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.

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.

Molecular Recognition of Citroflavonoids Naringin and Naringenin at the Active Site of the HMG-CoA Reductase and DNA Topoisomerase Type II Enzymes of Candida spp. and Ustilago maydis.

Two agents from natural sources, citroflavonoids naringin and naringenin, can target enzymes in pathogenic yeasts responsible for hospital infections and crop failure. The aim of this study was to examine the molecular recognition site for naringin and naringenin on the HMGR and TOPOII enzymes of eleven Candida species and one phytopathogen, U. maydis, and evaluate yeast susceptibility to these flavonoids. The HMGR and TOPOII enzymes were analyzed in silico. The alignment of the two enzymes in the twelve pathogenic organisms with the corresponding enzyme of Homo sapiens revealed highly conserved amino acid sequences. Modeling studies of the enzymes indicated highly conserved structures. According to molecular docking simulations, both citroflavonoids recognized the amino acid residues of the active site of the enzymes. Binding energy values were higher for naringin (-10.75 and -9.38 kcal/mol, respectively) than simvastatin on HMGR (-9.9) and curcumin on TOPOII (-8.37). The appraisal of twenty-nine virtual mutations provided evidence of probable mechanisms of resistance (high binding energy) or susceptibility (low energy) to the drugs and emphasized the role of key residues. An in vitro susceptibility evaluation of the twelve yeasts demonstrated that the two flavonoids have similar or better MIC values than those reported for the reference compounds, obtaining the lowest with Candida dubliniensis (2.5 µg/ml) and U. maydis (5 µg/ml). Based on the present findings, naringin and naringenin could possibly be effective for treating diseases caused by pathogenic yeasts of the Candida species and U. maydis, presumably by inhibition of their HMGR and TOPOII enzymes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00980-0.

Inhibitors of DNA topoisomerases I and II applied to Candida dubliniensis reduce growth, viability, the generation of petite mutants and toxicity, while acting synergistically with fluconazole.

The increasing resistance of Candida species to azoles emphasizes the urgent need for new antifungal agents with novel mechanisms of action. The aim of this study was to examine the effect of three DNA topoisomerase inhibitors of plant origin (camptothecin, etoposide and curcumin) on the growth of Candida dubliniensis. The phylogenetic analysis showed a close relationship between the topoisomerase enzymes of C. dubliniensis and Candida albicans. The alignment of the amino acid sequences of topoisomerase I and II of yeasts and humans evidenced conserved domains. The docking study revealed affinity of the test compounds for the active site of topoisomerase I and II in C. dubliniensis. Curcumin and camptothecin demonstrated a stronger in vitro antifungal effect than the reference drugs (fluconazole and itraconazole). Significant synergistic activity between the topoisomerase inhibitors and fluconazole at the highest concentration (750 µM) was observed. Fluconazole induced the petite phenotype to a greater degree than the topoisomerase inhibitors, indicating a tendency to generate resistance. Lower toxicity was found for such inhibitors versus reference drugs on Galleria mellonella larva. The topoisomerase inhibitors exhibited promising antifungal activity, and the DNA topoisomerase enzymes of C. dubliniensis proved to be an excellent model for evaluating new antifungal compounds.

Etoposide and Camptothecin Reduce Growth, Viability, the Generation of Petite Mutants, and Recognize the Active Site of DNA Topoisomerase I and II Enzymes in Candida glabrata.

Candidemia, one of the most common invasive fungal infections in hospitalized patients, can lead to death and huge financial losses. Candida albicans is the main causative agent of this disorder and Candida glabrata occupies the second or third place, for which new therapeutic alternatives must be found. The objective of the present study was to evaluate the inhibitory effect of etoposide and camptothecin (inhibitors of deoxyribonucleic acid (DNA) topoisomerase) on the C. glabrata CBS138 strain. Etoposide and camptothecin showed better or similar MIC (minimum inhibitory concentration) (5 and 2.5 µg/mL, respectively), with respect to fluconazole (8 µg/mL) and itraconazole (4 µg/mL). They also suppressed colony formation during the 12-h test. On the other hand, petite colonies were less formed by exposing C. glabrata to etoposide or camptothecin (indicating low toxicity), with respect fluconazole and itraconazole. Such colonies are phenotypically observed as limited growth in medium containing a non-fermentable carbon source, and are genotypically characterized by a partial or total loss of mitochondrial DNA (mtDNA) fragments. Using PCR techniques and cell staining with 4',6-diamidino-2-phenylindole (DAPI), loss of mtDNA was detected only in yeast cells treated with fluconazole. Additionally, molecular docking studies with etoposide and camptothecin showed recognition in the active site of the Topo I and II enzymes from C. glabrata. Since etoposide and camptothecin showed good inhibitory activity and low toxicity on C. glabrata; they should certainly be of interest for the treatment of C. glabrata infections and the design and development of new antifungal compounds derived from these drugs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00942-6.

Synthesis and highly potent anti-inflammatory activity of licofelone- and ketorolac-based 1-arylpyrrolizin-3-ones.

Since NSAIDs are commonly used anti-inflammatory agents that produce adverse effects, there have been ongoing efforts to develop more effective and less toxic compounds. Based on the structure of the anti-inflammatory pyrrolizines licofelone and ketorolac, a series of 1-arylpyrrolizin-3-ones was synthesized. Also prepared was a series of substituted pyrroles, mimicking similar known anti-inflammatory agents. The anti-inflammatory activity of the test compounds was determined with a phorbol ester (TPA)-induced murine ear edema protocol. For the most active derivatives, 19b-c/20b-c, the anti-inflammatory effect was the same as that of the reference compound (indomethacin) and was dose-dependent. These compounds have an aryl ring at the C-1 position and a methoxycarbonyl group at the C-2 position of the pyrrolizine framework, which represent plausible pharmacophore groups with anti-inflammatory activity. The anti-inflammatory activity of 1-substituted analogs containing a five- or six-membered heterocycles was lower but still good, while that of the pyrroles was only moderate. Although the docking studies suggests that the effect of analogs 19a-c/20a-c is associated with the inhibition of cyclooxygenase-2, experimental assays did not corroborate this idea. Indeed, a significant inhibition of NO was found experimentally as a plausible mechanism of action.

Dibutyltin(IV) Complexes Derived from L-DOPA: Synthesis, Molecular Docking, Cytotoxic and Antifungal Activity.

A series of organotin(IV) complexes was herein prepared and characterized. A one-pot synthetic strategy afforded reasonable to high yields, depending on the nature of the ligand. All new complexes were fully characterized by spectroscopic techniques, consisting of IR, MS and NMR (1H, 13C and 119Sn). The in vitro cytotoxicity tests demonstrated that the organotin complexes produced a greater inhibition, versus cisplatin (the positive control), of the growth of six human cancer cell lines: U-251 (glioblastoma), K-562 (chronic myelogenous leukemia), HCT-15 (colorectal), MCF-7 (breast), MDA-MB-231 (breast) and SKLU-1 (non-small cell lung). The potency of this cytotoxic activity depended on the nature of the substituent bonded to the aromatic ring. All complexes exhibited excellent IC50 values. The test compounds were also screened in vitro for their antifungal effect against Candida glabrata and Candida albicans, showing minimum inhibitory concentration (MIC) values lower than those obtained for fluconazole. A brine shrimp bioassay was performed to examine the toxic properties. Molecular docking studies demonstrated that the organotin(IV) complexes bind at the active site of topoisomerase I in a similar manner to topotecan, sharing affinity for certain amino acid side chains (Ile535, Arg364 and Asp533), as well as for similar DNA regions (DA113, DC112 and DT10).

Synthesis, Molecular Docking, and Antimycotic Evaluation of Some 3-Acyl Imidazo[1,2-a]pyrimidines.

A series of 3-benzoyl imidazo[1,2-a]pyrimidines, obtained from N-heteroarylformamidines in good yields, was tested in silico and in vitro for binding and inhibition of seven Candida species (Candida albicans (ATCC 10231), Candida dubliniensis (CD36), Candida glabrata (CBS138), Candida guilliermondii (ATCC 6260), Candida kefyr, Candida krusei (ATCC 6358) and Candida tropicalis (MYA-3404)). To predict binding mode and energy, each compound was docked in the active site of the lanosterol 14α-demethylase enzyme (CYP51), essential for fungal growth of Candida species. Antimycotic activity was evaluated as the 50% minimum inhibitory concentration (MIC50) for the test compounds and two reference drugs, ketoconazole and fluconazole. All test compounds had a better binding energy (range: -6.11 to -9.43 kcal/mol) than that found for the reference drugs (range: 48.93 to -6.16 kcal/mol). In general, the test compounds showed greater inhibitory activity of yeast growth than the reference drugs. Compounds 4j and 4f were the most active, indicating an important role in biological activity for the benzene ring with electron-withdrawing substituents. These compounds show the best MIC50 against C. guilliermondii and C. glabrata, respectively. The current findings suggest that the 3-benzoyl imidazo[1,2-a]pyrimidine derivatives, herein synthesized by an accessible methodology, are potential antifungal drugs.

An Efficient Strategy for the Synthesis of 1-(Trifluoromethylsulfonamido)propan-2-yl Esters and the Evaluation of Their Cytotoxic Activity.

An efficient method for the synthesis of 1-(trifluoromethylsulfonamido)propan-2-yl benzoates is described, the products of the reaction were characterized by heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple bond correlation (HMBC) and NMR experiments. The overall process began with the activation of the oxazoline ring by triflic anhydride, followed by the opening of the five-membered ring in the 5-methyl-2-phenyl-4,5-dihydrooxazole system. The cytotoxic activity of the new trifluoromethyl sulfonamides was evaluated with six cancer cell lines and human gingival fibroblasts, posteriorly analyzing the influence on cytotoxicity exerted by the withdrawing and donor substituents at the para-position of the phenyl ring. Compounds 3b-e showed cytotoxic activity, with IC50 values ranging from 17-17.44 µM for the cell lines tested, finding the highest effect for compound 3e.

New Organotin (IV) Compounds Derived from Dehydroacetic Acid and Thiosemicarbazides: Synthesis, Rational Design, Cytotoxic Evaluation, and Molecular Docking Simulation.

Organotin complexes were prepared through a one-pot reaction with three components by reacting thiosemicarbazide or 4-methyl-3-thiosemicarbazide or 4-phenylthiosemicarbazide, dehydroacetic acid (DHA) and dibutyl, diphenyl, dicyclohexyl, and bis[(trimethylsilyl)methyl]tin(IV) oxides; all complexes were characterized by infrared (IR), ultraviolet-visible (UV-vis), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. The 119Sn NMR revealed chemical shifts corresponding to a pentacoordinated environment in solution. The X-ray crystallography of the two complexes evidenced the formation of monomeric complexes with a pentacoordinated geometry around tin via three donor atoms from the ligand, the sulfur of the thiol, the nitrogen of the imine group, and the oxygen of the pyran ring. The geometries of the five-coordinated complexes 3a (Bu2SnL3), 3c (Ph2SnL3), and 3d (Cy2SnL3) acid were intermediate between square pyramidal and trigonal bipyramidal, and complex 1a (Bu2SnL1) adopted a bipyramidal trigonal geometry (BPT). The sulforhodamine B assay assessed the cytotoxicity of organotin(IV) complexes against the MDA-MB-231 and MCF-7 (human mammary adenocarcinoma) cell lines and one normal COS-7 (African green monkey kidney fibroblast). The IC50 values evidenced a significant antiproliferative effect on cancer cells; the complexes were more potent than the positive cisplatin control and the corresponding ligands, dehydroacetic acid thiosemicarbazone (L1), dehydroacetic acid-N(4)-methylthiosemicarbazone (L2), and dehydroacetic acid-N(4)-phenylthiosemicarbazone (L3). The IC50 values also indicated that the organotin(IV) complexes were more cytotoxic against the triple-negative breast cell line MDA-MB-231 than MCF-7, inducing significant morphological alterations. The interactions of organotin(IV) 1c (Ph2SnL1), 1d (Cy2SnL1), and 1e (((CH3)3SiCH2)2SnL1) were evaluated with ss-DNA by fluorescence; intensity changes of the fluorescence were indicative of the displacement of ethidium bromide (EB), confirming the interaction of the organotin(IV) complexes with ss-DNA; the results showed a DNA binding affinity. The thermodynamic parameters obtained through isothermal titration calorimetry showed that the interaction of 1c (Ph2SnL1), with ss-ADN, was exothermic. Molecular docking studies also demonstrated that the organotin(IV) complexes were intercalated in DNA by conventional hydrogen bonds, carbon-hydrogen bonds, and π-alkyl interactions. These complexes furthermore showed a greater affinity towards DNA than cisplatin.

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.