Celso Almeida

Marilones A–C, phthalides from the sponge-derived fungus Stachylidium sp.

Summary The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia sp. cf. C. flammea. Culture on a biomalt medium supplemented with sea salt led to the isolation of three new phthalide derivatives, i.e., marilones A–C (1–3), and the known compound silvaticol (4). The skeleton of marilones A and B is most unusual, and its biosynthesis is suggested to require unique biochemical reactions considering fungal secondary metabolism. Marilone A (1) was found to have antiplasmodial activity against Plasmodium berghei liver stages with an IC50 of 12.1 µM. Marilone B (2) showed selective antagonistic activity towards the serotonin receptor 5-HT2B with a K i value of 7.7 µM.

Marilines A-C: Novel phthalimidines from the sponge-derived fungus Stachylidium sp

A marine-derived fungus of the genus Stachylidium was isolated from the sponge Callyspongia cf. C. flammea. Chemical investigation of the bioactive fungal extract led to the isolation of the novel phthalimidine derivatives marilines A(1) (1a), A(2) (1b), B (2), and C (3). The absolute configurations of the enantiomeric compounds 1a and 1b were assigned by a combination of experimental circular dichroism (CD) investigations and quantum chemical CD calculations. The skeleton of marilines is most unusual, and its biosynthesis is suggested to require uncommon biochemical reactions in fungal secondary metabolism. Both enantiomers, marilines A(1) (1a) and A(2) (1b), inhibited human leukocyte elastase (HLE) with an IC(50) value of 0.86 μM.

Stachylines A-D from the sponge-derived fungus Stachylidium sp.

The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia cf. C. flammea. Four new, putatively tyrosine-derived and O-prenylated natural products, stachylines A-D (1-4), were obtained from the fungal extract. The structures of 1-4 were elucidated on the basis of extensive spectroscopic analyses. The absolute configuration of compound 2 was established by Moshers method. Stachyline A (1) possesses a rare terminal oxime group and occurs as an interchangeable mixture of E/Z-isomers.

Hydroxylated Sclerosporin Derivatives from the Marine-derived Fungus Cadophora malorum

The marine-derived fungus Cadophora malorum was isolated from the green alga Enteromorpha sp. Growth on a biomalt medium supplemented with sea salt yielded an extract, from which we have isolated sclerosporin and four new hydroxylated sclerosporin derivatives, namely, 15-hydroxysclerosporin (2), 12-hydroxysclerosporin (3), 11-hydroxysclerosporin (4), and 8-hydroxysclerosporin (5). The compounds were evaluated in various biological activity assays. Compound 5 showed a weak fat-accumulation inhibitory activity against 3T3-L1 murine adipocytes.

Endolides A and B, Vasopressin and Serotonin-Receptor Interacting N-Methylated Peptides from the Sponge-Derived Fungus Stachylidium sp.

The marine-derived fungus Stachylidium sp. was isolated from the sponge Callyspongia sp. cf. C. flammea. Culture on a biomalt medium supplemented with sea salt led to the isolation of two new, most unusual N-methylated peptides, i.e., the tetrapeptides endolide A and B (1 and 2). Both of these contain the very rare amino acid 3-(3-furyl)-alanine. In radioligand binding assays endolide A (1) showed affinity to the vasopressin receptor 1A with a Ki of 7.04 μM, whereas endolide B (2) exhibited no affinity to the latter receptor, but was selective toward the serotonin receptor 5HT2b with a Ki of 0.77 μM.

Graminin B, a furanone from the fungus Paraconiothyrium sp.

Members of the furanone structural class of natural oxygenated heterocycles have been reported from fungi of the genera Cephalosporium (for example, gregatins and graminin A), Aspergillus (huaspenone A and B or the unnamed tetronic acid derivatives from A. panamensis) and Penicillium (penicilliol).1–6 The structure initially proposed of 3-acyl-4-methoxyfuran-2(5H)-one was later revised to a structure of 4-acyl-5-methoxyfuran-3(2H)-one after the total synthesis of racemic and (þ )-gregatin B by Takaiwa and Yamashita.7,8 Very recently, a serendipitous synthesis of (þ )gregatin B9 and total syntheses of the gregatins A–D and aspertetronin A10 led to a second structural revision establishing a central core of 4-(methoxycarbonyl)furan-3(2H)-one as the right structure for the members of this family of secondary metabolites (Figure 1). As part of a continuing program to identify novel pharmaceutical lead structures from natural sources, Fundación MEDINA has been investigating new antibiotic agents from its proprietary library of over 100 000 strains of actinomycetes and fungi. The fermentation extract of a fungal strain was selected for further purification based on its antibiotic activity. A phylogenetic placement of this strain, based on its ITS/28S, identified the fungus as a species of Paraconiothyrium and closely related to Paraconiothyrium hawaiensis. Herein we report the isolation and structural elucidation of graminin B (1), a new furanone derivative obtained from fermentation broths of this species, and its activity against Escherichia coli and methicillin-resistant Staphylococcus aureus. Graminin B was isolated from acetone extracts of Paraconiothyrium sp. (CF-217411) by fractionation on SP207SS resin followed by repeated semi-preparative HPLC. The molecular formula of 1 was deduced to be C18H26O4 by accurate mass measurement (ESI-TOF, Supplementary Figure S4), requiring six degrees of unsaturation. The 13C NMR and 1H/13C HSQC spectroscopic analysis indicated the presence of 18 carbon resonances, including four resulting from methyl groups and four from sp2 methines, whereas further five signals resulting from methylene groups and five resonances were assigned to quaternary carbons (see Table 1 and Supplementary Figure S2). The 1H NMR (Supplementary Figure S1) and 1H/1H COSY spectra of 1 displayed two spin systems, one composed by aliphatic protons from H3-1 through H2-5, and another between the four olefinic protons (H-10 through H-13) and the aliphatic protons H2-14 and H3-15. The HMBC spectrum (Supplementary Figure S3) exhibited correlations from H2-5 to C-6 (dC 200.9) and C-7 (dC 107.0) (Figure 2), deduced to belong to a furanone ring. The methoxyl group H3-18, with a proton resonance in 1H NMR at d 3.81, displayed long-range heteronuclear correlations to the carbonyl C-17 (dC 164.3) of the esther group, confirming the presence of a methoxycarbonyl group in the molecule (Figure 1). The presence of a double bond between C-6 (dC 200.9) and C-7 (dC 107.0) in the furanone moiety was deduced on the basis of similar 13C NMR chemical shifts with other members of the same structural class.10 The methyl group H3-16 and the methine H-10 from the second spin system chain also showed key heteronuclear long-range correlations (Figure 2), to C-9 and to the ketone C-8 (dC 200.7), deduced to be part of the furanone moiety. An E geometry was assigned to the 10D double bond based on the existence of a coupling constant of 15.4 Hz between H-10 and H-11, whereas a coupling constant of 10.6 Hz between H-12 and H-13 secured a Z configuration for the 12D olefin. These data indicated that 1 has a structure similar to the structure of graminin A,3 the only differences between both compounds being the absence of 4D insaturation in the structure of 1 and the geometry of the 12D double bond. The trivial name graminin B is therefore proposed for compound 1. The absolute configuration at the only chiral center of the furanone ring was assumed to be R after comparison of the optical rotation value ([a]20 D 96) with those reported for the synthetic enantiomers ( )-gregatin A and (þ )aspertetronin A.10

Biosynthesis of Phenylnannolone A, a Multidrug Resistance Reversal Agent from the Halotolerant Myxobacterium Nannocystis pusilla B150

The myxobacterial strain Nannocystis pusilla B150 synthesizes the structurally new polyketides phenylnannolone A–C. Apart from some common volatiles and siderophores, these are the first natural products from the genus Nannocystis. Phenylnannolone A shows inhibitory activity towards the ABCB1 gene product P‐glycoprotein and reverses daunorubicin resistance in cancer cells. To decipher the biochemical reactions leading to the formation of phenylnannolone A, the putative biosynthetic genes were identified (phn1, phn2). Phn2 is a polyketide synthase (PKS) with an NRPS‐like loading module, and its domain order is consistent with the phenylnannolone A structure. The functionality and substrate selectivity of the loading module were determined by means of a γ‐18O4‐ATP pyrophosphate exchange and a phosphopantetheine ejection assay. A specific activation of cinnamic acid by the AMP‐ligase was detected. Phn1 is a putative butyryl‐CoA carboxylase (BCC), providing ethylmalonyl‐CoA for the formation of the ethyl‐substituted part of phenylnannolone A. Phn1 is the first BCC found in biosynthetic genes for an ethyl‐substituted natural compound. Biosynthesis of phenylnannolone A, putatively encoded by phn1 and phn2, thus utilizes the first biosynthetic machinery in which both a BCC and a PKS are involved.

Corallorazines from the myxobacterium Corallococcus coralloides.

The myxobacterium Corallococcus coralloides is the producer of the antibiotic compound corallopyronin A, which is currently in preclinical evaluation. To obtain suitable amounts of this antibiotic, the production strain C. coralloides B035 was cultured in large volumes, which in the addition to the isolation of the target molecule facilitates the detection of additional metabolites of this myxobacterial strain (corallorazines A-C). Corallorazine A is a new structural type of dipeptide composed of a dehydroalanine and a glycine moiety that are linked via a semiaminal bond, thus forming a piperazine ring. The latter is further connected via an amide bond to an unusual aliphatic acyl chain.

Unprecedented Polyketides from a Marine Sponge-Associated Stachylidium sp.

From the marine sponge-derived fungus Stachylidium sp. six novel phthalide-related compounds, cyclomarinone (1), maristachones A-E (2-5), and marilactone (6), were isolated. The structure of compound 1 comprises a hydroxycyclopentenone ring instead of the furanone ring characteristic for phthalides and represents a new carbon arrangement within polyketides. In the epimeric compounds 5a and 5b the phthalide (=isobenzofuranone) nucleus is modified to an isobenzofuran ring with ketal and acetal functionalities. Biosynthetically the structural skeletons of cyclomarinone (1) and maristachones A (2), C (4), D (5a), and E (5b) are most unusual due to the presence of an additional carbon atom when compared to the basic polyketide skeleton. This special biosynthetic feature also holds true for the likewise isolated polyketide marilactone (6).

Non-geminal Aliphatic Dihalogenation Pattern in Dichlorinated Diaporthins from Hamigera fusca NRRL 35721

Two new epimeric dihalogenated diaporthins, (9 R *)-8-methyl-9,11-dichlorodiaporthin (2) and (9 S *)-8-methyl-9,11-dichlorodiaporthin (3), have been isolated from the soil fungus Hamigera fusca NRRL 35721 alongside the known regioisomeric isocoumarin 8-methyl-11,11-dichlorodiaporthin (1). Their structures were elucidated by high-resolution mass spectrometry and NMR spectroscopy combined with molecular modeling. Compounds 1-3 are the first isocoumarins and the first halogenated metabolites ever reported from the Hamigera genus. The new compounds 2 and 3 display a non-geminal aliphatic dichlorination pattern unprecedented among known fungal dihalogenated aromatic polyketides. A bifunctional methyltransferase/aliphatic halogenase flavoenzyme is proposed to be involved in the biosynthesis of dichlorinated diaporthins 1-3. These metabolites are weakly cytotoxic.