Marine microorganisms are recognized as important sources of pharmacologically active metabolites [1–3]. In particular, a growing number of marine-derived fungi have been reported to produce novel bioactive secondary metabolites. In an earlier part of this chemical investigation we described the isolation and structure elucidation of several new antiviral naphthalenone derivatives from the EtOAc extract of an ascomycetous fungus [4]. On further investigation of this fungus, we describe herein the isolation, structure elucidation and the biological activity of the novel 12-membered macrolide, balticolid (1).

Fungal metabolites that contain twelve-membered lactone rings like recifeiolide from Cephalosporium recifei [5], cladospolides A-C from Cladosporium cladosporioides [6] and Cladosporium tenuissimum [7], patulolides A-C from Penicillium urticae [8,9], pandangolides 1–4 from Cladosporium herbarum [10] and chloriolide from Chloridium virescens var. chlamydosporum [11] have already been reported.

Balticolid (1) was obtained as colorless oil. Its molecular formula was determined to be C₁₂H₁₆O₄ by ESIMS at m/z 225 [M + H]⁺ and by HRESIMS at m/z 471.2001 for C₂₄H₃₂O₈Na as [2M + Na]⁺, suggesting the existence of five degrees of unsaturation. The UV spectrum showed absorption bands consistent with a conjugated carbonyl group. Inspection of the ¹H and ¹³C NMR data (Table 1) together with DEPT and HMQC spectral data revealed the presence of one methyl, three methylenes, six methines (four olefinic) and two quaternary carbons (carbonylic).

In addition, the IR spectrum showed the presence of a hydroxyl group (3435 cm⁻¹) and trans disubstituted alkenes (1662 cm⁻¹). An absorption band appeared at 1716 cm⁻¹, and is due to the conjugated ketone, the absorption band of the 12-membered lactone potentially merged with the aforementioned as a single absorption band. The ¹H NMR splitting pattern of 1 showed coupling between the protons at δ_H 1.34 and 5.11 which indicates the presence of a CH₃-CH-O group. The ¹H-¹H COSY and ROESY spectra revealed two structural elements, one of C-2 to C-6 and the second from the unsaturated methine carbon C-8 to methyl group C-12. HMBC correlations observed from methylene protons H₂-2 (δ_H 2.68, 2.62) and deshielded oxymethine proton H-11(δ_H 5.11) to carboxy carbon C-1 established the location of the ester linkage (Table 1). The HMBC correlation of H-3 (δ_H 4.54) with C-1, C-2 together with molecular formula C₁₂H₁₆O₄ indicated the presence of a methine group and a hydroxyl group at C-3. The presence of a methyl group at C-11 was deduced by the HMBC correlation of H₃-12 (δ_H 1.34) with C-9 (δ_C 148.1), C-10 (δ_C 39.6) and C-11 (δ_C 72.1). The geometries of C4-C5 and C8-C9 double bonds were assigned E, E on the basis of ¹H-¹H coupling constants (J_H4-H5 = 15.9 Hz, J_H8-H9 = 16.1 Hz respectively) leading to the gross structure of 1 as shown (Figure 1). Therefore, compound 1 was established as (4E,8E)-3-hydroxy-11-methyloxacyclododeca-4,8-diene-1,7-dione and named as balticolid.

The orientation of hydroxyl group at C-3 (δ_H 69.1) was assigned by taking into account the ¹H-¹H coupling constant values of H-3 (δ_H 1.34) with all the neighboring protons (J_H2a-H3 = 3.4 Hz, J_H2b-H3 = 4.7 Hz, J_H3-H4 = 2.8 Hz). On the basis of vicinal coupling constant values for other protons a 3D structure for 1 was constructed as shown in Figure 2. These findings were also supported by cross peaks in ROESY spectrum and difference NOEs experiments.

Furthermore, the absolute configuration of balticolid (1) at C-3 was deduced by modified high-field ¹H NMR Mosher method [12–14]. In order to determine the absolute configuration at C-3, balticolid (1) was treated with (R) and (S)-α-methoxy-α-trifluoromethyl-phenylacetyl chloride (MTPA-Cl) in the presence of 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide to give the (S)-and (R)-MTPA esters respectively. In the ¹H NMR spectrum of the (S)-MTPA ester, the proton signals assigned to H-2a, and H-2b were observed at a higher field than those of the (R)-MTPA ester, and H-4, H-5, H-6a and H-6b were observed at a lower field than those of the (R)-MTPA ester. The bulk effect on chemical shifts induced by esterification with the chiral reagents was calculated using Δδ = (δ_S – δ_R), where δ_S and δ_R are the shifts (in ppm) of diagnostic protons neighboring the chiral center in balticolid (1) of the (S) and (R) Mosher’s esters respectively. The absolute configuration of 1 was obtained by positioning the protons with positive Δδ values on the right side and those with negative Δδ values on the left side in the model described by Ohtani et al. [13] which clearly showed that the absolute configuration at C-3 was R (Figure 3).

The relative configuration of 1 at C-11 was elucidated from the interactions observed in difference NOEs experiments. By applying a decoupling field to methine proton H-8 (δ_H 5.99) there was enhancement of H-5 (δ_H 5.75), H-6a (δ_H 3.43) and H-10a (δ_H 2.52) indicating that these protons are situated on the same face while H-6b is on the opposite face. Furthermore, enhancement of proton H-4 (δ_H 5.73) and H-11 (δ_H 5.11) upon irradiation of H-2b (δ_H 2.68) and H-9 (δ_H 6.78) were observed respectively (Figure 2) indicating that these protons are situated on the opposite face or β-oriented in 1. Therefore, the absolute configurations of both chiral centers of 1 were assigned as 3R, 11R.

Compound 1 was tested at non-cytotoxic concentrations for antiviral activity against influenza A virus and Herpes simplex virus (HSV) type I. Balticolid was only found to exhibit inhibitory activity against herpes simplex virus with an IC₅₀ value of 0.45 μM compared to 0.44 μM/aciclovir. The compound showed no remarkable antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida maltosa at 200 μg/disc in the agar-diffusion assay. Further structural modifications to optimize physico-chemical properties, by total and semi-synthetic approach could result in more potent molecules with desirable pharmacokinetic properties. Thorough structure activity relationship (SAR) studies of balticolid derivatives might result in new drug-lead candidates which can be exploited as future drugs.