New C15 Acetogenins from Two Species of Laurencia from the Aegean Sea

The chemical diversity of the approximately 1,200 natural products isolated from red algae of the genus Laurencia, in combination with the wide range of their biological activities, have placed species of Laurencia in the spotlight of marine chemists’ attention for over 60 years. The chemical investigation of the organic (CH2Cl2/MeOH) extracts of Laurencia microcladia and Laurencia obtusa, both collected off the coasts of Tinos island in the Aegean Sea, resulted in the isolation of 32 secondary metabolites, including 23 C15 acetogenins (1–23), 7 sesquiterpenes (24–30) and 2 diterpenes (31 and 32). Among them, six new C15 acetogenins, namely 10-acetyl-sagonenyne (2), cis-sagonenyne (3), trans-thuwalenyne C (4), tinosallene A (11), tinosallene B (12) and obtusallene XI (17), were identified and their structures were elucidated by extensive analysis of their spectroscopic data. Compounds 1–3, 5–11, 13 and 15–32 were evaluated for their antibacterial activity against Staphylococcus aureus and Escherichia coli.


Introduction
Red algae of the genus Laurencia can be found in temperate to tropical shores around the world, inhabiting littoral to sublittoral zones and spreading to depths down to 65 m. The plasticity of their distinction markers has led to debates and frequent taxonomic revisions of the genus, which currently includes 137 accepted species [1]. Species of the genus Laurencia have been extensively investigated for over 60 years, resulting in the isolation of more than 1200 natural products, mainly sesquiterpenes, diterpenes, triterpenes and C 15 acetogenins [2,3]. Nevertheless, they are still considered a fascinating source of new natural products, usually halogenated, that exhibit a variety of biological activities, including cytotoxic [4,5], antimicrobial [6], antiviral [7], antiparasitic [8], anti-inflammatory [9] and antifouling [10].
In the framework of our ongoing research on new bioactive natural products from the genus Laurencia, we investigated the chemical profiles of Laurencia microcladia Kützing and Laurencia obtusa (Hudson) Lamouroux, both collected off the coasts of Tinos Island in the Aegean Sea. Herein, we report the isolation and structure elucidation of 32 compounds (1-32) (Figure 1), among which six (2, 3, 4, 11, 12 and 17) are new natural products. In addition, we report the evaluation of the antibacterial activities of compounds 1-3, 5-11, 13 and 15-32 against Staphylococcus aureus and Escherichia coli.
Compound 2 was obtained as a colorless oil. Based on HR-APCIMS and NMR data, its molecular formula was calculated as C 19 H 26 Br 2 O 5 . The presence of three quaternary, nine methine, four methylene and three methyl carbon atoms was deduced by the HSQC and HMBC spectra (Tables 1 and 2). In addition, the chemical shift of the quaternary carbon at δ C 81.5, along with the resonances of three tertiary carbons at δ C 77.3, 112.2 and 141.2, were indicative of a terminal enyne moiety. Furthermore, the presence of two acetoxy groups was evident. The remaining signals in the 1 H NMR spectrum of 2 were reminiscent of C 15 acetogenins, frequently isolated from species of Laurencia. Indeed, analysis of the HSQC, HMBC and COSY spectra revealed that 2 featured a tetrahydropyran ring resulting from the formation of an ether bridge between C-9 and C-13, with the two acetoxy groups positioned at C-7 and C-10 ( Figure 2). The geometry of the ∆ 3 double bond was determined as E on the basis of the coupling constant value (J = 15.8 Hz) measured between the olefinic methines H-3 and H-4, as well as the chemical shift of the acetylenic proton H-1 (δ H 2.83). The assignment of the relative configuration of the asymmetric centers of 2 relied on the measured coupling constants and observed NOE cross-peaks ( Figure 3). Specifically, the NOE interactions of H-9/H-13, H-9/H-11α and H-11β/H-12, along with the coupling constant of H-12/H-13 (J = 10.0 Hz), established the axial orientation of H-9, H-11α, H-12 and H-13 and the relative configurations at C-9, C-12 and C-13. The equatorial orientation of H-10 and the relative configuration at C-10 were deduced based on the small coupling constants of H-10, which appeared as a broad singlet peak, and the NOE enhancement of H-9/H-10. Thus, the relative configuration of the stereogenic centers of 2 was established as 9R*,10R*,12R*,13S*, and as such compound 2 was identified as 10-acetyl-sagonenyne.  Compound 3, which exhibited a pseudomolecular ion peak at m/z 451.0111 with isotopic ion peaks at m/z 453.0092 and 455.0070 (HR-APCIMS) corresponding to C 17 H 25 Br 2 O 4 , was isolated as a colorless oil. The NMR spectroscopic features (Tables 1 and 2), pointing to a C 15 acetogenin bearing an acetoxy group, closely resembled those of metabolite 1. Analysis of the 2D NMR spectra of 3 suggested that it was the geometric isomer of 1. Indeed, the chemical shift of the acetylenic proton H-1 (δ H 3.12) and the coupling constant value measured between the olefinic methines H-3 and H-4 (J = 10.9 Hz) assigned the geometry of the double bond of the terminal -enyne system of 3 as Z, while the remaining NOE enhancements and coupling constants observed were in accordance with those of compound 1. Therefore, compound 3 was identified as cis-sagonenyne.
Compound 4 was obtained as a colorless oil. Its molecular formula was deduced as C 15 H 21 Br 2 O on the basis of its HR-APCIMS and NMR data. Comparison of its 1 H and 13 C NMR data (Tables 1 and 2) with those of thuwalenyne C [40] revealed that the main difference was the geometry of the ∆ 3 double bond, which was determined as E, as suggested by the coupling constant measured between H-3 and H-4 (J = 16.0 Hz) and the chemical shift of the acetylenic proton H-1 (δ H 2.79). The geometry of the ∆ 6 double bond was assigned as Z on the basis of the resonance of the doubly allylic methylene carbon C-5 at δ C 30.7 [41]. Even though compound 4 proved unstable before the acquisition of NOESY spectrum, the high similarity observed in the chemical shifts and coupling constants of H-9 (3.44 ddd (6.8, 6.8, 0.8) vs. 3.45 ddd (7.3, 7.3, 0.6)), H-10 (3.67 brs vs. 3.68 brs), H-12 (3.98 ddd (12.5, 10.3, 4.7) vs. 3.98 ddd (12.3, 9.9, 4.5)) and H-13 (3.33 ddd (10.3, 9.0, 2.3) vs. 3.33 ddd (9.9, 9.9, 2.1)) of 4 in comparison to those of thuwalenyne C [40] unambiguously suggested the same relative configuration. Therefore, compound 4 was identified as trans-thuwalenyne C. Tinosallene A (11), isolated as a yellow oil, displayed the molecular formula C 15 H 20 Br 2 O 2 , as indicated by the HR-APCIMS and NMR data. The HSQC and HMBC spectra indicated the presence of 15 carbons, including one methyl, four methylenes, nine methines and one non-protonated carbon (Tables 1 and 2). The signals of a bromoallene moiety (δ H/C 6.09/74.5, 202.9 and 5.39/100.2) and two olefinic methines (δ H/C 5.82/129.9 and 5.85/128.0), as well as of five deshielded carbons bearing halogen or oxygen atoms (δ C 63.2, 75.4, 75.4, 80.8 and 85.4) were evident. Since the allene moiety and the isolated double bond accounted for three of the five degrees of unsaturation, the molecular structure of 11 was determined as bicyclic. The NMR spectroscopic features of 11 (Tables 1 and 2) resembled those of thuwalallene E [40]. Specifically, the COSY correlations identified an extended spin system spanning from C-3 to C-15, while the HMBC correlations from H-4 to C-10 and from H-12 to C-9 identified the oxocane and tetrahydrofuran rings, respectively ( Figure 2). The NOE cross-peaks of H-3/H-10 and H-9/H-10, in conjunction with the simultaneous absence of NOE correlations between H-4 and H-9, as well as between H-10 and H-12, determined the relative configurations at C-4, C-9, C-10 and C-12 as 4R*,9S*,10S*,12R* ( Figure 3). The absolute configuration of the bromoallene functionality was established as 2S on the basis of the positive value of its measured optical rotation, according to the empirical rule proposed by Lowe about the absolute configuration of chiral allenes [42,43].  (12), obtained as a yellow oil, had the same molecular formula (C 15 H 20 Br 2 O 2 ) as 11, as established from its HR-APCIMS and NMR data. In addition, the 1 H NMR data of 12 shared a high degree of similarity to those of compound 11, with the exception of H-9, H-11 and H-12 (Tables 1 and 2). Analysis of the 2D NMR spectra of 12, which proved to be unstable, indicated the same planar structure as that of tinosallene A (11). However, the correlations of H-3/H-10, H-9/H-10 and H-10/H-12 observed in the NOESY spectrum of 12 established the relative configurations at C-4, C-9, C-10 and C-12 as 4R*,9S*,10S*,12S* (Figure 3), thereby identifying 12 as the epimer of 11 at C-12. Moreover, the positive value of the optical rotation measured for compound 12 was indicative of the 2S configuration of the bromoallene moiety.
Obtusallene XI (17), obtained as a white solid, displayed the molecular formula C 15 H 18 Br 3 ClO 2 , as indicated by its HR-APCIMS measurements. The presence of one nonprotonated, eleven methine, two methylene and one methyl carbon atoms was confirmed by the HSQC and HMBC experiments (Tables 1 and 2). Further interpretation of its NMR data revealed a bromoallene moiety (δ H/C 6.05/74.  (Table S1). Among them, iso-laurenisol (24) and bromolaurenisol (25) were active against S. aureus, with minimum inhibition concentrations (MICs) of 16 and 4 µg/mL, respectively, while rogiolenyne B (7) was mildly active against the same strain, with a MIC value of 128 µg/mL. In contrast, all of the compounds tested were proven inactive against E. coli.

General Experimental Procedures
Optical rotations were measured on a Krüss model P3000 polarimeter (A. KRÜSS Optronic GmbH, Hamburg, Germany) with a 0.5 dm cell. UV spectra were obtained on a Perkin Elmer Lambda 40 spectrophotometer (PerkinElmer Ltd., Buckinghamshire, UK). IR spectra were obtained on a Bruker Alpha II spectrometer (Bruker Optik GmbH, Ettlingen, Germany). The 1D and 2D NMR spectra were recorded on Bruker AC 200, DRX 400, Avance III 600 and Avance NEO 700 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometers, using standard Bruker pulse sequences at room temperature. Chemical shifts are given on a δ (ppm) scale using TMS as the internal standard. High-resolution APCI mass spectra were measured on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Low-resolution EI mass spectra were measured on a Hewlett-Packard 5973 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Low-resolution CI mass spectra were measured on a Thermo Electron Corporation DSQ mass spectrometer (Thermo Electron Corporation, Austin, TX, USA) using a direct-exposure probe with CH 4 as the reagent gas. Normal-and reversed-phase column chromatography separations were performed with Kieselgel Si 60 (Merck, Darmstadt, Germany) and Kieselgel RP-18 (Merck, Darmstadt, Germany), respectively. HPLC separations were conducted on a Pharmacia LKB 2248 liquid chromatograph (Pharmacia LKB Biotechnology, Uppsala, Sweden) equipped with an RI-102 Shodex refractive index detector (ECOM spol. s r.o., Prague, Czech Republic), using the following columns: (i) Econosphere Silica 10u (250 × 10 mm, Grace, Columbia, MD, USA) or (ii) Chiralcel OD 10 µm (250 × 10 mm, Daicel Chemical Industries Ltd., Osaka, Japan). TLC was performed with Kieselgel 60 F 254 aluminum-backed plates (Merck, Darmstadt, Germany) and spots were visualized after spraying with 15% (v/v) H 2 SO 4 in MeOH reagent and heating at 100 • C for 1 min.

Biological Material
Specimens of Laurencia microcladia Kützing and Laurencia obtusa (Hudson) Lamouroux were collected from the coasts of Agia Kyriaki and Agios Sostis, respectively, at Tinos Island in the Aegean Sea, at a depth of 0.5-2 m in September 2011. Voucher specimens of the algae have been deposited at the Herbarium of the Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens (ATPH/MP0290 and ATPH/MP0291, respectively).

Evaluation of Antibacterial Activity
The bacterial strains S. aureus ATCC-25923 and E. coli NCTC-10418 were generously provided by Prof. S. Gibbons (University of East Anglia). The antibacterial activity evaluation was performed as previously described [44]. Briefly, both strains were cultured on nutrient agar and incubated at 37 • C for 24 h prior to the determination of MIC values. Compounds 1-3, 5-11, 13 and 15-32 were dissolved in DMSO and subsequently diluted in Mueller−Hinton broth (MHB) to give an initial concentration of 512 µg/mL. For each strain, bacterial inocula equivalent to the 0.5 McFarland turbidity standard were prepared in normal saline and diluted to a final inoculum density of 5 × 10 5 cfu/mL. MHB supplemented with 10 mg/L Mg 2+ and 20 mg/L Ca 2+ (125 µL/well) was dispensed into wells 1−11 of each row of 96-well microtiter plates. The compound solution (125 µL) was added to the first well of each row and serially diluted across the row, leaving well 11 empty for growth control. The final volume was dispensed into well 12, which being free of MHB or inoculum served as the sterility control. The inoculum (125 µL/well) was added to wells 1−11 of each row, and the microtiter plates were incubated at 37 • C for 18 h. The lowest concentration at which no bacterial growth was observed was recorded as the MIC. The observation was confirmed by the addition of a 5 mg/mL methanolic solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 20 µL/well) and further incubation at 37 • C for 20 min. Bacterial growth was indicated by a color alteration from yellow to dark blue. Vancomycin and chloramphenicol were used as positive controls for S. aureus and E. coli strains, respectively. The highest concentration of DMSO remaining after dilution (3.125% v/v) caused no inhibition of bacterial growth. All samples were tested in triplicate.

Conclusions
The chemical investigations of the organic extracts of L. microcladia and L. obtusa collected off the coasts of Tinos Island in the Aegean Sea led to the isolation and structure elucidation of six new (2-4, 11, 12 and 17) and 26 known (1, 5-10, 13-16 and 18-32) secondary metabolites. Among the new compounds, 2, 3 and 4 possessing a 9,13-epoxy ring belong to the small group of tetrahydropyran C 15 acetogenins, which includes only seven more representatives to date. Tinosallenes A (11) and B (12) are stereoisomers of thuwalallene E, with these three being the only C 15 acetogenins bearing a 4,10:9,12-bisepoxy system. Obtusallene XI (17) belongs to the obtusallene family, a rare class of twelve-membered ring C 15 acetogenins, consisting of approximately 25 representatives characterized by a 4,14-epoxy core and a bromoallene terminal functionality, while incorporating one or two more ether rings into their structures. Iso-laurenisol (24) and bromolaurenisol (25) were active against S. aureus, while all metabolites tested were proven inactive against E. coli.

Data Availability Statement:
The data presented in this study are available in the present article and the supplementary material.