Action of pyrazolopyrimidine derivatives on Trypanosoma rangeli culture forms

The capacity of 54 different pyrazolo(3,4-d)- or pyrazolo(4,3-d)pyrimidine derivatives to inhibit the multiplication of Trypanosoma rangeli culture forms was evaluated. Among pyrazolo(3,4-d)pyrimidines, 14 derivatives showed trypanostatic activity, 4-aminopyrazolo-(3,4-d)pyrimidine (APP) being the most active, with 4-hydroxypyrazolo(3,4-d)pyrimidine (HPP) lacking trypanostatic activity. 7-Hydroxy-3-beta-D-ribofuranosylpyrazolo(4,3-d)pyrimidine (FoB) was as active as 7-amino-3-beta-D-ribofuranosylpyrazolo(4,3-d)pyrimidine (FoA), both compounds being five-fold less inhibitory than APP. It can be concluded that, regarding T. rangeli, the chemical analogy to hypoxanthine or inosine of pyrazolo(3,4-d)- and pyrazolo(4,3-d)pyrimidine, respectively, is not absolutely critical, as different modifications on the heterocyclic ring did not abolish the inhibitory activity of these compounds.     

Macrophage cytostatic effect on Trypanosoma musculi involves an L-arginine-dependent mechanism

Peritoneal macrophages from mice infected with an extracellular parasite, Trypanosoma musculi were effective in inhibiting parasite proliferation in vitro. This trypanostatic activity could be suppressed by NG monomethyl-L-arginine (NGMMA), a specific inhibitor of a biochemical pathway synthesizing L-citrulline and inorganic nitrogen oxides from L-arginine. Macrophages exerted this in vitro antiproliferative effect from the 10th day of infection on and this activity was maximum around 14th day of infection. Nitrite production paralleled development of macrophage trypanostatic activity. Macrophages collected from BCG-infected mice or treated with IFN-gamma in vitro also exerted a trypanostatic activity which was suppressed by NGMMA. A trypanostatic activity suppressed by NGMMA was also exerted by splenic macrophages from T. musculi-infected mice. Trypanostatic activity of IFN-gamma-treated macrophages was reduced by addition of anti-TNF-alpha showing the participation of TNF-alpha in IFN-gamma-mediated macrophage trypanostatic activity. Nitric oxide (NO) gas inhibited T. musculi proliferation. Addition of excess iron reversed the trypanostatic effect of both macrophages and NO gas. All these data showed that, as reported for a broad spectrum of microorganisms, activated macrophages displayed an antimicrobial effect on trypanosomes through the L-arginine: NO pathway that could participate in controlling infection in T. musculi-infected mice before appearance of antibody-dependent mechanisms. NO production by activated macrophages could trigger iron loss from critical target enzymes in trypanosomes.     

Design,synthesis and evaluation of hydrazine and acyl hydrazone derivatives of 5-pyrrolidin-2-one as antifungal agents

Twenty-eight 5-pyrrolidine-2-ones decorated by hydrazine or acyl hydrazones groups have been designed, synthesized and evaluated as antifungal agents on a panel of twelve fungal strains and three non albicans candida yeasts species which have demonstrated reduced susceptibility to commonly used antifungal drugs. Half of the target compounds exhibited good to high antifungal activities on at least one strain with MIC₅₀ lower than the control antifungal agent – hymexazol or ketoconazole. 5-Arylhydrazino-pyrrolidin-2-ones were found active and the –NH-NH- linker proved to be essential to maintain the antifungal potential. Compound 2a is a broad-spectrum antifungal, active on 60% of the tested strains. Replacing the hydrazine linker by an acylhydrazone one narrowed the spectrum of activity but pyroglutamylaryl hydrazones, mainly aromatic ones, exhibited good activity, adequate “fungicide-like” properties and were devoted of cytotoxicity.     

Antifungal, cytotoxic and SAR studies of a series of N-alkyl, N-aryl and N-alkylphenyl-1,4-pyrrolediones and related compounds

The synthesis, in vitro evaluation and SAR studies of 67 maleimides and derivatives acting as antifungal agents are reported. A detailed SAR study supported by theoretical calculations led us to determine that: an intact maleimido ring appears to be necessary for a strong antifungal activity, dissimilarly affected by the substituents in positions 2 and 3. The best activities were shown by 2,3-nonsubstituted followed by 2,3 dichloro- and 2-methyl-substituted maleimides. They all were fungicide rather than fungistatic enhancing the importance of their antifungal activity. 2,3-Dimethyl and 2,3-diphenyl-maleimides possessed marginal or null activity. The presence of a flexible connecting chain in N-phenylalkyl maleimides appears not to be essential for antifungal activity, although its length shows a correlation with the antifungal behavior, displaying maleimides with alkyl chains of n=3 and n=4 the best antifungal activities in most fungi. Different substituents on the benzene ring did not have a clear influence on the activity. Values of chemical potential properties as well as of energy do not sufficiently discriminate between active and inactive compounds. Nevertheless, it was found that, although logP alone is not strong enough to properly predict the antifungal activity, the comparison of its values for compounds within the same sub-type, showed an enhancement of antifungal activity along with an increment of lipophilicity. In addition, the LUMO's electronic clouds of the highly active compounds showed to be concentrated on the imido ring, indicating that their carbon atoms are potential sites for nucleophilic attack. Same results were obtained from MEPs. Most of the active compounds did not show cytotoxic activity against human cancer cell lines and no one possessed hemolytic activity, indicating that their activity is selective to pathogenic fungi and that they are not toxic at MIC concentrations.     

水果中抗病原真菌物质成分的提取分离方法及应用前景

诱发因子如草酸,拮抗菌产生的抗素等,以及采后热处理,紫外线处理可以诱导水果产生抗病性,此外有的水果具有先天性抗病成分,对这些活性成分进行分析,并用之于水果采后病害的生物防治,为水果的防腐保鲜提供了新的方法,追踪这些抗病物质的产生和分布,需要采用一些专门的分析方法,目前采用的方法是将果实组织破碎,离心,通过萃取,层析或电泳技术进行纯化,然后用核磁共振,X－射线衍射及各种波谱分析技术进行结构鉴定,抗真菌物质的活性可以用薄层层层析生物学方法,孢子萌发或纸碟法进行鉴定,而抗真菌活性物质的定位则用组织化学法,电子显微术,放射自显著技术和免疫印迹技术也广泛用物于组织化学分析。     

Antifungal effect and action mechanism of antimicrobial peptide polybia‐CP

The incidence of life‐threatening invasive fungal infections increased significantly in recent years. However, the antifungal therapeutic options are very limited. Antimicrobial peptides are a class of potential lead chemical for the development of novel antifungal agents. Antimicrobial peptide polybia‐CP was purified from the venom of the social wasp Polybia paulista. In this study, we synthesized polybia‐CP and determined its antifungal effects against a series of Candidian species. Our results showed that polybia‐CP has potent antifungal activity and fungicidal activity against the tested fungal cells with a proposed membrane‐active action mode. In addition, polybia‐CP could induce the increase of cellular reactive oxygen species production, which would attribute to its antifungal activity. In conclusion, the present study suggests that polybia‐CP has potential as an antifungal agent or may offer a new strategy for antifungal therapeutic option. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

水果中抗病原真菌物质成分的提取分离方法及应用前景

诱发因子如草酸、拮抗菌产生的抗菌素等,以及采后热处理、紫外线处理可以诱导水果产生抗病性,此外有的水果具有先天性抗病成分。对这些活性成分进行分析,并用之于水果采后病害的生物防治,为水果的防腐保鲜提供了新的方法。追踪这些抗病物质的产生和分布,需要采用一些专门的分析方法。目前采用的方法是将果实组织破碎,离心,通过萃取、层析或电泳技术进行纯化,然后用核磁共振、X_射线衍射及各种波谱分析技术进行结构鉴定。抗真菌物质的活性可以用薄层层析生物学方法、孢子萌发法或纸碟法进行鉴定。而抗真菌活性物质的定位则用组织化学法。电子显微术、放射自显影技术和免疫印迹技术也广泛用于组织化 学分析。     

Colorimetric broth microdilution method for the antifungal screening of plant extracts against yeasts

Screening plant extracts for antifungal activity is increasing due to demand for new antifungal agents, but the testing methods present many challenges. Standard broth microdilution methods for antifungal susceptibility testing of available antifungal agents are available now, but these methods are optimised for single compounds instead of crude plant extracts. In this study we evaluated the standard NCCLS method as well as a modification which uses spectrophotometric determination of the end-points with a plate reader. We also evaluated another standard method, the EUCAST method, which is a similar microdilution assay to the NCCLS method, but uses a larger inoculum size and a higher glucose concentration in the medium as well as spectrophotometric end-point determination. The results showed that all three methods had some drawbacks for testing plant extracts and thus we modified the NCCLS broth microdilution method by including a colorimetric indicator-resazurin for end-point determination. This modified method showed good reproducibility and clear-cut end-point, plus the end-point determination needed no instruments. It enabled us to evaluate the activity of a selection of extracts from six Combretaceous plants against three Candida spp. and thus provided pharmacological evidence for some traditional uses of these plants while assisting the identification of the active ingredients.     

Antimicrobial activity of 2,4-dihydro-[1,2,4]triazol-3-one derivatives

Antibacterial and antifungal activity of 2,4-dihydro- [1,2,4]triazol-3-one derivatives were examined by the disc-diffusion method (growth inhibition zone diameter in agar medium). The MIC's for the most active agents were determined. Of all the tested compounds, aminomethyl derivatives of 2,4-dihydro-[1,2,4]triazol-3-one exhibit activity against the majority of microorganisms studied.     

In vitro activity of a liposomal nystatin formulation (Nyotran) against Cryptococcus neoformans

The in vitro antifungal activity of a new liposomal nystatin formulation (NISTL, Nyotran, Aronex Ltd., EE.UU.) was evaluated by a microdilution method with RPMI based on the M27A document of the National Committee for Clinical Laboratory Standards (NCCLS) against 22 isolates of Cryptococcus neoformans. This antifungal activity was compared with those of other seven antifungal agents, such as nystatin (NIST), amphotericin B deoxycholate, liposomal amphotericin B, amphotericin B lipid complex, amphotericin B colloidal dispersion, fluconazole, and itraconazole. NISTL was more active in vitrothan NIST, showing MIC values 2-3 fold smaller in 90% of the isolates. The results obtained suggest that this new formulation would be very helpful for the treatment of cryptococcosis.     

Synthesis and conformational analysis of His-Phe-Arg-Trp-NH2 and analogues with antifungal properties

The synthesis, in vitro evaluation, and conformational study of His-Phe-Arg-Trp-NH2 and related derivatives acting as antifungal agents are reported. Among them, His-Phe-Arg-Trp-NH2 and His-Tyr-Arg-Trp-NH2 exhibited antifungal activity against Cryptococcus neoformans. Antifungal activity of these compounds appears to be closely related to the alpha-MSH effect. A conformational and electronic study allows us to propose a biologically relevant conformation for these tetrapeptides acting as antifungal agents. In addition, these theoretical calculations permit us to determine the minimal structural requirements to produce the antifungal response and may provide a guide for the design of compounds with this biological activity.     

In-vitro studies with four new antifungal agents: BAY n 7133, bifonazole (BAY h 4502), ICI 153,066 and Ro 14-4767/002

Four new antifungal agents were compared in vitro with miconazole and ketoconazole. The agents were BAY n 7133 and ICI 153,066, two orally active triazoles, and bifonazole (BAY h 4502) and Ro 14-4767/002, both topical agents. While all four were found to be broad spectrum antifungal agents they also demonstrated certain gaps in their spectra. In general, Ro 14-4767/002 was the most active agent tested whereas bifonazole and BAY n 7133 were the least active. Noteworthy activities included that of Ro 14-4767/002 against Candida albicans, the dermatophytes and Sporothrix schenckii and that of ICI 153,066 against Torulopsis glabrata.     

Synthesis of disulfide esters of dialkylaminocarbothioic acid as potent, non-detergent spermicidal agents

S,S'-[disulfanediylbis(dialkylaminopropane-2,1-diyl)]bis- (dialkylaminothiocarbamate) (14-31) were prepared and evaluated for the spermicidal activity and antifungal activity. Dialkyldithiocarbamates (1-5) were reacted with epichlorohydrin to give 1-dialkylaminocarbothioic acid S-[(2,3-epithio)propyl]ester (7-11), these on further reaction with a secondary amine gave S,S'-[disulfanediylbis(dialkylaminopropane-2,1-diyl)]bis- (dialkylaminothiocarbamate) (14-31). Some of these compounds (16, 19-21, 23, 30, 31) were found to be very potent spermicidal agents with marginal antifungal activity. Two compounds (20, 21) were 25 times more active than nonoxynol-9 (N-9), the spermicide currently in the market.     

Structure-antifungal activity relationship of His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 and analogues

The synthesis, in vitro evaluation and conformational study of His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH(2) and analogues acting as antifungal agents are reported. Among them, His-Phe-Lys-Trp-Gly-Arg-Phe-Val-NH(2) exhibited a moderate but significant antifungal activity against Cryptococcus neoformans, Candida albicans and Candida tropicalis. A theoretical study allows us to propose a biologically relevant conformation for these octapeptides acting as antifungal agents. In addition, these theoretical calculations allow us to determine the minimal structural requirements to produce the antifungal response and can provide a guide for the design of compounds with this biological activity.     

Synthesis and in vitro activity of 2-thiazolylhydrazone derivatives compared with the activity of clotrimazole against clinical isolates of Candida spp

In this paper, we report on the synthesis of a novel series of 2-thiazolylhydrazone derivatives and the influence of the substituents on the thiazole ring on antifungal activity. All synthesized compounds were screened for their in vitro activities against 22 clinical isolates of Candida spp., representing six different species, compared to clotrimazole as a reference compound. Some of the tested compounds were found to possess significant antifungal activity when compared to clotrimazole, in particular compound 14 which exhibited higher potency against most of the Candida spp. considered. The compounds that were most active as anti-Candida agents were also submitted to cytotoxic screening by the Trypan Blue dye exclusion assay and in general they were shown to induce low cytotoxic effects.     

Molecular targeted treatments for fungal infections: the role of drug combinations

Invasive mycoses are associated with a high mortality rate, and their incidence is increased in immunologically deficient patients. From a diagnostic and therapeutic perspective, these infections represent a significant challenge to medicine. In addition to new antifungal agents, drug combinations are an important therapeutic resource, which might be exploited clinically, owing to the multiplicity of fungal targets against which currently available agents are active. In this review, we examine the experimental data regarding the combination of conventional antifungal agents with cytokines, antibacterial agents, calcineurin inhibitors and drugs under development characterized by novel mechanisms of action.     

Influencia del grupo ecológico sobre la sensibilidad in vitro de los hongos dermatofitos a los antifúngicos

BackgroundDermatophytes can be divided into geophilic (soil), zoophilic (animals) and anthropophilic (humans) strains, depending on the source of the keratin that they use for nutritional purposes.AimsThe in vitro susceptibility of clinical isolates of dermatophyte fungi has been studied in the 3 ecological groups with several antifungal agents for the topical management of dermatophytoses in order to determine their relationship with the ecological group.MethodsA standardised dilution micromethod in a liquid medium was used for the determination of the in vitro antifungal activity of 9 topical antifungal drugs: amorolfine (AMR), bifonazole (BFZ), clotrimazole, econazole, ketoconazole, miconazole, oxiconazole, terbinafine (TRB) and tioconazole. The in vitro activity was obtained against 124 clinical isolates of dermatophyte moulds from the anthropophilic, zoophilic and geophilic ecological groups.ResultsThe in vitro antifungal activity was different depending on the ecological group, although a species-dependent profile was also observed.ConclusionsAzole derivatives showed a similar antifungal profile, being more active against anthropophilic dermatophytes > zoophilic > geophilic. Activity of TRB and AMR was different from that of azole derivatives (zoophilic > anthropophilic > geophilic). A higher in vitro antifungal activity against the 3 ecological groups was observed with TRB and AMR, whilst BFZ was the less active drug.     

Sensitivity of some strains of the genus Epidermophyton to different antifungal agents

The inhibitory activity of some antifungal agents against 17 strains of genus Epidermophyton have been studied. The behaviour of Epidermophyton stockdaleae against antifungal agents tested is clearly different from that observed in the strains belonging to Epidermophyton floccosum, since all of the latter were sensitive to the antifungal agents used and the geophilic species showed resistance to griseofulvin, isoconazole, natamycin and nystatin, and intermediate sensitivity to ketoconazole and miconazole. The low sensitivity of E. stockdaleae (and other geophilic dermatophytes) to antifungal agents could be a problem in the current therapy if further studies or case reports demonstrate their pathogenic role.     

Structure-activity relationship study: short antimicrobial peptides.

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an alpha-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an alpha-helical structure correlated with the antimicrobial activity and a beta-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of L-amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the beta-sheet structure retained antifungal activity in the same condition.