Synthesis, In Silico Study, and In Vitro Antifungal Activity of New 5-(1,3-Diphenyl-1H-Pyrazol-4-yl)-4-Tosyl-4,5-Dihydrooxazoles.

The increase in multi-drug resistant Candida strains has caused a sharp rise in life-threatening fungal infections in immunosuppressed patients, including those with SARS-CoV-2. Novel antifungal drugs are needed to combat multi-drug-resistant yeasts. This study aimed to synthesize a new series of 2-oxazolines and evaluate the ligands in vitro for the inhibition of six Candida species and in silico for affinity to the CYP51 enzymes (obtained with molecular modeling and protein homology) of the same species. The 5-(1,3-diphenyl-1H-pyrazol-4-yl)-4-tosyl-4,5-dihydrooxazoles 6a-j were synthesized using the Van Leusen reaction between 1,3-diphenyl-4-formylpyrazoles 4a-j and TosMIC 5 in the presence of K2CO3 or KOH without heating, resulting in short reaction times, high compound purity, and high yields. The docking studies revealed good affinity for the active site of the CYP51 enzymes of the Candida species in the following order: 6a-j > 4a-j > fluconazole (the reference drug). The in vitro testing of the compounds against the Candida species showed lower MIC values for 6a-j than 4a-j, and for 4a-j than fluconazole, thus correlating well with the in silico findings. According to growth rescue assays, 6a-j and 4a-j (like fluconazole) inhibit ergosterol synthesis. The in silico toxicity assessment evidenced the safety of compounds 6a-j, which merit further research as possible antifungal drugs.

Inhibitors of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Decrease the Growth, Ergosterol Synthesis and Generation of petite Mutants in Candida glabrata and Candida albicans.

Candida glabrata and Candida albicans, the most frequently isolated candidiasis species in the world, have developed mechanisms of resistance to treatment with azoles. Among the clinically used antifungal drugs are statins and other compounds that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), resulting in decreased growth and ergosterol levels in yeasts. Ergosterol is a key element for the formation of the yeast cell membrane. However, statins often cause DNA damage to yeast cells, facilitating mutation and drug resistance. The aim of the current contribution was to synthesize seven series of compounds as inhibitors of the HMGR enzyme of Candida ssp., and to evaluate their effect on cellular growth, ergosterol synthesis and generation of petite mutants of C. glabrata and C. albicans. Compared to the reference drugs (fluconazole and simvastatin), some HMGR inhibitors caused lower growth and ergosterol synthesis in the yeast species and generated fewer petite mutants. Moreover, heterologous expression was achieved in Pichia pastoris, and compounds 1a, 1b, 6g and 7a inhibited the activity of recombinant CgHMGR and showed better binding energy values than for α-asarone and simvastatin. Thus, we believe these are good candidates for future antifungal drug development.

Pyrrole-Based Enaminones as Building Blocks for the Synthesis of Indolizines and Pyrrolo[1,2-a]pyrazines Showing Potent Antifungal Activity.

As a new approach, pyrrolo[1,2-a]pyrazines were synthesized through the cyclization of 2-formylpyrrole-based enaminones in the presence of ammonium acetate. The enaminones were prepared with a straightforward method, reacting the corresponding alkyl 2-(2-formyl-1H-pyrrol-1-yl)acetates, 2-(2-formyl-1H-pyrrol-1-yl)acetonitrile, and 2-(2-formyl-1H-pyrrol-1-yl)acetophenones with DMFDMA. Analogous enaminones elaborated from alkyl (E)-3-(1H-pyrrol-2-yl)acrylates were treated with a Lewis acid to afford indolizines. The antifungal activity of the series of substituted pyrroles, pyrrole-based enaminones, pyrrolo[1,2-a]pyrazines, and indolizines was evaluated on six Candida spp., including two multidrug-resistant ones. Compared to the reference drugs, most test compounds produced a more robust antifungal effect. Docking analysis suggests that the inhibition of yeast growth was probably mediated by the interaction of the compounds with the catalytic site of HMGR of the Candida species.

Synthesis and Molecular Docking Studies of Alkoxy- and Imidazole-Substituted Xanthones as α-Amylase and α-Glucosidase Inhibitors.

Current antidiabetic drugs have severe side effects, which may be minimized by new selective molecules that strongly inhibit α-glucosidase and weakly inhibit α-amylase. We have synthesized novel alkoxy-substituted xanthones and imidazole-substituted xanthones and have evaluated them for their in silico and in vitro α-glucosidase and α-amylase inhibition activity. Compounds 6c, 6e, and 9b promoted higher α-glucosidase inhibition (IC50 = 16.0, 12.8, and 4.0 µM, respectively) and lower α-amylase inhibition (IC50 = 76.7, 68.1, and >200 µM, respectively) compared to acarbose (IC50 = 306.7 µM for α-glucosidase and 20.0 µM for α-amylase). Contrarily, derivatives 10c and 10f showed higher α-amylase inhibition (IC50 = 5.4 and 8.7 µM, respectively) and lower α-glucosidase inhibition (IC50 = 232.7 and 145.2 µM, respectively). According to the structure-activity relationship, attaching 4-bromobutoxy or 4'-chlorophenylacetophenone moieties to the 2-hydroxy group of xanthone provides higher α-glucosidase inhibition and lower α-amylase inhibition. In silico studies suggest that these scaffolds are key in the activity and interaction of xanthone derivatives. Enzymatic kinetics studies showed that 6c, 9b, and 10c are mainly mixed inhibitors on α-glucosidase and α-amylase. In addition, drug prediction and ADMET studies support that compounds 6c, 9b, and 10c are candidates with antidiabetic potential.

Divergent Pd-catalyzed Functionalization of 4-Oxazolin-2-ones and 4-Methylene-2-oxazolidinones and Synthesis of Heterocyclic-Fused Indoles.

Palladium-catalyzed functionalization was presently performed on two building blocks: 4-oxazolin-2-ones and 4-methylene-2-oxazolidinones. Direct Heck arylation of 4-oxazolin-2-ones led to a series of 5-aryl-4-oxazolin-2-ones, including analogues with N-chiral auxiliary, in an almost quantitative yield. The Pd(II)-catalyzed homocoupling reaction of 4-oxazolin-2-ones provided novel heterocyclic across-ring dienes. Meanwhile, the intramolecular cross-coupling of N-aryl-4-methylene-2-oxazolidinones furnished a series of oxazolo[3,4-a]indol-3-ones. Further functionalization of 4-methylene-2-oxazolidinones afforded substituted indoles and heterocyclic-fused indoles with aryl, bromo, carbinol, formyl, and vinyl groups. A computational study was carried out to account for the behavior of the formylated derivatives. The currently developed methodology was applied to a new formal total synthesis of ellipticine.

Antifungal Activity of Fibrate-Based Compounds and Substituted Pyrroles That Inhibit the Enzyme 3-Hydroxy-methyl-glutaryl-CoA Reductase of Candida glabrata (CgHMGR), Thus Decreasing Yeast Viability and Ergosterol Synthesis.

Due to the emergence of multidrug-resistant strains of yeasts belonging to the Candida genus, there is an urgent need to discover antifungal agents directed at alternative molecular targets. The aim of the current study was to evaluate the capacity of three different series of synthetic compounds to inhibit the Candida glabrata enzyme denominated 3-hydroxy-methyl-glutaryl-CoA reductase and thus affect ergosterol synthesis and yeast viability. Compounds 1c (α-asarone-related) and 5b (with a pyrrolic core) were selected as the best antifungal candidates among over 20 synthetic compounds studied. Both inhibited the growth of fluconazole-resistant and fluconazole-susceptible C. glabrata strains. A yeast growth rescue experiment based on the addition of exogenous ergosterol showed that the compounds act by inhibiting the mevalonate synthesis pathway. A greater recovery of yeast growth occurred for the C. glabrata 43 fluconazole-resistant (versus fluconazole-susceptible) strain and after treatment with 1c (versus 5b). Given that the compounds decreased the concentration of ergosterol in the yeast strains, they probably target ergosterol synthesis. According to the docking analysis, the inhibitory effect of 1c and 5b could possibly be mediated by their interaction with the amino acid residues of the catalytic site of the enzyme. Since 1c displayed higher binding energy than α-asarone and 5b, it is the best candidate for further research, which should include structural modifications to increase its specificity and potency. The derivatives could then be examined with in vivo animal models using a therapeutic dose. IMPORTANCE Within the context of the COVID-19 pandemic, there is currently an epidemiological alert in health care services due to outbreaks of Candida auris, Candida glabrata, and other fungal species multiresistant to conventional antifungals. Therefore, it is important to propose alternative molecular targets, as well as new antifungals. The three series of synthetic compounds herein designed and synthesized are inhibitors of ergosterol synthesis in yeasts. Of the more than 20 compounds studied, two were selected as the best antifungal candidates. These compounds were able to inhibit the growth and synthesis of ergosterol in C. glabrata strains, whether susceptible or resistant to fluconazole. The rational design of antifungal compounds derived from clinical drugs (statins, fibrates, etc.) has many advantages. Future studies are needed to modify the structure of the two present test compounds to obtain safer and less toxic antifungals. Moreover, it is important to carry out a more in-depth mechanistic approach.

Cascade synthesis of indolizines and pyrrolo[1,2-a]pyrazines from 2-formyl-1-propargylpyrroles.

A straightforward synthesis of indolizines and pyrrolo[1,2-a]pyrazines was performed through a cascade condensation/cyclization/aromatization reaction of substituted 2-formyl-N-propargylpyrroles with active methylene compounds such as nitromethane, alkyl malonates, methyl cyanoacetate and malononitrile. Under basic conditions, the reaction proceeded satisfactorily to provide the corresponding 6,7-disubstituted indolizines. The condensation of the pyrrolic analogues with ammonium acetate gave rise to pyrrolo[1,2-a]pyrazines in high yields. N-Allenyl-2-formylpyrroles behaved as more reactive substrates than 2-formyl-N-propargylpyrroles, furnishing the expected indolizines in higher yields. Hence, an allenyl-containing intermediate was probably generated as the reactive species in the reaction mechanism of some N-propargyl pyrroles prior to the cyclization reaction.

Three-Component Synthesis of 2-Amino-3-cyano-4H-chromenes, In Silico Analysis of Their Pharmacological Profile, and In Vitro Anticancer and Antifungal Testing.

Chromenes are compounds that may be useful for inhibiting topoisomerase and cytochrome, enzymes involved in the growth of cancer and fungal cells, respectively. The aim of this study was to synthesize a series of some novel 2-amino-3-cyano-4-aryl-6,7-methylendioxy-4H-chromenes 4a-o and 2-amino-3-cyano-5,7-dimethoxy-4-aryl-4H-chromenes 6a-h by a three-component reaction, and test these derivatives for anticancer and antifungal activity. Compounds 4a and 4b were more active than cisplatin (9) and topotecan (7) in SK-LU-1 cells, and more active than 9 in PC-3 cells. An evaluation was also made of the series of compounds 4 and 6 as potential antifungal agents against six Candida strains, finding their MIC50 to be less than or equal to that of fluconazole (8). Molecular docking studies are herein reported, for the interaction of 4 and 6 with topoisomerase IB and the active site of CYP51 of Candida spp. Compounds 4a-o and 6a-h interacted in a similar way as 7 with key amino acids of the active site of topoisomerase IB and showed better binding energy than 8 at the active site of CYP51. Hence, 4a-o and 6a-h are good candidates for further research, having demonstrated their dual inhibition of enzymes that participate in the growth of cancer and fungal cells.

Regioselective Mercury(I)/Palladium(II)-Catalyzed Single-Step Approach for the Synthesis of Imines and 2-Substituted Indoles.

An efficient synthesis of ketimines was achieved through a regioselective Hg(I)-catalyzed hydroamination of terminal acetylenes in the presence of anilines. The Pd(II)-catalyzed cyclization of these imines into the 2-substituted indoles was satisfactorily carried out by a C-H activation. In a single-step approach, a variety of 2-substituted indoles were also generated via a Hg(I)/Pd(II)-catalyzed, one-pot, two-step process, starting from anilines and terminal acetylenes. The arylacetylenes proved to be more effective than the alkyl derivatives.

Highly selective Diels-Alder and Heck arylation reactions in a divergent synthesis of isoindolo- and pyrrolo-fused polycyclic indoles from 2-formylpyrrole.

A highly regio-, chemo- and stereoselective divergent synthesis of isoindolo- and pyrrolo-fused polycyclic indoles is herein described, starting from 2-formylpyrrole and employing Diels-Alder and Heck arylation reactions. 3-(N-Benzyl-2-pyrrolyl)acrylates and 4-(pyrrol-2-yl)butenones underwent a highly endo-Diels-Alder cycloaddition with maleimides to furnish octahydropyrrolo[3,4-e]indoles, which served as precursors in the regioselective synthesis of aza-polycyclic skeletons via an intramolecular Heck arylation reaction. Through the latter reaction, the 3-(N-benzyl-2-pyrrolyl)acrylates give rise to 3-(pyrrolo[2,1-a]isoindol-3-yl)acrylates. A further oxidative aromatization of the polycyclic intermediates provides the corresponding polycyclic pyrrolo-isoindoles and isoindolo-pyrrolo-indoles. A theoretical study on the stereoselective Diels-Alder reactions, carried out by calculating the endo/exo transition states, revealed the assistance of non-covalent interactions in governing the endo stereocontrol.

Selective synthesis of trisubstituted pyrroles through the reactions of alkynyl Fischer carbene complexes with oxazolones.

An efficient and simple synthesis of novel trisubstituted 1H-pyrroles 4a-qvia 1,3-dipolar cycloaddition of Δ3-trifluoromethyloxazolones 2a-d with both chromium and tungsten alkynyl Fischer carbene complexes (1a-h) is described. An unexpected and unreported -CF3 group elimination process was observed in the pyrrole structure. Our experimental and theoretical data suggested that the metal fragment may be responsible for this phenomenon. The dipolar cycloaddition proceeded efficiently to produce a single regioisomer, which was unambiguously established through NMR and single-crystal X-ray diffraction studies. Nevertheless, the reaction of alkynyl carbenes bearing an α,ß,γ,δ-unsaturated moiety with excess oxazolone 2a produced a polycyclic compound 6 speculatively formed through a cascade reaction involving 1,6-, 1,4- and 1,2-nucleophilic addition steps.

Enantiopure 4-oxazolin-2-ones and 4-methylene-2-oxazolidinones as chiral building blocks in a divergent asymmetric synthesis of heterocycles.

Enantiopure 3-((R)- and 3-((S)-1-phenylethyl)-4-oxazoline-2-ones were evaluated as chiral building blocks for the divergent construction of heterocycles with stereogenic quaternary centers. The N-(R)- or N-(S)-1-phenylethyl group of these compounds proved to be an efficient chiral auxiliary for the asymmetric induction of the 4- and 5-positions of the 4-oxazolin-2-one ring through thermal and MW-promoted nucleophilic conjugated addition to Michael acceptors and alkyl halides. The resulting adducts were transformed via a cascade process into fused six-membered carbo- and heterocycles. The structure of the reaction products depended on the electrophiles and reaction conditions used. Alternative isomeric 4-methylene-2-oxazolidinones served as chiral precursors for a versatile and divergent approach to highly substituted cyclic carbamates. DFT quantum calculations showed that the formation of bicyclic pyranyl compounds was generated by a diastereoselective concerted hetero-Diels-Alder cycloaddition.

Novel prenyloxy chalcones as potential leishmanicidal and trypanocidal agents: Design, synthesis and evaluation.

The available drugs for treating Leishmaniasis and American trypanosomiasis have high toxicity and multiple side effects, among other problems. More effective and less toxic treatments are urgently needed. A series of chalcones that contained a prenyloxy or geranyloxy substituent was synthesized and characterized. Each substituent was attached to the A ring in some compounds and to the B ring in others, with additional substituents placed on the chalcone moiety. The present aim was to evaluate the effect of the substitution pattern on leishmanicidal and trypanocidal activity. When tested at a single concentration, the compounds exerting a metabolic inhibition close to or exceeding 50% for Leishmania mexicana were 11, 17 and 12, and for Trypanosoma cruzi were 11, 17, 15 and 26. Upon determining the selectivity index (SI =IC50/CC50), the values were 80.9, 1.24 and 55.12 for 11, 17 and 12 (respectively) versus L. mexicana, and 75.1, 1.43, 27.36 and 33.52 for 11, 17, 15 and 26 (respectively) versus T. cruzi. Structural isomers 11 and 17 showed activity for both the L. mexicana and T. cruzi strains, though the greater cytotoxic activity of 17 led to a lower SI. Compounds 12, 15 and 26 were species specific. For T. cruzi, the SI was higher for 11, 15 and 26 than for the reference drugs nifurtimox and benznidazole. The examination of promastigote morphology after exposing L. mexicana and T. cruzi to 11 revealed a decrease in cell density. The current findings suggest that 11 could be a useful lead compound for further SAR studies.

Simvastatin and other inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase of Ustilago maydis (Um-Hmgr) affect the viability of the fungus, its synthesis of sterols and mating.

BACKGROUND: The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgr) catalyzes the synthesis of mevalonate, a key compound for the synthesis of cholesterol in humans and ergosterol in fungi. Since the Hmgr enzymes of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida glabrata are similar to the Hmgr enzymes of mammals, fungal Hmgr enzymes have been proposed as a model for studying antifungal agents. AIMS: To examine the correlation between inhibiting Um-Hmgr enzyme and the viability, sterols synthesis and mating in Ustilago maydis. METHODS: Using in silico analysis, the ORF codifying for Um-Hmgr was identified and the protein characteristics were deduced. The effect of the competitive inhibitors of Um-Hmgr on the viability of this basidiomycota, the synthesis of its sterols, and its mating were evaluated. RESULTS: The Umhmgr gene (XP_011389590.1) identified putatively codifies a protein of 1443 aa (ca. MW=145.5kDa) that has a possible binding domain in the endoplasmic reticulum (ER) and high identity with the Hmgr catalytic domain of humans and other yeasts. The inhibition of Um-Hmgr caused a decrease of viability and synthesis of sterols, and also the inhibition of mating. The activity of Um-Hmgr is mainly located in the membrane fraction of the fungus. CONCLUSIONS: Given our results we believe U. maydis is a valid model for studying synthetic inhibitors with lipid-lowering or antifungal activity. Additionally, we propose the Hmgr enzyme as an alternative molecular target to develop compounds for treating both phytopathogenic and pathogenic human fungi.

Inhibition of recombinant enzyme 3-hydroxy-3-methylglutaryl-CoA reductase from Candida glabrata by α-asarone-based synthetic compounds as antifungal agents.

Due to increasing resistance of Candida species to antifungal drugs, especially azoles, new drugs are needed. The proposed compounds 3 and 4 are analogous to α-asarone (2), a naturally occurring potent inhibitor of HMGR with hypolipidemic and antifungal activity. We used the recombinant enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase of Candida glabrata (CgHMGR) as a model to test the effectiveness of the test compounds. Compounds 3 and 4 demonstrated inhibitory kinetics, having lower IC50 values (42.65 µM and 28.77 µM, respectively) than compound 2 (>100 µM). The docking studies showed better binding energies for compounds 3 and 4 (-5.35 and -6.1 kcal/mol, respectively) than for compound 2 (-4.53 kcal/mol). These findings suggest that the tested compounds are better than their natural analogue. Plaque assays were performed on the C. glabrata strain CBS138 by applying ergosterol or cholesterol to evaluate the possible reversal of the inhibition induced by compounds 2, 3 and 4. Inhibition was easily suppressed in all three cases, recovering the viability of C. glabrata. These results reveal that the CgHMGR model is excellent for testing antifungals. Compound 4 produced the best effect and is herein proposed as a new potent antifungal agent.

Synthesis of exo-Imidazolidin-2-one Dienes, Their Isomerization, and Selectivity in Diels-Alder Cycloadditions.

An efficient and alternative synthesis of exo-imidazolidin-2-one dienes is described. A condensation reaction was carried out with bis-imino derivatives, diacetyl, and triphosgene, affording symmetrically N, N-disubstituted dienes. The use of alkyl methyl α-diketones led to the formation of nonsymmetrical dienes, which underwent isomerization to provide more stable inner-outer-ring dienes under Lewis acid conditions. Evaluation was made of the reactivity as well as regio- and stereoselectivity of these dienes in Diels-Alder reactions. They proved to be highly reactive and selective. DFT calculations of the transition states accounted for their behavior.

Synthesis of Polyheterocyclic Pyrrolo[3,4-b]pyridin-5-ones via a One-Pot (Ugi-3CR/aza Diels-Alder/N-acylation/aromatization/SN2) Process. A Suitable Alternative towards Novel Aza-Analogues of Falipamil.

We describe the one-pot synthesis of twenty polyheterocyclic pyrrolo[3,4-b]pyridin-5-ones via a cascade process (Ugi-3CR/aza Diels-Alder/N-acylation/aromatization) in 20 to 95% overall yields, as well as four pharmacologically promising analogues via an improved cascade process (Ugi-3CR/aza Diels-Alder/N-acylation/aromatization/SN2): two piperazine-linked pyrrolo[3,4-b]pyridin-5-ones in 33 and 34%, and a couple of Falipamil aza-analogues in 30 and 35% overall yields. It is worth highlighting the good substrate scope found, because final products are furnished with alkyl, aryl, and heterocyclic substituents. The use of chain-ring tautomerizable isocyanides (as key reagents for the Ugi-type three component reaction) allowed for a rapid and efficient assembly of the polysubstituted oxindoles, which were used in situ toward the complex products, conferring features like robustness, sustainability, and the one-pot approach to this synthetic methodology.

Theoretical study of the regioselective cyclization of enaminones in the construction of benzofurans and indoles.

A theoretical study was undertaken regarding the regioselective Lewis acid-promoted intramolecular cyclization of novel enaminones 1-3 leading to the corresponding benzofurans 4-5 and indoles 6. The density functional theory (DFT) and hard and soft acids and bases (HSAB) principle provided data to describe the electronic effects of the substituents in the reactivity of the benzene ring and the enaminone moiety. The condensed and local Fukui functions for nucleophilic and electrophilic attacks of the reactants accounted for the experimentally observed preference, in regard to precursors 1-3, of the cyclization between the C6' carbon (rather than the C2' carbon) of the benzene ring and the C3 center of the enaminone moiety.

Condensation of Diacetyl with Alkyl Amines: Synthesis and Reactivity of p-Iminobenzoquinones and p-Diiminobenzoquinones.

Condensation reactions between diacetyl and α-branched primary alkylamines under mild and neutral conditions provided a mixture of 2,5-dimethylbenzoquinone(alkylimines), 2,5-dimethylbenzoquinone(bis-alkyldiimines), and N,N'-dialkyl-2,5-dimethylbenzene-1,4-diamines, which were efficiently separated as pure products by column chromatography. Both 2,5-dimethylbenzoquinone(alkylimines) and 2,5-dimethylbenzoquinone(bis-alkyldiimines) underwent an interchange of the alkylimino group when treated with anilines, followed by reductive aromatization, to provide diarylamines and 1,4-dianilinobenzenes, respectively. Evaluation was also made of the reactivity and selectivity of these compounds in the presence of anilines, thiophenols and alkylhalides.

Synthesis of novel polysubstituted N-benzyl-1H-pyrroles via a cascade reaction of alkynyl Fischer carbenes with α-imino glycine methyl esters.

An efficient and simple synthesis of novel and densely substituted N-benzyl-1H-pyrroles 6a-r is described by a 1,4-addition/isomerization/ring closure/demetalation cascade process of alkynyl Fischer carbene complexes 1a-f and 2a and α-imino glycine methyl esters 3a, b, d, g, h, and k promoted with LDA.