Synthesis, Fungitoxic Activity against Botrytis cinerea and Phytotoxicity of Alkoxyclovanols and Alkoxyisocaryolanols.

Clovane and isocaryolane derivatives have been proven to show several levels of activity against the phytopathogenic fungus Botrytis cinerea. Both classes of sesquiterpenes are reminiscent of biosynthetic intermediates of botrydial, a virulence factor of B. cinerea. Further development of both classes of antifungal agent requires exploration of the structure-activity relationships for the antifungal effects on B. cinerea and phytotoxic effects on a model crop. In this paper, we report on the preparation of a series of alkoxy-clovane and -isocaryolane derivatives, some of them described here for the first time (2b, 2d, 2f-2h, and 4c-4e); the evaluation of their antifungal properties against B. cinerea, and their phytotoxic activites on the germination of seeds and the growth of radicles and shoots of Lactuca sativa (lettuce). Both classes of compound show a correlation of antifungal activity with the nature of side chains, with the best activity against B. cinerea for 2d, 2h, 4c and 4d. In general terms, while 2-alkoxyclovan-9-ols (2a-2e) exert a general phytotoxic effect, this is not the case for 2-arylalkoxyclovan-9-ols (2f-2i) and 8-alkoxyisocaryolan-9-ols (4a-4d), where stimulating effects would make them suitable candidates for application to plants.

Synthesis of Trichodermin Derivatives and Their Antimicrobial and Cytotoxic Activities.

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds 9, 13, and 15, which presented average IC50 values of 2 µg/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes).

Natural Compounds That Modulate the Development of the Fungus Botrytis cinerea and Protect Solanum lycopersicum.

Botrytis cinerea is the causal agent of gray mold disease and is responsible for the loss of millions of dollars in crops in worldwide. Currently, this pathogen exhibits increasing resistance to conventional fungicides; therefore, better control methods and novel compounds with a more specific mechanism of action but without biocidal effects, are required. In this work, several natural compounds to control B. cinerea were analyzed in vitro. Detected effects were dependent on the stage of fungus development, and 3-phenyl-1-propanol displayed the most potent inhibition of in vitro germination, germ tube development, and sporulation. However, it had lower protection of leaves and postharvest fruit in plant infection. Isoeugenol and 1-phenylethanol exhibited lower inhibition of in vitro germination and sporulation, but at the highest concentrations, they inhibited germ tube elongation. Although the lowest rates of foliage infection were recorded using isoeugenol and 3-phenyl-1-propanol, 1-phenylethanol significantly decreased the disease in postharvest tomato fruit, with an efficacy like Mancozeb, but at 18 times lower micromolar concentration. All compounds resulted in high cell viability after spores were removed from the treatment solution exhibited high cell viability, suggesting a non-biocidal effect. The diversity of in vitro and in-plant effects seems to indicate a different mechanism of action.

Further mulinane and azorellane diterpenoids isolated from Mulinum crassifolium and Azorella compacta.

The new mulinane diterpenoids 1 and 2 were isolated from the EtOAc extract of Mulinum crassifolium, while the rearranged mulinane 5, which was isolated for the first time from a natural source, was isolated from Azorella compacta. Compounds 1-2 were prepared by semi-synthesis thorough acetylation of the diterpene 17-acetoxymulinic acid (3). A mechanism of reaction was proposed, while the structures of the new compounds were elucidated on the basis of comprehensive spectroscopic analysis and computational methods.

Phytotoxic activity and metabolism of Botrytis cinerea and structure-activity relationships of isocaryolane derivatives.

Research has been conducted on the biotransformation of (8S,9R)-isocaryolan-9-ol (4a) and (1S,2S,5R,8S)-8-methylene-1,4,4-trimethyltricyclo[6.2.1.0(2,5)]undecan-12-ol (5a) by the fungal phytopathogen Botrytis cinerea. The biotransformation of compound 4a yielded compounds 6-9, while the biotransformation of compound 5a yielded compounds 10-13. The activity of compounds 4a and 5a against B. cinerea has been evaluated. (8R,9R)-Isocaryolane-8,9-diol (6), a major metabolite of compound 4a, shows activity compared to its parent compound 4a, which is inactive. The effect of isocaryolanes 3, 4a, and 5a, together with their biotransformation products 6-8, 10, and 14-17, on the germination and radicle and shoot growth of Lactuca sativa (lettuce) has also been determined. Compounds 7-13 are described for the first time.

HPLC analysis of midodrine and desglymidodrine in culture medium: evaluation of static and shaken conditions on the biotransformation by fungi.

A high-performance liquid chromatography (HPLC) method is presented for the simultaneous determination of midodrine and desglymidodrine (DMAE) in Czapek-Dox culture medium, to be used in biotransformation studies by fungi. The HPLC analysis was conducted using a Lichrospher 100 RP18 column, acetonitrile-40 mmol/L formic acid solution (60:40, v/v) as mobile phase, and ultraviolet detection at 290 nm. The sample preparation was conducted by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 0.4-40.0 µg/mL for midodrine (r ≥ 0.9997) and DMAE (r ≥ 0.9998). Within-day and between-day precision and accuracy were evaluated by relative standard deviations (≤ 8.2%) and relative errors (-7.3 to 7.4%), respectively. The validated method was used to assess midodrine biotransformation by the fungi Papulaspora immersa Hotson SS13, Botrytis cinerea UCA 992 and Botrytis cinerea 2100 under static and shaken conditions. Under shaken conditions, the biotransformation of midodrine to DMAE was more efficient for all studied fungi, especially for the fungus Botrytis cinerea 2100, which converted 42.2% of midodrine to DMAE.

Biotransformation of bioactive isocaryolanes by Botrytis cinerea.

The metabolism of the fungistatic agent (8R,9R)-8-methoxyisocaryolan-9-ol (4) by the fungus Botrytis cinerea has been investigated. Biotransformation of compound 4 yielded compounds 5 and 6-9. No dihydrobotrydial is observed after 4 days of incubation of compound 4. Separate biotransformation of (8R,9R)-isocaryolane-8,9-diol (5) yielded compounds 7-11. The evaluation of the fungistatic activity against B. cinerea of compounds 4, 5, and 6 is reported. (4R,8R,9R)-8-Methoxyisocaryolane-9,15-diol (6), a major metabolite of (8R,9R)-8-methoxyisocaryolan-9-ol (4), shows a much reduced biological activity when compared with the parent compound. Isocaryolane derivatives 6-11 are described for the first time.

Azaphilones from the endophyte Chaetomium globosum.

Six new azaphilones, 5'-epichaetoviridin A (7), 4'-epichaetoviridin F (9), 12ß-hydroxychaetoviridin C (10), and chaetoviridins G-I (11-13), and six known azaphilones, chaetoviridins A-E (1-5) and 4'-epichaetoviridin A (8), were isolated from the endophytic fungus Chaetomium globosum cultivated in PDB medium for 21 days. The structure elucidation and the assignment of the relative configurations of the new natural products were based on detailed NMR and MS spectroscopic analyses. The structure of compound 1 was confirmed by single-crystal X-ray diffraction analysis. The absolute configurations of compounds 4, 7, 8, and 12 were determined using Mosher's method. The antibiotic activity of the compounds was evaluated using an in vivo Caenorhabditis elegans infection model.

Diketopiperazines produced by endophytic fungi found in association with two Asteraceae species.

Diketopiperazine (DKP) derivatives, named colletopiperazine, fusaperazine C and E as well as four known DKPs were isolated from cultures of Colletotrichum gloeosporioides, Penicillium crustosum, both endophytic fungi isolated from Viguiera robusta, and a Fusarium spp., an endophyte of Viguiera arenaria, respectively. Their structures were established on the basis of their spectroscopic data. Conformational analysis of two known DKPs showed that folded conformations were as energetically stable as the extended one.

Sesquiterpenes from the wood of Juniperus lucayana.

Bioassay-guided fractionation of ethanolic extract from the wood of Juniperus lucayana afforded three sesquiterpenes named 3-hydroxypseudowiddran-6(7)-en-4-ol (1), 15-hydroxyallo-cedrol (2) and 12-hydroxywiddrol (3) together with six known sesquiterpenes (4-9) and two known flavonoids (10 and 11). Their structures were established on the basis of comprehensive spectroscopic analyses, including 2D NMR spectroscopy and mass spectrometry. The structures of compounds were identified as 1alpha,4beta,11alpha,11beta-tetramethylbicyclo[5,4,0]undec-6(7)-en-3alpha, 4alpha-diol (1), 4beta-hydroxymethyl-5,5,9beta-trimethyltricyclo[4.3.0.2(1.4)]undecan-3alpha-ol (2) and 4beta-hydroxymethyl-7alpha,11alpha,11beta-trimethylbicyclo [5.4.0]undec-1-en-4alpha-ol (3). The major compounds isolated were evaluated for their antifungal activity against Botrytis cinerea. Widdrol (7) was the most active, reaching the 71% inhibition level on mycelial growth after 6 days.

Quantitative structure-activity relationship studies for the prediction of antifungal activity of N-arylbenzenesulfonamides against Botrytis cinerea.

The Botrytis cinerea is one of the most interesting fungal pathogens. It can infect almost every plant and plant part and cause early latent infections which damage the fruit before ripening. The QSAR is an alternative method for the research of new and better fungicides against B. cinerea. This paper describes the results of applying a topological sub-structural molecular design (TOPS-MODE) approach for predicting the antifungal activity of 28 N-arylbenzenesulfonamides. The model described 86.1% of the experimental variance, with a standard deviation of 0.223. Leave-one-out and leave-group-out cross validation was carried out with the aim of evaluating the predictive power of the model. The values of their respective squared correlations coefficients were 0.754 and 0.741. The TOPS-MODE approach was compared with three other predictive models, but none of these could explain more than 72.8% of the variance with the same number of variables. In addition, this approach enabled the assessment of the contribution of different bonds to antifungal activity, thereby making the relationships between structure and biological activity more transparent. It was found that the fungicidal activity of the chemicals analyzed was increased by the presence of a sulfonamide group bonded to two aromatics rings, making this group the most important of the molecule. The majority of the substituents present in the aromatic rings have an electron withdrawing effect and they contribute to a smaller degree than the sulfonamide group to the property under study. The aromatic moiety plays an important role in this activity; its contribution changes with different substituents. Generally, the nitro group has a positive and great contribution to the biological property but when this group is involved in some compounds in ortho effect with the SO2 moiety of the sulfonamide group a lower value of contribution is observed for both groups.

The antifungal activity of widdrol and its biotransformation by Colletotrichum gloeosporioides (penz.) Penz. & Sacc. and Botrytis cinerea Pers.: Fr.

Widdrol (1) was tested against the necrotrophic plant pathogens Botrytis cinerea and Colletotrichum gloeosporioides. While 1 was found to be inactive against C. gloeosporioides, it showed a selective and effective control of B. cinerea, significantly inhibiting the mycelial growth of the fungus at concentrations of 100 ppm and above. In addition, the biotransformation of 1 by both fungi was studied. Incubation with C. gloeosporioides and B. cinerea afforded four and one biotransformation products (2-6), respectively. Biotransformation with C. gloeosporioides was highly regioselective, yielding for the most part oxidation products at C-10: 10-oxowiddrol (2), 10beta-hydroxywiddrol (3), 10alpha-hydroxywiddrol (4), and 14alpha-hydroxywiddrol (5). The structures of all products were determined on the basis of their spectroscopic data, including coupling constants, two-dimensional NMR analysis (heteronuclear multiple quantum coherence, heteronuclear multiple bond correlation, and nuclear Overhauser enhancement spectroscopy), and nuclear Overhauser effect. The biotransformation products were then tested against B. cinerea and found to be inactive. These results shed further light on the structural modifications, which may be necessary to develop selective fungal control agents against B. cinerea.

A topological substructural molecular design to predict soil sorption coefficients for pesticides.

A TOPological Sub-structural MOlecular DEsign (TOPS-MODE) approach was used to predict the soil sorption coefficients for a set of pesticide compounds. The obtained model accounted for more than 85% of the data variance and demonstrated the importance of the dipole moment, the standard distance, the polarizability, and the hydrophobicity in describing the property under study. In addition, we compared this new model to a previous one using different descriptors such as WHIM and molecular connectivity indices. Finally, the TOPS-MODE was used to calculate the contribution of different fragments to the soil sorption coefficient of the compounds studied. The present approximation proved to be a good method for studying the soil sorption coefficient for pesticides, but it could also be extended to other series of chemicals.