Marilones A–C, phthalides 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 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 Ki value of 7.7 µM.

Phthalides exhibit an equally broad spectrum of bioactivity, including modulation of the central nervous system, protection against brain eschemia, modulation of platelet aggregation and cardiac function, inhibition of smooth muscle cell proliferation, anti-angina activity, and smooth muscle relaxation, as well as antibacterial, antifungal, antiviral and phytotoxic activity [1]. The medically most important member of this family of natural products is mycophenolic acid, initially isolated from Penicillium brevicompactum, and used in the form of its derivative mycophenolate mofetil as an immunosuppressant drug [10].
During our search for new natural products produced from the marine-derived fungus Stachylidium sp., several phthalide derivatives, i.e., marilones A-C, were isolated from a culture on agar-BMS media supplemented with artificial sea salt (Scheme 1). Albeit phthalide-like structures are not rare, the structural skeleton of marilones A and B is most unusual, and its biosynthesis is suggested to require unique reactions in fungal secondary metabolism. Marilone A (1) exhibited antiplasmodial activity against Plasmodium berghei with an IC 50 of 12.1 µM. Marilone B (2) showed a specific antagonistic effect on the serotonin receptor 5-HT 2B with a K i value of 7.7 µM.

Results and Discussion
The molecular formula of 1 was deduced by accurate mass measurement (HRMS-EI) to be C 21 H 28 O 4 , requiring eight degrees of unsaturation. The 13 C NMR and DEPT135 spectra contained 21 carbon resonances, including six resulting from methyl groups, three from sp 2 methines, and one from an sp 3 methine, whereas a further three signals resulted from methylene groups, and eight resonances were assigned to quaternary carbons ( Table 1, Table 2 and Supporting Information File 1). The 1 H NMR spectrum of 1 displayed a singlet resonance for the aromatic methine (6-CH) at δ 6.95 indicating, together with UV and 13 C NMR data, the presence of a penta-substituted benzene ring. The methyl group 10-CH 3 (δ C 8.8) was linked to C-4 of the aromatic ring due to heteronuclear long range correlations of the methyl protons with C-3, C-4 and C-5. The methoxy group 9-OCH 3 (δ H 3.98) had a heteronuclear long range correlation to C-3 of the aromatic ring, thus, clearly delineating its position. Besides the aromatic proton 6-H, the 1 H NMR spectrum showed two further resonance signals in the downfield shifted region (δ H 5.52 and 5.09) indicating, together with 13 C NMR and 1 H/ 13 C HMBC data, the presence of a geranyl substituent. The C-1' to C-10' part of the molecule was deduced from two proton coupling spin systems observed in the 1 H/ 1 H COSY spectrum, namely 1'-H 2 to 2'-H (J = 6.6 Hz) and 4'-H 2 to 6'-H through 5'-H 2 . 1 H/ 13 C HMBC data showed correlations from 9'-H 3 to C-2', C-3' and C-4', and from methyl protons 8'-H 3 and 10'-H 3 to C-6'and C-7', disclosing a geranyl fragment. Based on literature comparisons we established the configuration of Δ 2'/3' as E [11]. The aromatic quaternary carbon C-5 (δ C 164.2) had a carbon resonance that indicated a connection to an oxygen atom. The monoterpenyl substituent was established to be connected to C-5 through an oxygen atom, based on the heteronuclear long range correlations of 1'-H 2 (δ H 4.69, 4.74) to C-5.
The 1 H/ 13 C HMBC spectrum exhibited a correlation from 6-H to C-8. Furthermore, the 13 C NMR resonance of C-8 at δ 77.0 was found to be characteristic for a carbon bound to an oxygen atom. The 1 H/ 1 H COSY spectrum showed a coupling of 8-H with 11-H 3 (J = 6.6 Hz), and the 1 H/ 13 C HMBC spectrum contained correlations from 8-H to C-6, C-7 and C-2 of the penta-substituted aromatic ring, as well as to the carbonyl carbon C-1. Ring double bond equivalents required a second ring within compound 1, and together with heteronuclear correlations of 8-H to C-1 and the carbon resonance of C-1 at δ C 168.2 indicating the presence of a carbonyl group, this gave evidence for a C-8-methylated phthalide skeleton, i.e., the C-1 to C-11 part of the structure. Since the resonance signal for 6-H did not show heteronuclear long range correlations to that of the carbonyl C-1, but instead correlated with the sp 3 methine C-8, the carbonyl group was assigned at C-1. In this way, a phthalide-nucleus identical to that of the known natural product nidulol was formed [6]. To further prove that the carbonyl group is positioned at C-1 and not at C-8 of 1, 1 H NMR spectra of 1 were compared with those of nidulol and silvaticol (4) (and derivatives, see Supporting Information File 1). The latter are known regioisomeric phthalides with the carbonyl group at C-1 and C-8, respectively. Differences in 1 H NMR resonances can be discerned especially for 6-H, resonating at δ H 6.59 (CDCl 3 ) for nidulol and δ H 7.04 (CDCl 3 ) for silvaticol [6]. The 1 H NMR spectrum of 1 (δ H 6.54 in CDCl 3 ) was shown to be similar to that of nidulol and the nidulol derivative 5-(3',3'-dimethylallyloxy)-7-methoxy-6-methylphthalide with 6-H resonating at δ H 6.62 (see Supporting Information File 1) [5]. For compound 1 the trivial name marilone A is suggested.
The molecular formula of 2 was deduced by accurate mass measurement (HRMS-EI) to be C 11 H 12 O 4 , requiring six sites of unsaturation. The NMR spectral data (see Table 1, Table 2 and Supporting Information File 1) indicated that compound 2 is identical to 1, except for the missing geranyl moiety attached to the hydroxy group at C-5. We propose the trivial name marilone B for compound 2.
The molecular formula of 3, was deduced by accurate mass measurement (HRMS-EI) to be C 20 H 26 O 4 , requiring eight degrees of unsaturation. The spectroscopic data of 3 revealed that the compound is also very similar to 1 ( Table 1, Table 2 and Supporting Information File 1). In contrast to compound 1,  however, resonance signals for a methylene group, i.e., 1-CH 2 (δ C 68.9) were found in the NMR spectra, instead of those for a methine (8-CH) and methyl group (11-CH 3 ) as in 1. The resonance signal for 6-H did not have a heteronuclear long range correlation to C-1, but correlated with the carbonyl carbon C-8. Hence, the location of carbonyl group in 3 was assigned to C-8, thus, forming a phthalide-nucleus as present in silvaticol (4). For compound 3 the name marilone C is suggested.
Spectroscopic data of 4 were determined to be identical to those of silvaticol (Supporting Information File 1) [6].
Compounds 1 and 2 possess a single chiral center at C-8. The measurement of the specific optical rotation for these compounds yielded values close to zero, and furthermore, the CD measurements showed hardly any CD effect for the referred compounds. This was expected at around 260 nm due to the proximity of the chiral center to the chromophoric penta-substituted benzene ring. We thus assumed the presence of racemic mixtures for these chiral compounds. Extensive trials to sepa-rate the enantiomers, employing three different HPLC chiral stationary phases, were unsuccessful. However, the presence of racemic mixtures was proven for the analogous, nitrogencontaining compounds, i.e., phthalimidine derivatives isolated from the same fungus (Almeida et al., unpublished data). Compounds 1-4 were further evaluated for antiviral activity, for inhibition of protein kinases and proteases, for growth inhibition of antibiotic-resistant Mycobacterium tuberculosis as well as further microbial pathogens, for activity in an antidiabetic activity assay panel, in a 3T3-L1 murine adipocyte assay, and in a NF-κB protein complex assay, but they exhibited no activity (see detailed description in Supporting Information File 1).

Marilones
Phthalide derivatives are compounds of the polyketide metabolism, which are common in nature [1]. Secondary metabolites 1 and 2 discovered in the marine-derived Stachylidium sp. were found to be derivatives of the natural product nidulol, whilst compound 3 was a derivative of silvaticol (4) (see Supporting Information File 1), formerly described from the fungus Aspergillus silvaticus [6]. Nidulol and silvaticol (4) are regioisomeric compounds and differ in terms of the position of the carbonyl group, which is either placed peri to the aromatic hydrogen, as in 3 and 4, or it is positioned peri to the aromatic methoxy moiety, e.g., in 1 and 2. The Stachylidium species investigated here is thus able to produce both types of phthalides, which are suspected to differ significantly in terms of their biosynthesis (Supporting Information File 1; Figure  S15).
Whereas compound 3 is simply the O-prenylated form of silvaticol (4), the nidulol derivatives 1 and 2 are distinguished by an additional methyl substituent (11-CH 3 ) at C-8. In terms of biosynthesis, i.e., polyketide metabolism, this substitution is most unusual for phthalides and, to the best of our knowledge, it was only found once in dimethoxydimethylphthalide (DDP) [12].
Biosynthetic studies focusing on phthalide structures, e.g., for mycophenolic acid [13], nidulol and silvaticol [9], were previously performed by means of feeding experiments with labeled precursors, evidencing the tetraketide nature of the phthalide nucleus (Supporting Information File 1; Figure S15). Compounds 1-4, possess a basic skeleton which is related to that of the well-known tetraketide 3-methyl-orsellinic acid [14].
Closing of the lactone ring would for compounds 1 and 2 require the oxidation of C-8 to obtain a hydroxy group, which could subsequently form a lactone with the C-1 carboxy group (Supporting Information File 1; Figure S15A). In contrast to that for 3 and 4, a reduction of the C-1 carboxy group to an alcoholic function and an oxidation of C-8 to a carboxylic function would be required (Supporting Information File 1; Figure  S15 B).
Most intriguing, however, is that in compounds 1 and 2 the acetate-derived methyl group 8-CH 3 in the methyl-orsellinic acid precursor would be replaced by an ethyl group. Thus, the biosynthesis seems to require either a propionate starter unit (see C in Figure S15; Supporting Information File 1) or a methylation (e.g., via a SAM-dependent methyl-transferase) at C-8 (see D in Figure S15; Supporting Information File 1). A third possibility would be the loss of a carbon atom from a pentaketide intermediate. To our knowledge, to date propionate as a starter unit was only described for pseurotin A and austrocorticinic acid in fungal polyketide metabolism [15,16]. Feeding experiments are under way in order to determine the building blocks for these molecules.
It is worthwhile to mention that marilones were produced solely on solid biomalt medium (BMS) supplemented with sea salt, whereas in other media such as Czapek or YPM no phthalides were formed.

Experimental
General experimental procedures. Optical rotations were measured on a Jasco DIP 140 polarimeter. UV and IR spectra were obtained with a Perkin-Elmer Spectrum BX instrument. All NMR spectra were recorded in MeOD or (CD 3 ) 2 CO on a Bruker Avance 300 DPX spectrometer. Spectra were referenced to residual solvent signals with resonances at δ H/C 3.  (Table 1 and

Supporting Information
Supporting Information File 1 Spectroscopic data and other relevant information for compounds 1-4.