OSMAC Strategy Integrated with Molecular Networking for Accessing Griseofulvin Derivatives from Endophytic Fungi of Moquiniastrum polymorphum (Asteraceae).

Three endophytic fungi isolated from Moquiniastrum polymorphum (Less.) G. Sancho (Asteraceae) were cultivated using the one strain many compounds (OSMAC) strategy to evaluate the production of griseofulvin derivatives. Extracts obtained were analyzed by HPLC-MS/MS and the chromatographic and spectrometric data used to elaborate a feature-based molecular network (FBMN) through the GNPS platform. This approach allowed the observation of differences such as medium-specific and strain-specific production of griseofulvin derivatives and variations of cytotoxic activity in most extracts. To evaluate the efficiency of the OSMAC approach allied with FBMN analysis in the prospection of compounds of biotechnological interest, griseofulvin and 7-dechlorogriseofulvin were isolated, and the relative concentrations were estimated in all culture media using HPLC-UV, allowing for the inference of the best strain-medium combinations to maximize its production. Malt extract-peptone broth and Wickerham broth media produced the highest concentrations of both secondary metabolites.

Antibacterial activity of griseofulvin analogues as an example of drug repurposing.

Griseofulvin is a well-known antifungal drug that was launched in 1962 by Merck & Co. for the treatment of dermatophyte infections. However, according to predictions using the Way2Drug computational drug repurposing platform, it may also have antibacterial activity. As no confirmation of this prediction was found in the published literature, this study estimated in-silico antibacterial activity for 42 griseofulvin derivatives. Antibacterial activity was predicted for 33 of the 42 compounds, which led to the conclusion that this activity might be considered as typical for this chemical series. Therefore, experimental testing of antibacterial activity was performed on a panel of Gram-positive and Gram-negative micro-organisms. Antibacterial activity was evaluated using the microdilution method detecting the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC). The tested compounds exhibited potent antibacterial activity against all the studied bacteria, with MIC and MBC values ranging from 0.0037 to 0.04 mg/mL and from 0.01 to 0.16 mg/mL, respectively. Activity was 2.5-12 times greater than that of ampicillin and 2-8 times greater than that of streptomycin, which were used as the reference drugs. Similarity analysis for all 42 compounds with the (approximately) 470,000 drug-like compounds indexed in the Clarivate Analytics Integrity database confirmed the significant novelty of the antibacterial activity for the compounds from this chemical class. Therefore, this study demonstrated that by using computer-aided prediction of biological activity spectra for a particular chemical series, it is possible to identify typical biological activities which may be used for discovery of new applications (e.g. drug repurposing).

Antifungal Activity of Griseofulvin Derivatives against Phytopathogenic Fungi in Vitro and in Vivo and Three-Dimensional Quantitative Structure-Activity Relationship Analysis.

With environmental pollution, residual hazards accumulate and severe drug resistance and many other problems appear; some highly toxic drugs have been banned, and antifungal agents are far from satisfactory. Natural products play an important role in the discovery and development of new pesticides. The natural product griseofulvin (1) has been known as an antifungal agent in the treatment of dermatomycoses for decades. In this study, a series of new griseofulvin derivatives were synthesized with good yields. Their structures were characterized by 1H and 13C nuclear magnetic resonance and high-resolution mass spectrometry (electrospray ionization). The antifungal activities of griseofulvin analogues were first evaluated against five phytopathogenic fungi ( Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria solani, and Fusarium solani) in vitro. Of significance is that most of them showed excellent antifungal activities against C. gloeosporioides. The antifungal activities of the four best compounds (6a, 6c, 6e, and 6f) against C. gloeosporioides were further investigated in vivo using infected apples. The results suggested that compounds 6c, 6e, and 6f [half-maximal inhibitory concentration (IC50) = 47.25 ± 1.46, 49.44 ± 1.50, and 53.63 ± 1.74 µg/mL, respectively] were better than thiophanate-methyl (IC50 = 69.66 ± 6.07 µg/mL). Furthermore, comparative molecular field analysis was performed on the basis of the antifungal activity results of all 22 of the compounds against C. gloeosporioides in vitro. The three-dimensional coefficient contour plots revealed that the suitable bulky and electronegative acyl-substituted groups seem to be more favorable for increasing activity at the 4' position of griseofulvin. The structure-activity relationships were also discussed. Griseofulvin derivatives can be used for the development of highly effective and safe agricultural fungicides.

Griseofulvin Derivative and Indole Alkaloids from Penicillium griseofulvum CPCC 400528.

A new griseofulvin derivative, 4'-demethoxy-4'-N-isopentylisogriseofulvin (1), three new indole alkaloids, 2-demethylcyclopiamide E (2), 2-demethylsperadine F (3), and clopiamine C (4), and five known metabolites (5-9) were isolated from Penicillium griseofulvum CPCC 400528. Compound 1 is the first reported griseofulvin analogue with an N-isopentane group and the first example of a naturally occurring N-containing griseofulvin analogue. Their structures and absolute configurations were elucidated through extensive spectroscopic analyses, calculated ECD, and single-crystal X-ray diffraction (Cu Kα). The possible biogenetic pathway of 1-3 was proposed. Compounds 1, 2, and 5 exhibited anti-HIV activities with IC50 values of 33.2, 20.5, and 12.6 µM, respectively.

Asymmetric Michael-aldol tandem reaction of 2-substituted benzofuran-3-ones and enones: a facile synthesis of griseofulvin analogues.

A highly enantioselective Michael-aldol tandem reaction with respect to prochiral 2-substituted benzofuran-3-ones and enones by a facile primary amine catalyst was investigated. The approach provides rapid access to the desired pharmaceutically active griseofulvin analogues.

GF-15, a novel inhibitor of centrosomal clustering, suppresses tumor cell growth in vitro and in vivo.

In contrast to normal cells, malignant cells are frequently aneuploid and contain multiple centrosomes. To allow for bipolar mitotic division, supernumerary centrosomes are clustered into two functional spindle poles in many cancer cells. Recently, we have shown that griseofulvin forces tumor cells with supernumerary centrosomes to undergo multipolar mitoses resulting in apoptotic cell death. Here, we describe the characterization of the novel small molecule GF-15, a derivative of griseofulvin, as a potent inhibitor of centrosomal clustering in malignant cells. At concentrations where GF-15 had no significant impact on tubulin polymerization, spindle tension was markedly reduced in mitotic cells upon exposure to GF-15. Moreover, isogenic cells with conditional centrosome amplification were more sensitive to GF-15 than parental controls. In a wide array of tumor cell lines, mean inhibitory concentrations (IC(50)) for proliferation and survival were in the range of 1 to 5 µmol/L and were associated with apoptotic cell death. Importantly, treatment of mouse xenograft models of human colon cancer and multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival. These results show the in vitro and in vivo antitumor efficacy of a prototype small molecule inhibitor of centrosomal clustering and strongly support the further evaluation of this new class of molecules.

Transformations of griseofulvin in strong acidic conditions--crystal structures of 2'-demethylgriseofulvin and dimerized griseofulvin.

The structure of griseofulvic acid, C16H15ClO6, at 100 K has orthorhombic (P2(1)2(1)2) symmetry. It is of interest with respect to biological activity. The structure displays intermolecular O-H...O, C-H...O hydrogen bonding as well as week C-H...pi and pi...pi interactions. In strong acidic conditions the griseofulvin undergoes dimerization. The structure of dimerized griseofulvin, C34H32C12O12 x C2H6O x H2O, at 100 K has monoclinic (P2(1)) symmetry. The molecule crystallized as a solvate with one ethanol and one water molecule. The dimeric molecules form intermolecular O-H...O hydrogen bonds to solvents molecules only but they interact via week C-H...O, C-H...pi, C-Cl...pi and pi...pi interactions with other dimerized molecules.

Disparate SAR data of griseofulvin analogues for the dermatophytes Trichophyton mentagrophytes, T. rubrum, and MDA-MB-231 cancer cells.

Griseofulvin and 53 analogues of this compound have been tested against the pathogenic dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes as well as against the breast cancer cell line MDA-MB-231. The modifications to griseofulvin include the 4, 5, 6, 2', 3', and 4' positions. The SAR of the griseofulvin analogues toward the two fungi followed the same trend with the majority being less active than griseofulvin and none had more than twice the potency of the parent compound. A comparison of the antifungal and the anticancer SAR revealed distinct differences, as the majority of analogues showed increased activity against the cancer cell line MDA-MB-231, highlighted by 2'-benzyloxy-2'-demethoxy-griseofulvin, which showed low activity against both fungi but was among the most potent compounds against MDA-MB-231 cancer cells. Tubulin has been proposed as the target of griseofulvin in both fungal and mammalian cells, but the differences revealed by this SAR study strongly suggest that the mode-of-action of the compound class toward fungi and mammalian cancer cells is different.

Two new derivatives of griseofulvin from the mangrove endophytic fungus Nigrospora sp. (strain No. 1403) from Kandelia candel (L.) Druce.

Two new compounds, methyl 3-chloro-6-hydroxy-2-(4-hydroxy-2-methoxy-6-methylphenoxy)-4-methoxybenzoate (1) and (2 S,5' R,E)-7-hydroxy-4,6-dimethoxy-2-(1-methoxy-3-oxo-5-methylhex-1-enyl)-benzofuran-3(2H)-one (2), together with four known compounds, griseofulvin (3), dechlorogriseofulvin (4), bostrycin (5), and deoxybostrycin (6), were isolated from the marine endophytic fungus NIGROSPORA sp. (No. 1403) collected from the South China Sea. The structures were elucidated by spectroscopic methods, 1D, 2D NMR, and HREIMS. Compounds 5 and 6 showed moderate antitumor and moderate antimicrobial activity.

Synthesis and structure-activity relationship of griseofulvin analogues as inhibitors of centrosomal clustering in cancer cells.

Griseofulvin was identified as an inhibitor of centrosomal clustering in a recently developed assay. Centrosomal clustering is an important cellular event that enables bipolar mitosis for cancer cell lines harboring supernumerary centrosomes. We report herein the synthesis and SAR of 34 griseofulvin analogues as inhibitors of centrosomal clustering. The variations in the griseofulvin structure cover five positions, namely the 4, 5, 2', 3', and 4' positions. Modification of the 4 and 5 positions affords inactive molecules. The enol ether must be at the 2' position, and the 4' position needs to be sp(2) hybridized. The most active analogues were the 2'-benzyloxy and 2'-(4-methylbenzyloxy) analogues as well as the oxime of the former with a 25-fold increase of activity compared to griseofulvin. Comparison of the results obtained in this work with prior reported growth inhibition data for dermatophytic fungi showed both similarities and differences.

Syntheses and antifungal activity of dl-griseofulvin and its congeners. II.

Griseofulvin derivatives, dl-6'-demethyl-6'-ethylgriseofulvin (dl-5) and dl-6'demethyl-6'phenylgriseofulvin (dl-6) were prepared by application of a synthetic method developed by us. Antifungal activity of these derivatives decreased in the order of dl-griseofulvin (dl-1) >> dl-6(inactive). The reaction of these derivatives with ethanethiol gave two types of compounds, 2'-(ethylthio)griseofulvin (15) and 4'-(ethylthio)isogriseofulvin (16). The relationship between the ratios of isolated yield of 15 and 16 and antifungal the activity of griseofulvin derivatives is discussed.

Syntheses and antifungal activity of dl-griseofulvin and its congeners. III.

Several congeners (1b-g), with novel substituents on the benzene ring of griseofulvin, were prepared by the application of a synthetic method developed by us. Antifungal activity of these congeners decreased in order of dl-griseofulvin (1a) = 1d > 1b, c >> 1e-f (inactive). The relationship between the antifungal activity and the position or kind of substituents on the benzene ring of griseofulvin is discussed.

Toxigenic molds in water-damaged buildings: dechlorogriseofulvins from Memnoniella echinata.

An investigation of a cluster of cases of pulmonary hemosiderosis in infants in Cleveland, OH, led to the isolation of many isolates of Stachybotrys atra and two isolates of a related toxigenic fungus, Memnoniella echinata. M. echinata produces two cytotoxic trichothecene mycotoxins, trichodermol (1a) and trichodermin (1b), as well as several griseofulvins. Dechlorogriseofulvin (2a) and epidechlorogriseofulvin (2b) were the major compounds isolated. This is the first report of a fungus outside the Penicillium genus producing griseofulvins.

Structure modification and biological activity of some griseofulvin derivatives.

Griseofulvin is an orally acting anti-fungal antibiotic with very limited water solubility. Five chemical modifications were made on the griseofulvin structure in order to evaluate these changes on the antifungal and water solubility properties. Antifungal activity was measured against Tricophyton mentagrophytes, T. rubrum, T. terrestre, and Microsporum canis. The oxime of griseofulvin was the most potent of the five compounds tested, but it was only of equal or less potency than griseofulvin. The somewhat increased water solubility of some of these compounds was offset by the lower anti-fungal potency of the structural modification.

Synthesis and biological activities of griseofulvin analogs.

In order to elucidate the structure-activity relationship of griseofulvin (1), (+/-)-6-demethyl analog (3), 2-demethoxy-6-demethyldihydro analog (4), (+/-)-dechloro-6-ethyl analog (5), (+/-)-dechloro-6-epi-ethyl analog (6), (+/-)6-ethyl analog (7) and (+/-)-6-epi-ethyl analog (8) were synthesized by a Diels-Alder cycloaddition of alkylidene ketones (16, 17 18, 19 and 20) with modified 1,3-butadienes (21 or 22). Their biological activities were examined against fungi.

Syntheses and antifungal activities of dl-griseofulvin and its congeners. I.

dl-Griseofulvin (1a) was prepared by two synthetic pathways. New 6'-congeners (3 and 4) of griseofulvin were also prepared. Their antifungal activities were evaluated and compounds 3 and 4 were found to be less active than 1a. Molecular calculations on 1a, dl-epigriseofulvin (1b), 3 and 4 were undertaken.

Regio- and stereoselective hydrogenation of 2'-demethoxy-2'-methyldehydrogriseofulvin, a symmetrical substrate, to (+)-2'-demethoxy-2'-methylgriseofulvin with a cell-free system of Streptomyces cinereocrocatus.

Enzymatic hydrogenation of 2'-demethoxy-2'-methyldehydrogriseofulvin (5) with a cell-free system of Streptomyces cinereocrocatus afforded (+)-2'-demethoxy-2'-methylgriseofulvin (6). The structure of 6 was determined on the basis of comparisons of the proton nuclear magnetic resonance spectrum, mass spectrum, and circular dichroism with those of a standard specimen which was synthesized chemically. The results demonstrated that when the 2'-position of (-)-dehydrogriseofulvin was substituted with a methyl group, its hydrogenation with the cell-free system occurred stereoselectively at the 5',6'-position.

The in vitro liberation and the bioavailability of different brands of griseofulvin in plasma and urine in man.

The bioavailability of griseofulvin in three different brands, two microfine forms (Gricin = G, Likuden = L), and one ultramicrofine form (Gris-PEG = GP), was determined in plasma and urine in six healthy volunteers in a crossover study and compared with in vitro liberation data. GP shows a higher AUCo infinity (140 +/- 24 mumol . h . l-1) and Cmax (4.5 +/- 0.1 mumol . h-1) than the microsize brands of griseofulvin (AUCo infinity:58 +/- 7, and 45 +/- 6 mumol . h . l-1; Cmax:1.7 +/- 0.2, and 1.5 +/- 0.2 mumol . l-1; G and L, resp.), but the same tmax at the third hour. There results correspond with the in vitro liberation data. Contradictory results of the bioavailability are found by determining the amount of 6-Demethylgriseofulvin eliminated in urine. The elimination of this main metabolite after dosing with L is lower (0.18 +/- 0.02 mmol) than those of the other two brands, which do not differ (0.31 +/- 0.04; 0.32 +/- 0.02 mmol, G and GP, resp.). It is concluded that the determination of bioavailability only by means of the eliminated amount of a metabolite in urine may produce false results.

Fat contents of meals and bioavailability of griseofulvin in man.

The bioavailability of griseofulvin was followed in twelve healthy volunteers by measuring the urinary excretion of the major metabolite 6-demethylgriseolfulvin, after each volunteer had ingested one 500 mg griseofulvin tablet under (1) fasting conditions, (2) immediately after a typical low-fat and (3) high-fat Nigerian meals. An increase of about 70 and 120% absorption occurred with the ingestion of the low-fat and high-fat meals respectively compared to the fasting state (P less than 0.01). The maximum excretion rates of the free metabolite (Vmax.) were also significantly increased (P less than 0.01) following consumption of low and high fat meals. Our results thus suggest that the higher the fat content of the meals the higher the enhancement of the bioavailability of griseofulvin in man.