An Efficient and Scalable "Second Generation" Total Synthesis of the Marine Polyketide Limaol Endowed with Antiparasitic Activity.

The cluster of four skipped exo-methylene substituents on the "northern" wing of limaol renders this dinoflagellate-derived marine natural product unique in structural terms. This arguably non-thermodynamic array gains kinetic stability by virtue of populating local conformations which impede isomerization to a partly or fully conjugated polyene. This analysis suggested that the difficulties encountered during the late stages of our first total synthesis of this polyketide had not been caused by an overly fragile character of this unusual substructure; rather, an unfavorable steric microenvironment about the spirotricyclic core was identified as the likely cause. To remedy the issue, the protecting groups on this central fragment were changed; in effect, this amendment allowed all strategic and practical problems to be addressed. As a result, the overall yield over the longest linear sequence was multiplied by a factor of almost five and the material throughput increased more than eighty-fold per run. Key-to-success was a gold-catalyzed spirocyclization reaction; the reasons why a Brønsted acid cocatalyst is needed and the origin of the excellent levels of selectivity were delineated. The change of the protecting groups also allowed for much improved fragment coupling processes; most notably, the sequence of a substrate-controlled carbonyl addition reaction followed by Mitsunobu inversion that had originally been necessary to affix the southern tail to the core could be replaced by a reagent controlled asymmetric allylation. Finally, a much-improved route to the "northern" sector was established by leveraging the power of asymmetric hydrogenation of a 2-pyrone derivative. Limaol was found to combine appreciable antiparasitic activity with very modest cytotoxicity.

Design and Synthesis of 1,3-Diarylpyrazoles and Investigation of Their Cytotoxicity and Antiparasitic Profile.

Herein, we report a series of 1,3-diarylpyrazoles that are analogues of compound 26/HIT 8. We previously identified this molecule as a 'hit' during a high-throughput screening campaign for autophagy inducers. A variety of synthetic strategies were utilized to modify the 1,3-diarylpyrazole core at its 1-, 3-, and 4-position. Compounds were assessed in vitro to identify their cytotoxicity properties. Of note, several compounds in the series displayed relevant cytotoxicity, which warrants scrutiny while interpreting biological activities that have been reported for structurally related molecules. In addition, antiparasitic activities were recorded against a range of human-infective protozoa, including Trypanosoma cruzi, T. brucei rhodesiense, and Leishmania infantum. The most interesting compounds displayed low micromolar whole-cell potencies against individual or several parasitic species, while lacking cytotoxicity against human cells.

Promising hit compounds against resistant trichomoniasis: Synthesis and antiparasitic activity of 3-(ω-aminoalkoxy)-1-benzyl-5-nitroindazoles.

A series of 11 3-(ω-aminoalkoxy)-1-benzyl-5-nitroindazoles (2-12) has been prepared starting from 1-benzyl-5-nitroindazol-3-ol 13, and evaluated against sensitive and resistant isolates of the sexually transmitted protozoan Trichomonas vaginalis. Compounds 2, 3, 6, 9, 10 and 11 showed trichomonacidal profiles with IC50 < 20 µM against the metronidazole-sensitive isolate. Moreover, all these compounds submitted to cytotoxicity assays against mammalian cells exhibited low non-specific cytotoxic effects, except compounds 3 and 9 which displayed moderate cytotoxicity (CC50 = 74.7 and 59.1 µM, respectively). Those compounds with trichomonacidal effect were also evaluated against a metronidazole-resistant culture. Special mention deserve compounds 6 and 10, which displayed better IC50 values (1.3 and 0.5 µM respectively) than that of the reference drug (IC50 MTZ = 3.0 µM). The high activity of these compounds against the resistant isolate reinforces the absence of cross-resistance with the reference drug. The remarkable trichomonacidal results against resistant T. vaginalis isolates suggest the interest of 3-(ω-aminoalkoxy)-1-benzyl-5-nitroindazoles to be considered as good prototypes to continue in the development of new drugs with enhanced trichomonacidal activity, aiming to increase the non-existent drugs to face clinical resistance efficiently for those patients in whom therapy with 5-nitroimidazoles is contraindicated.

Lactate dehydrogenase and malate dehydrogenase: Potential antiparasitic targets for drug development studies.

Parasitic diseases remain a major public health concern for humans, claiming millions of lives annually. Although different treatments are required for these diseases, drug usage is limited due to the development of resistance and toxicity, which necessitate alternative therapies. It has been shown in the literature that parasitic lactate dehydrogenases (LDH) and malate dehydrogenases (MDH) have unique pharmacological selective and specificity properties compared to other isoforms, thus highlighting them as viable therapeutic targets involved in aerobic and anaerobic glycolytic pathways. LDH and MDH are important therapeutic targets for invasive parasites because they play a critical role in the progression and development of parasitic diseases. Any strategy to impede these enzymes would be fatal to the parasites, paving the way to develop and discover novel antiparasitic agents. This review aims to highlight the importance of parasitic LDH and MDH as therapeutic drug targets in selected obligate apicoplast parasites. To the best of our knowledge, this review presents the first comprehensive review of LDH and MDH as potential antiparasitic targets for drug development studies.

p-Trifluoromethyl- and p-pentafluorothio-substituted curcuminoids of the 2,6-di[(E)-benzylidene)]cycloalkanone type: Syntheses and activities against Leishmania major and Toxoplasma gondii parasites.

A series of the title curcuminoids with structural variance in the heteroatom of the cycloalkanone and the p-substituents of the phenyl rings were tested for their activities against Leishmania major and Toxoplasma gondii parasites. The majority of them showed high activities against both parasite forms with EC50 values in the sub-micromolar concentration range. Bis(p-pentafluorothio)-substituted 3,5-di[(E)-benzylidene]piperidin-4-one 1b was not just noticeable antiparasitic, but also exhibited a considerable selectivity for L. major promastigotes over normal Vero cells. While derivatives differing only in the p-phenyl substituents being CF3 or SF5 showed similar antiparasitic activities, the cyclic ketone hub was more decisive both for the anti-parasitic activities and the selectivities for the parasites vs. normal cells. QSAR calculations confirmed the observed structure-activity relations and suggested structural variations for a further improvement of the antiparasitic activity. Docking studies based on DFT calculations revealed L. major pteridine reductase 1 as a likely molecular target protein of the title compounds.

Design, Synthesis and Antiparasitic Evaluation of Click Phospholipids.

A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against Leishmania infantum and Leishmania donovani intracellular amastigotes, against Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure-activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative 27 substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two L. infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC50) against THP-1 macrophages ranged between 50 and 100 µM. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases.

Coumarin-Tagged Dinuclear Trithiolato-Bridged Ruthenium(II)⋠Arene Complexes: Photophysical Properties and Antiparasitic Activity.

The synthesis, characterization, photophysical and biological properties of 13 new conjugate coumarin-diruthenium(II)⋅arene complexes against Toxoplasma gondii are presented. For all conjugate organometallic unit/coumarins, an almost complete loss of fluorescence efficacy was observed. However, the nature of the fluorophore, the type of bonding, the presence and length of a linker between the coumarin dye and the ruthenium(II) moiety, and the number of dye units influenced their biological properties. The in vitro activity against a transgenic T. gondii strain grown in human foreskin fibroblasts (HFF) leads to IC50 values for T. gondii ß-gal from 105 to 735 nM. Of note is that nine compounds displayed lower IC50 than the standard drug pyrimethamine. One compound applied at its IC50 did not affect B-cell proliferation but had an impact on T-cell proliferation in murine splenocyte cultures. Transmission electron microscopy of T. gondii ß-gal-infected HFF showed that treatment predominantly affected the parasites' mitochondrion.

Expanding the scope of synthetic 1,2,4-trioxanes towards Trypanosoma cruzi and Leishmania donovani.

A series of synthetic 1,2,4-trioxanes related to artemisinin was tested against L. donovani and T. cruzi parasites. This screening identified some active compounds, with key common structural features. Interestingly, these selected trioxanes were efficient against both parasites, and achieved antiparasitic activities comparable or superior than those presented by the corresponding reference drugs, artemisinin and artesunate. This study represents the first example of synthetic trioxanes evaluated on T. cruzi and provides possible candidates for developing new drugs for the treatment of leishmaniasis and Chagas disease.

Synthesis, in vitro and in vivo biological evaluation of dihydroartemisinin derivatives with potential anti-Toxoplasma gondii agents.

In this study, four series of dihydroartemisinin derivatives were designed, synthesized, and evaluated for anti-toxoplasma gondii activity, and calculated the selectivity index (SI). It was the higher the SI, the better the effect of this compound against Toxoplasma gondii. Our goal was to filter out compounds that were bigger SI than the lead compound. The compound with the highest SI was selected for the anti-toxoplasmosis test in mice in vivo. Among the synthesized compounds, the (3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-trimethyl-decahydro-12H-3,12-epoxy[1,2]di-oxepino[4,3 -i]isochromen-10-yl-(te-rt-butoxycarbonyl)-l-alaninate (A2) exhibited the most potent anti-T. gondii activity and low cytotoxicity (SI: 6.44), yielding better results than the lead compound DHA (SI: 1.00) and the clinically used positive-control drug spiramycin (SI: 0.72) in vitro. Furthermore, compound A2 had better growth inhibitory effects on T. gondii in vivo than spiramycin did and significantly reduced the number of tachyzoites in the peritoneal cavity of mice (P < 0.01). The evaluation of the data generated in the T. gondii mouse infection model indicates that compound A2 treatment was a good inhibitor of T. gondii in vivo and that it was effective in relieving the liver damage induced by T. gondii. In addition, the results of a docking study revealed that A2 could become a better T. gondii calcium-dependent protein kinase1 (TgCDPK1) inhibitor. For this reason, compound A2 has potential as an anti-parasitic drug. Further studies are required to elucidate the mechanism of the action of compound A2, as well as to develop drug delivery systems for patients.

Novel Linezolid analogues with antiparasitic activity against Hymenolepis nana.

The stereoselective synthesis and anti- Hymenolepis nana activity of six Linezolid-type compounds, obtained by chemical modification of l-Alanine, are reported in this work. The synthetic strategy was to prepare diasteromeric N,N-dibenzylamino oxazolidinones 1 and 2, and coupling with 4-(4-bromophenyl)morpholine (3) to obtain N,N-dibenzylamino Linezolid analogues 4 and 5. A hydrogenolysis reaction over 4 and 5 resulted in amino-free Linezolid analogues 6 and 7, which were acetylated to reach diasteromeric Linezolid analogues 8 and 9. The six Linezolid analogues 4-9 show in vitro antiparasitic activity against Hymenolepis nana cestode, but not against several bacterial strains. Interestingly, compounds 6, 7 and 9 exhibit high potency, having shorter paralysis and death times after exposure (6-10 and 18-21 min, respectively), shorter than those found with antihelmintic compound Praziquantel (20 and 30 min) at 20 mg/mL. In addition, a cytocompatibility assay of 6-9 with human cells (ARPE-19 cells) demonstrate a non-cytotoxic effect at 0.4 mM. These results show the pharmacological potential of the newly reported Linezolid-type analogues as antiparasitic agents against Hymenolepis nana.

New Antiparasitic Bis-Naphthoquinone Derivatives.

A series of bis-naphthoquinone derivatives prepared by condensation of aryl aldehydes with lawsone were tested for antiparasitic activities against Toxoplasma gondii and Trypanosoma brucei parasites. Monofluorophenyl derivative 1a, 3,4-difluorophenyl analog 1c and furyl compound 1l exhibited significant activity against T. gondii cells and appear to be new promising drug candidates against this parasite. The 3,4,5-trifluorophenyl derivative 1g and the isovanillyl derivative 1j displayed selective activity against Leishmania major amastigotes.

Lactoferrin and Its Derived Peptides: An Alternative for Combating Virulence Mechanisms Developed by Pathogens.

Due to the emergence of multidrug-resistant pathogens, it is necessary to develop options to fight infections caused by these agents. Lactoferrin (Lf) is a cationic nonheme multifunctional glycoprotein of the innate immune system of mammals that provides numerous benefits. Lf is bacteriostatic and/or bactericidal, can stimulate cell proliferation and differentiation, facilitate iron absorption, improve neural development and cognition, promote bone growth, prevent cancer and exert anti-inflammatory and immunoregulatory effects. Lactoferrin is present in colostrum and milk and is also produced by the secondary granules of polymorphonuclear leukocytes, which store this glycoprotein and release it at sites of infection. Lf is also present in many fluids and exocrine secretions, on the surfaces of the digestive, respiratory and reproductive systems that are commonly exposed to pathogens. Apo-Lf (an iron-free molecule) can be microbiostatic due to its ability to capture ferric iron, blocking the availability of host iron to pathogens. However, apo-Lf is mostly microbicidal via its interaction with the microbial surface, causing membrane damage and altering its permeability function. Lf can inhibit viral entry by binding to cell receptors or viral particles. Lf is also able to counter different important mechanisms evolved by microbial pathogens to infect and invade the host, such as adherence, colonization, invasion, production of biofilms and production of virulence factors such as proteases and toxins. Lf can also cause mitochondrial and caspase-dependent regulated cell death and apoptosis-like in pathogenic yeasts. All of these mechanisms are important targets for treatment with Lf. Holo-Lf (the iron-saturated molecule) can contain up to two ferric ions and can also be microbicidal against some pathogens. On the other hand, lactoferricins (Lfcins) are peptides derived from the N-terminus of Lf that are produced by proteolysis with pepsin under acidic conditions, and they cause similar effects on pathogens to those caused by the parental Lf. Synthetic analog peptides comprising the N-terminus Lf region similarly exhibit potent antimicrobial properties. Importantly, there are no reported pathogens that are resistant to Lf and Lfcins; in addition, Lf and Lfcins have shown a synergistic effect with antimicrobial and antiviral drugs. Due to the Lf properties being microbiostatic, microbicidal, anti-inflammatory and an immune modulator, it represents an excellent natural alternative either alone or as adjuvant in the combat to antibiotic multidrug-resistant bacteria and other pathogens. This review aimed to evaluate the data that appeared in the literature about the effects of Lf and its derived peptides on pathogenic bacteria, protozoa, fungi and viruses and how Lf and Lfcins inhibit the mechanisms developed by these pathogens to cause disease.

Further insights of selenium-containing analogues of WC-9 against Trypanosoma cruzi.

As a continuation of our project aimed at searching for new chemotherapeutic agents against American trypanosomiasis (Chagas disease), new selenocyanate derivatives were designed, synthesized and biologically evaluated against the clinically more relevant dividing form of Trypanosoma cruzi, the etiologic agent of this illness. In addition, in order to establish the role of each part of the selenocyanate moiety, different derivatives, in which the selenium atom or the cyano group were absent, were conceived, synthesized and biologically evaluated. In addition, in order to study the optimal position of the terminal phenoxy group, new regioisomers of WC-9 were synthesized and evaluated against T. cruzi. Finally, the resolution of a racemic mixture of a very potent conformationally rigid analogue of WC-9 was accomplished and further tested as growth inhibitors of T. cruzi proliferation. The results provide further insight into the role of the selenocyanate group in its antiparasitic activity.

Antiparasitic activities of new lawsone Mannich bases.

A series of new lawsone Mannich bases derived from salicylaldehydes or nitrofurfural were prepared and tested for their activities against Leishmania major, Toxoplasma gondii, and Trypanosoma brucei brucei parasites. The hydrochloride salts 5a and 6a of the Mannich bases 2a and 3a, derived from unsubstituted salicylaldehyde and long-chained alkyl amines, were selectively and strongly active against T. gondii cells and appear to be new promising drug candidates against this parasite. Compound 6a showed an even higher activity against T. gondii than the known lawsone Mannich base 1b. Compound 4a, derived from salicylaldehyde and 2-methylaminopyridine, was also distinctly active against T. gondii cells. The derivatives 3a (salicyl derivative), 3b (3,5-dichloro-2-hydroxyphenyl derivative), and 3d (5-nitrofuranyl derivative) as well as the hydrochlorides 6a and 6b were also efficacious against T. b. brucei cells with compounds 3a and 3b being more selective for T. b. brucei over Vero cells when compared with the known control compound 1b. The derivatives 5a, 5c, 6a, and 6c proved to be up to five times more active than 1b against L. major promastigotes and up to four times more efficacious against L. major amastigotes.

Synthesis and In Vitro Growth Inhibition of 2-Allylphenol Derivatives Against Phythopthora cinnamomi Rands.

Phytophthora cinnamomi is a phytopathogen that causes extensive damage in different crops, and therefore, produces important economic losses all around the world. Chemical fungicides are a key factor for the control of this disease. However, ecological and environmental considerations, as well as the appearance of strains that are resistant to commercial fungicides, have prompted the quest for new antifungal agents which are of low ecological impact. In this work, a series of new 2-allylphenol derivatives was synthesized, and their structures were confirmed by FT-IR, NMR, and MS. Some of the synthesized compounds, more specifically nitro derivatives, exhibit strong growth inhibition of P. cinnamomi with EC50 as low as 10.0 µg/mL. This level of activity is similar to that exhibited by METALAXYL MZ 58 WP, a commonly-used commercial fungicide; therefore, these compounds might be of agricultural interest due to their potential use as fungicides against P. cinnamomi. The results indicate that this activity depends on the chemical structures of the 2-allylphenol derivatives, and that it is strongly enhanced in molecules where nitro and hydroxyl groups adopt a -para configuration. These effects are discussed in terms of the electronic distribution of the aromatic ring induced by substituent groups.

Fluorescent-fipronil: Design and synthesis of a stable conjugate.

Fipronil is a phenyl pyrazole molecule widely used across the world as both insecticide and veterinary drug. The main goal of this work was to synthesize a fluorescently labeled fipronil derivative for cellular imaging without affecting its intrinsic properties. We selected fluorescein as fluorescent probe and we investigated different strategies for stable chemical ligation between both entities, such as thiourea and direct peptide bond. While thiourea bond displayed low stability, direct peptide bond was difficult to achieve due to problems of steric hindrance. The best result was obtained by conjugation using click chemistry, which allowed to obtain fipronil stably labeled with the fluorescent probe.

Pyrazinoates as antiparasitic agents against Trypanosoma cruzi.

This work reports a repurposing study of pyrazinoic acid (1) and methyl (2), ethyl (3) and 2-chloroethyl (4) ester derivatives with antimycobacterial activity, in assays against Trypanosoma cruzi. The compounds and benznidazole, the standard antitrypanosoma drug, were evaluated in concentrations ranging from 100 to 6.25 µg/mL. The results showed that compounds 2 and 3 (EC50 = 182 and 447 µM) significantly reduced the infection rate of the parasite into the mammalian cells at 100 µg/mL (p < 0.05) in a similar way to benznidazole. In addition, all the compounds also significantly reduced the number of intracellular parasites (compound 1 at 50 µg/mL, and compounds 2-4 at 100 µg/mL, p < 0.05) in comparison to the control. Compounds 1 and 2 were more effective than benznidazole at 50 µg/mL (p < 0.001). Moreover, compounds 1-4 did not show significant cytotoxicity against THP-1, J774, and HeLa cells (>1000 µM), indicating that they possess considerable selectivity against the parasites. This report represents the first study of such compounds against T. cruzi, indicating the potential of pyrazinoates as antiparasitic agents.

Lead selection of antiparasitic compounds from a focused library of benzenesulfonyl derivatives of heterocycles.

A library of 89 synthetic benzenesulfonyl derivatives of heterocycles with drug-like properties was assayed for in vitro antiparasitic activity and the results were added to our previously reported derivatives for a comprehensive SAR evaluation. Four compounds showed an IC50 between 0.25 and 3µM against Leishmania donovani and low cytotoxicity. Compound G{16} (1-(2,3,5,6-tetramethylphenylsulfonyl)-2-methylindoline), was particularly interesting with an IC50 similar to the reference drug miltefosine. Seven compounds showed an IC50 below 6µM against Trypanosoma cruzi, and three of them (E{3}, E{9} and G{3}) were identified as lead scaffolds for further optimization based on their activity-toxicity profile. Two promising structures (B{15} and G{15}) showed moderate inhibitory activity against Plasmodium falciparum. In general, the presence of a benzenesulfonyl moiety improves the antiparasitic activity of the heterocycles included in this study (with the exception of Trypanosoma brucei rhodesiense), validating the criteria used in the selection of the privileged structures and diversification used to generate this library. SAR analysis showed that the presence of lipophilic and electron withdrawing groups were favorable for the antiparasitic activity.

An Efficient One-Pot Catalyzed Synthesis of 2,4-Disubstituted 5-Nitroimidazoles Displaying Antiparasitic and Antibacterial Activities.

A one-pot regioselective bis-Suzuki-Miyaura or Suzuki-Miyaura/Sonogashira reaction on 2,4-dibromo-1-methyl-5-nitro-1H-imidazole under microwave heating was developed. This method is applicable to a wide range of (hetero)arylboronic acids and terminal alkynes. Additionally, this approach provides a simple and efficient way to synthesize 2,4-disubstituted 5-nitroimidazole derivatives with antibacterial and antiparasitic properties.

Chroman-4-One Derivatives Targeting Pteridine Reductase 1 and Showing Anti-Parasitic Activity.

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.