazoxanide, using a DCC-activated amidation. Compounds 1-5 were tested in vitro against a panel of five protozoa: 2 amitochondriates (Giardia intestinalis, Trichomonas vaginalis) and 3 kinetoplastids (Leishmania mexicana, Leishmania amazonensis and Trypanosoma cruzi). All chimeras showed broad spectrum and potent antiprotozoal activities, with IC50 values ranging from the low micromolar to nanomolar order. Compounds 1-5 were even more active than metronidazole and nitazoxanide, two marketed first-line drugs against giardiasis. In particular, compound 4 (an indomethacin hybrid) was one of the most potent of the series, inhibiting G. intestinalis growth in vitro with an IC50 of 0.145μM. Compound 4 was 38-times more potent than metronidazole and 8-times more active than nitazoxanide. The in vivo giardicidal effect of 4 was evaluated in a CD-1 mouse model obtaining a median effective dose of 1.709μg/kg (3.53nmol/kg), a 321-fold and 1015-fold increase in effectiveness after intragastric administration over metronidazole and nitazoxanide, respectively. Compounds 1 and 3 (hybrids of ibuprofen and clofibric acid), showed potent giardicidal activities in the in vitro as well as in the in vivo assays after oral administration. Therefore, compounds 1-5 constitute promising drug candidates for further testing in experimental chemotherapy against giardiasis, trichomoniasis, leishmaniasis and even trypanosomiasis infections.