Fernando Carrasco

Background: The study of lichen species has accelerated significantly due to their promising potential as sources of novel bioactive compounds. Although usnic acid (UA) and atranorin (ATR) are well-documented as major metabolites in several lichen species, the biological activities exhibited by most studied species appear to derive from a synergistic effect among these compounds and other secondary metabolites. This work evaluates the antimicrobial and antioxidant activities of eight lichen extracts obtained with a MeOH: (CH₃)₂CO (1:1) mixture, providing a comprehensive overview of their biological profile and metabolite content (UA and ATR). Methods: HPLC-DAD was used to quantify UA and ATR in the studied extracts. Antimicrobial activity against Staphylococcus aureus , Escherichia coli and Candida spp. was evaluated using disk diffusion and broth microdilution assays. Different Antioxidant capacities were observed through DPPH radical scavenging (1364–2461 µg/mL), ABTS cation decolorization (11.5–15.9 mg TE/g) and ferric reducing antioxidant power (FRAP) assays (169-719.79 µM Fe²⁺/g), while total phenolic content (TPC) was determined using the Folin–Ciocalteu method (11.34–332.15 µg GAE/g). Results: Eight lichen species collected from San Cristóbal de Huascoy (Huaral Province) were evaluated. In all species under investigation, UA and ATR concentrations ranged from 0.024 to 1.2% and 0.018–0.46%, respectively. The lichen extracts exhibited significant antibacterial action against four Staphylococcus aureus strains (MIC = 0.1–0.35 mg/mL), showing a potency comparable to Gentamicin (0.1 mg/mL) but lower than standard UA (0.02 mg/mL). Furthermore, all extracts demonstrated to have a moderate antifungal activity against the evaluated Candida strains (MIC = 0.4–1.0 mg/mL). Given that both MBC/MIC and MFC/MIC ratios were below 4, the extracts were classified as bactericidal and fungicidal, respectively. Conclusion: Among all lichens tested, the Alectoria ochroleuca extract exhibited the second-highest total phenolic content (TPC) and moderate antioxidant performance. Overall, all tested lichen extracts displayed moderate antimicrobial and antioxidant profiles across all assays. The mixture of soluble metabolites in the MeOH: (CH₃)₂CO (1:1) extract, including UA and ATR, may exerts a synergistic effect driving the observed biological activities. Future studies isolating individual metabolites and evaluating their in vivo bioactivity are needed to confirm the pharmacological relevance of these lichen extracts.

In this study, sixteen secondary metabolites, including two chromones, four dibenzofurans, three lipids, three depsides, two aromatic compounds, a quinone, and a terpene, were detected in the methanol:acetone (1:1 v/v) extract of the lichen Hypotrachyna enderythraea (Zahlbr.) Hale, using High-Performance Liquid Chromatography coupled to Orbitrap Electrospray Ionization tandem Mass Spectrometry (HPLC-Orbitrap ESI tandem MS/MS). These metabolites were characterized by analysis of their exact molecular masses and corresponding fragmentation patterns. The retention times of the identified metabolites were compared with those of standard compounds, confirming the presence of naturally occurring bioactive compounds. Density Functional Theory (DFT) calculations were employed to investigate preferential deprotonation sites in representative polyprotic metabolites. All these findings may contribute to expanding the spectrum of compounds identified within the genus Hypotrachyna and to evaluating their potential biological activities.

Six new phenyl‐1 H ‐pyrazole‐carbaldehyde isonicotinylhydrazone derivatives 1-10 were synthesized and characterized by spectroscopic (FT‐IR, 1H NMR, and 13C NMR) techniques and electrospray ionization–mass spectrometry (ESI–MS). Homonuclear data from 2D‐NMR ( ¹ H‐ ¹ H NOESY) revealed that 3 and 5 adopt the transE isomeric form. The in vitro antitubercular activity of all the synthesized compounds was determined against Mycobacterium tuberculosis (sensitive H37RV and resistant TB DM97). With respect to the isoniazid (INH) standard drug (MIC = 0.91 μM), all the prepared compounds showed greater antitubercular activity with MIC values in the range of 0.38–0.82 μM against Mtb H37Rv‐sensitive strain. However, the preliminary results indicated that Compounds 1 – 10 tested against the TB DM97‐resistant strain showed low bioactivity. The optimized geometry of Compounds 1 – 10 has been determined by density functional theory (DFT) calculations. For Compounds 1 – 6 , the most stable isomers (in DMSO) exhibited an E geometrical configuration, while Compounds 7–10 exhibited a Z configuration. The global reactivity values indicate that Compounds 1 – 10 are slightly more reactive and less stable than the standard (INH). Additionally, they exhibit a strong binding capacity to biomolecules, comparable to INH. The MEP map reveals that the negative and positive potential sites are predominantly located on the C=O and HN–NH groups, respectively, similar to those observed for INH. Drug‐likeness and ADME properties were evaluated. All compounds showed adequate ADME properties and are drug‐like. Molecular docking studies (Compounds 1 – 10 ) into the active site of mycobacterial InhA showed an acceptable dissociation constant with improved binding energy (from −6.7 to −8.1 kcal mol ⁻¹ ) compared to isoniazid (−5.6 kcal mol ⁻¹ ). Molecular dynamics simulations revealed that the molecules cmp3 (3) and cmp5 (5) form stable ternary complexes with InhA through persistent hydrogen bonding and hydrophobic interactions. These compounds showed reduced active‐site fluctuations and superior binding free energies compared to INH. The findings represent a significant advancement in the discovery of potential antitubercular agents.

Introduction: Lichens are complex organisms conformed by a symbiotic association between algae and fungi; they are haven considerable interest owing to their pharmacological properties. Objectives: To determine chemical composition of the fatty acid (FA) and the percentage of usnic acid (UA), and to evaluate the in vitro antibacterial activity of the ethanolic extract obtained from the lichens Usnea sp., Thamnolia vermicularis subsp. solida and Ramalina asperula. Materials and Methods: Organic extracts from the three lichens were obtained using a rotary evaporator, soxhlet extractor and ultrasound equipment. The chemical composition of FA was determined by gas chromatography-flame ionisation detector, and the percentage of UA was quantified by UV-Vis spectroscopy. The in vitro antibacterial activity of the lichen ethanolic extracts was evaluated against E. coli ATCC 25922,Bacillus subtilis ATCC 11774, Staphylococcus aureus ATCC 43300 and S. aureus ATCC 29213 using the diffusion and microdilution methods. The minimum inhibitory concentration (MIC) was determined. Results: In the three tested lichens, the identified FAs were palmitic, stearic, oleic, linoleic, and linolenic acids; and the obtained percentage of UA was between 0.2 and 1.07 %. All the ethanolic extracts obtained from three lichens showed a high activity against B. subtilis (MIC = 0.5 mg/mL), while the ethanolic extracts obtained from T. vermicularis subsp. solida and R. asperula showed moderate activity against S. aureus ATCC 43300 (MIC = 3 and 1 mg/mL) and S. aureus ATCC29213 (MIC = 4 and 1 mg/mL). Conclusions: Our findings found for the three studied lichens confirmed the presence of some FAs and AU. These species provide an acceptable nutritional content and besides they can be considered as potential antibacterial agents.

Six novel phenylisoxazole semicarbazone derivatives 1–6 were synthesized by reaction of the corresponding phenylisoxazole‐3/5‐carbaldehyde derivatives with semicarbazide hydrochloride. The synthesized compounds were characterized by ESI‐MS, FT‐IR, and NMR (¹H, ¹³C) spectroscopic techniques. The two‐dimensional ¹H‐¹H NOESY NMR (in acetone‐d₆) data revealed that compound 1 exists in the E isomeric form. The computational study of the energetic, structural, and electronic properties, carried out at B3LYP/6‐311G++(d,p) level of theory, showed that the most stable conformer for the all synthesized compounds, in both gas and liquid (acetone and DMSO) phases, has a cisE geometrical configuration. This evidence found is in good agreement with the spectrometric results. The geometrical parameters, frontier molecular orbital (FMO), molecular electrostatic potential (MEP), Mulliken atomic charges, and natural bonding orbital (NBO) analysis were also performed at the same level of theory. Taking into account the relative enthalpies ΔH computed, we can establish for tautomeric structures of each of the compounds, the following stability order: I (cisE) > II (E′E) > III (cisE). The MEP descriptors indicate that the oxygen atom of the carbonyl group C=O is susceptible to electrophilic attack, while the hydrogen atoms of the amide and hydrazone fragments are sensitive to nucleophilic attack. The calculated HOMO‐LUMO gap energies E_g indicate that 5 (in gas phase) and 6 (in liquid phase) are the most stable and less reactive compounds, while 1 is the less stable and the most reactive compound. From the NBO analysis, it becomes evident that the presence of the hydrazone fragment produces stabilizing effects due to hyperconjugative interactions.

Ten new thiosemicarbazone derivatives, furan-2-carbaldehyde thiosemicarbazone (1), 3-methyl-furan-2-carbaldehyde thiosemicarbazone (2), 5-hydroxymethyl-furan-2-carbaldehyde thiosemicarbazone (3), 5-trifluoromethyl-furan-2-carbaldehyde thiosemicarbazone (4), 5-nitro-furan-2-carbaldehyde thiosemicarbazone (5), 5-phenyl-furan-2-carbaldehyde thiosemicarbazone (6), 5-(2-fluorophenyl)-furan-2-carbaldehyde thiosemicarbazone (7), 5-(4-methoxyphenyl)-furan-2-carbaldehyde thiosemicarbazone (8), 5-(1-naphthyl)-furan-2-carbaldehyde thiosemicarbazone (9), and 5-(1H-Pyrazol-5-yl)-furan-2-carbaldehyde thiosemicarbazone (10) were synthesized by condensing thiosemicarbazide with the respective furan-2-carbaldehyde in methanol. The prepared compounds were characterized by spectroscopic studies (FT-IR and NMR) and electrospray mass spectrometry. The molecular structures of 2, 6, 7, and 8 have also been determined by X-ray crystallography. Compounds 2, 6, and 7 crystallize in the E conformation about the N1-C6, N1-C11, and N1-C11 bonds, respectively, while 8 adopts the Z conformation about the N1-C12 bond with the presence of an intramolecular N2-HO2 hydrogen bond. All prepared thiosemicarbazone derivatives were evaluated for their in vitro antibacterial, antifungal, and antitumor activities against Staphylococcus aureus strains, Candida albicans/Candida tropicalis fungi, and seven human tumor cell lines (HuTu80, H460, DU145, M-14, HT-29, MCF-7, and LNCaP), respectively. The antioxidant activity was also studied by the DPPH assay. Compound 5 exhibited significant antibacterial activity against Staphylococcus aureus ATCC700699 (MIC=1μg/mL) compared to the nitrofurantoin and gentamicin reference drugs (MIC=125 and 10->100μg/mL, respectively). Compound 4 was ten times less active than amphotericin B (MIC=5μg/mL) against Candida albicans (ATCC90028 and ATCC10231), while 1 exhibited a moderate effect of scavenging of DPPH radical (IC₅₀=40.9μg/mL) in comparison to ascorbic acid reference compound (IC₅₀=22.0μg/mL). Among all the studied thiosemicarbazones, 5 showed a higher cytotoxic activity (IC₅₀=13.3627.73μ) in relation to the other tested compounds (IC₅₀=34.84>372.34μ) against all tested cell lines, except the LNCaP cell line, exhibiting its highest antiproliferative activity (IC₅₀=13.36μ) on the HuTu80cell line. Besides, 8 and 9 exhibited high antitumor activity (IC₅₀=13.31 and 7.69μ, respectively) against the LNCaP cells.

Eight new phenylisoxazole isoniazid derivatives, 3-(2-fluorophenyl)isoxazole-5-carbaldehyde isonicotinylhydrazone (1), 3-(2-methoxyphenyl)isoxazole-5-carbaldehyde isonicotinylhydrazone (2), 3-(2-chlorophenyl)isoxazole-5-carbaldehyde isonicotinylhydrazone (3), 3-(3-clorophenyl)isoxazole-5-carbaldehyde isonicotinylhydrazone (4), 3-(4-bromophenyl)isoxazole-5-carbaldehyde isonicotinylhydrazone (5), 5-(4-methoxiphenyl)isoxazole-3-carbaldehyde isonicotinylhydrazone (6), 5-(4-methylphenyl)isoxazole-3-carbaldehyde isonicotinylhydrazone (7), and 5-(4-clorophenyl)isoxazole-3-carbaldehyde isonicotinylhydrazone (8), have been synthesized and characterized by FT-IR, ¹H-NMR, ¹³C-NMR, and mass spectral data. The 2D NMR (¹H-¹H NOESY) analysis of 1 and 2 confirmed that these compounds in acetone-d₆ are in the trans(E) isomeric form. This evidence is supported by computational calculations which were performed for compounds 18, using DFT/B3LYP level with the 6-311++G(d,p) basis set. The in vitro antituberculous activity of all the synthesized compounds was determined against the Mycobacterium tuberculosis standard strains: sensitive H37Rv (ATCC-27294) and resistant TB DM97. All the compounds exhibited moderate bioactivity (MIC=0.340.41μM) with respect to the isoniazid drug (MIC=0.91μM) against the H37Rv sensitive strain. Compounds 6 (X=4-OCH₃) and 7 (X=4-CH₃) with MIC values of 12.41 and 13.06μM, respectively, were about two times more cytotoxic, compared with isoniazid, against the resistant strain TB DM97.

Eight new thiosemicarbazone derivatives, 6-(1-trifluoroethoxy)pyridine-3-carbaldehyde thiosemicarbazone (1), 6-(4′-fluorophenyl)pyridine-3-carbaldehyde thiosemicarbazone (2), 5-chloro-pyridine-3-carbaldehyde thiosemicarbazone (3), 2-chloro-5-bromo-pyridine-3-carbaldehyde thiosemicarbazone (4), 6-(3′,4′-dimethoxyphenyl)pyridine-3-carbaldehyde thiosemicarbazone (5), 2-chloro-5-fluor-pyridine-3-carbaldehyde thiosemicarbazone, (6), 5-iodo-pyridine-3-carbaldehyde thiosemicarbazone (7), and 6-(3′,5′-dichlorophenyl)pyridine-3-carbaldehyde thiosemicarbazone (8) were synthesized, from the reaction of the corresponding pyridine-3-carbaldehyde with thiosemicarbazide. The synthesized compounds were characterized by ESI-Mass, UV-Vis, IR, and NMR (¹H, ¹³C, ¹⁹F) spectroscopic techniques. Molar mass values and spectroscopic data are consistent with the proposed structural formulas. The molecular structure of 7 has been also confirmed by single crystal X-ray diffraction. In the solid state 7 exists in the E conformation about the N2-N3 bond; 7 also presents the E conformation in solution, as evidenced by ¹H NMR spectroscopy. The in vitro antitumor activity of the synthesized compounds was studied on six human tumor cell lines: H460 (lung large cell carcinoma), HuTu80 (duodenum adenocarcinoma), DU145 (prostate carcinoma), MCF-7 (breast adenocarcinoma), M-14 (amelanotic melanoma), and HT-29 (colon adenocarcinoma). Furthermore, toxicity studies in 3T3 normal cells were carried out for the prepared compounds. The results were expressed as IC₅₀ and the selectivity index (SI) was calculated. Biological studies revealed that 1 (IC₅₀ = 3.36 to 21.35 μM) displayed the highest antiproliferative activity, as compared to the other tested thiosemicarbazones (IC₅₀ = 40.00 to >582.26 μM) against different types of human tumor cell lines. 1 was found to be about twice as cytotoxic (SI = 1.82) than 5-fluorouracile (5-FU) against the M14 cell line, indicating its efficiency in inhibiting the cell growth even at low concentrations. A slightly less efficient activity was shown by 1 towards the HuTu80 and MCF7 tumor cell lines, as compared to that of 5-FU. Therefore, 1 can be considered as a promising candidate to be used as a pharmacological agent, since it presents significant activity and was found to be more innocuous than the 5-FU anticancer drug against the 3T3 mouse embryo fibroblast cells.