New Haloterpenes from the Marine Red Alga Laurencia papillosa: Structure Elucidation and Biological Activity

Analysis of the air-dried marine red alga Laurencia papillosa, collected near Ras-Bakr at the Suez gulf (Red Sea) in Egypt delivered five new halogenated terpene derivatives: aplysiolic acid (1), 7-acetyl-aplysiol (2), aplysiol-7-one (3), 11,14-dihydroaplysia-5,11,14,15-tetrol (5a), and a new maneonene derivative 6, named 5-epi-maneolactone. The chemical structures of these metabolites were characterized employing spectroscopic methods, and the relative and absolute configurations were determined by comparison of experimental and ab initio-calculated NMR, NOE, ECD, and ORD data, and by X-ray diffraction of 2 and 6. The antimicrobial activities of the crude extract and compounds 1–3, 5a and 6 were studied.

In the course of our research to isolate and investigate bioactive metabolites from Laurencia algae collected from the shallow waters of the Red Sea on Egyptian coasts, a series of four halogenated bioactive decalin derivatives was isolated, namely aplysiolic acid (1), 7-acetyl-aplysiol (2), aplysiol-7-one (3), 11,14-dihydroaplysia-5,11,14,15-tetrol (5a), and a further chloro compound 6 ( Figure 1). We named the latter 5-epi-maneolactone (6), because of the similarity with the maneonenes, bioactive C 15 acetogenins from L. obtusa [17,18]. Additionally, thyrsiferol [19], 10-hydroxykahukuene B [20], cholesterol, hexadecanoic acid, thymine, and uracil were gained from the dichloromethane-methanol extract of L. papillosa. The chemical structures of these compounds were determined by extensive 1D and 2D NMR and ESI HR mass measurements. Their relative and absolute configurations were derived by 1D and 2D NMR measurements, from ab initio calculations of ECD, ORD, 13 C NMR data, by geometry-derived NOE predictions, and by X-ray diffraction in case of 2 and 6. The antimicrobial activity of the crude extract and of the new compounds was studied using a set of diverse microorganisms.

Working up and Structure Identification
The dichloromethane-methanol extract of air-dried L. papillosa algae was fractionated by a series of chromatographic purification steps implying silica gel and size exclusion columns, to afford the haloterpenes 1-3, 5a-c, and 6 ( Figure 1), and additionally thyrsiferol [19], 10-hydroxykahukuene B [20], cholesterol, hexadecanoic acid, thymine, and uracil. The physico-chemical properties of the new compounds are listed in Table 1.    Table 1).
The 13 C NMR spectrum (Table 2) showed a total of 13 resonances, amongst them ten in the aliphatic, two in the olefinic, and one in the carbonyl region. The 1 H NMR pattern exhibited two singlets at δ 4.90 and 4.81, which were assigned by H,H COSY and HMQC spectra as exo-methylene protons (δ C 110.2). The respective quaternary carbon signal appeared at δ 148.5 and was assigned by HMBC correlations (Figure 2). The carbonyl group, together with one methyl, six methylene, and two methine groups and three fully substituted carbons, sums up to C 13 H 17 BrO. Respectively, 1 should contain two rings, and the remaining two hydrogen and oxygen atoms are forming hydroxyl groups. "-" means that there is no carbon and therefore also no shift, as seen in the structure. An alternative is just to omit the hyphen. Based on the H,H COSY correlations, two fragments were identified in 1, a 1,1,3trisubstituted propane (-CH 2 -CH 2 -CH-), and a 1,2,4-trisubstituted butane (-CH 2 -CH-CH 2 -CH 2 -) unit, respectively. The terminal methylene protons in the first fragment (δ 2.70, 2.15) showed 2 J and 3 J HMBC correlations with the olefinic carbons C-4 and C-13, confirming their direct neighborhood. One of these methylene protons (δ 2.70) showed a 4 J COSY signal with the exo-methylene protons. The methine proton H-1 (δ 4.72) exhibited 2 J and 3 J correlations towards C-10 (δ 43.0) and the methyl singlet of C-12 (δ 14.8), respectively, suggesting a connection of C-10 with C-1 and Me-12. If C-5 is hydroxylated (δ 76.2), the downfield shift of C-1 (δ 62.9) fits best on its substitution with bromine [21]. Among others, the methylene group CH 2 -3 showed a 3 J HMBC correlation towards C-5, which itself gave cross signals with the methylene protons H 2 -13 and Me-12, so that an exomethylenecyclohexane (ring A) was formed.

7-Acetyl-aplysiol
An additional optically active colorless solid had similar chromatographic properties as 1, but with noticeably less polarity. The ESI HR mass spectrum indicated the molecular formula C 14 H 21 BrO 2 with four double bond equivalents (DBE) as well. However, an OH group in 1 was formally exchanged against a methyl group. Respectively, a methyl singlet (δ H 2.13) and its corresponding carbon (28.3) were visible. The carboxyl signal in 1 (181.0) was exchanged in 2 against a keto carbonyl at δ C 211.8, but the remaining 1 H and 13 C NMR signals were very similar in both compounds. On the basis of long-range 2D NMR couplings ( Figure 2) and NOESY experiments (Figure 4), the structure was assigned as 2, which we named 7-acetyl-aplysiol. From this ketone, the acid 1 may be formed in a haloform reaction (Einhorn reaction). With respect to the negative optical rotation, the decalin system of 2 should have the same absolute all-(S)-configuration as 1, which also agrees with biosynthetic considerations. However, as some doubt remained after comparison of the calculated and experimental ECD spectra, a crystal of 2 was analyzed additionally by X-ray diffraction ( Figure S6), which reassured the all-(S)-configuration.

Aplysiol-7-one
A third closely related decalin gave the molecular formula C 12 H 17 BrO 2 by ESI HR, containing 4 DBE as well. The 1 H and 13 C NMR spectra of 3 resembled those of 1 and 2; however, the signals of CH-7 (in 1 and 2) and CH 3 -14 (in 2) were absent, and the ABX signal of CH 2 -6 in 1 and 2 had changed into an AB signal. As indicated by the HMBC correlations and the downfield shifts of CH 2 -6 and CH 2 -8, the carbonyl signal at δ C 209.0 was due to a ß-decalone. Furthermore, long-range 2D NMR couplings ( Figure 2) confirmed structure 3, which we named as aplysiol-7-one. In addition, the all-(S)-configuration was plausible for NOESY experiments ( Figure 4) and biosynthetic reasons; it was, however, not further analyzed due to an inseparable contamination by 10-hydroxykahukuene B [20].
2.1.4. 11,14-Dihydroaplysia-5,11,14,15-tetrols A further brominated terpenoide with moderate polarity was isolated as an optically active resin with color reactions similar as of 1 ( Table 1). The NMR spectrum (Table 3) of this compound showed 20 13 C signals. At high magnification, however, each of them appeared as a group of up to four signals in distances of <0.2 ppm (Figures S34 and S35), so that a mixture of four stereoisomers or otherwise closely related compounds was expected; this was confirmed by the 1 H NMR signal of the ∆ 12 double bond ( Figure S11) and also by analytical HPLC analyses, where three components in the ratio of~1:1:1 were separated. By HPLC/HRMS, two stereoisomers C 20 H 33 BrO 4 (5a,b) and a slightly more polar component C 20 H 33 BrO 5 (5c) were detected (Figures S12 and S13). The latter compound seems to be a 11,14-dihydroaplysiapentol; we need, however, to isolate further material to fully elucidate the structure (see Supporting Information). Table 3. 13 C (125 MHz) and 1 H NMR (300 MHz) data of dihydroaplysiatetrols (5a and 5b) in CD 3 OD. For the 13 C shifts, we used two digits behind the decimal point to differentiate between 5a and its epimer 5b); in Figure S15, the average of shifts for 5a and 5b was used with only one digit.
On this basis, the absolute configuration of the main component 5a was determined as (1S,5S,7S,10S,11R,14R) by correlation of NOE signals with ab-initio calculated atom distances (Table S6 and Figure S7); the other diastereomers could not be assigned, due to overlapping signals.

5-epi-Maneolactone
Compound 6 displayed neither UV absorbance nor fluorescence on TLC, but turned brownish gray with anisaldehyde/sulfuric acid on heating. The molecular weight was determined by ESI MS: two quasi-molecular ion peaks in the ratio of 1:0.33 at m/z 238 and 240 indicated the presence of one chlorine atom. The corresponding molecular formula C 12 H 11 ClO 3 was determined by ESI HRMS and indicated seven DBE ( Table 1). The IR spectrum of 6 displayed a characteristic vibration signal (ν = 2361 cm −1 ) of an acetylenic bond. Two absorption bands at ν =1781 and 1592 cm −1 indicated the presence of a lactone carbonyl and an olefinic double bond, respectively, so that three rings were expected.
The 1 H NMR methine signal at δ H 3.36 did not show an HSQC correlation; however, a large HMBC coupling with the CH carbon at δ C 86.5 and a smaller one with a C q at δ C 78.2 was noted. Together with strong long-range correlations with a cis-double bond (δ C 113.8, 139.2, J = 10.5 Hz), this indicated a terminal acetylene in conjugation with a double bond. According to further HMBC and COSY correlations, the (Z)-enyne unit was connected with the methines C-5 and C-6, and the latter additionally with CH-7 and CH-11 ( Figure 5). A detailed analysis resulted in a ((Z)-pent-2-en-4-ynyl)-cyclohexane, where the shift assignments of positions CH-7, -9, and -10 remained open due to their overlapping 1 H NMR signals (Table 4).  According to AntiBase [27], only two groups of metabolites with these structural features have been described before, the lembynes [2,28], and the maneonenes [17,18]. All were isolated from Laurencia spp. If chlorine was present, it was found in position C-5; also the shifts of 6 were explained best by chlorine at C-5. HMBC correlations of the upfield methine protons CH-6 and CH-11 with the signal at δ C 173.5 connected the ester carbonyl C-12 with C-11, and the correlation with the oxymethine CH-9 (or CH-7) via oxygen closed a lactone ring with C-12. The remaining oxygen atom bridges the oxymethine groups CH-10 with CH-7 or CH-9, so that all DBE were used. Further correlation analyses by means of COCON [29] delivered four structure options, with structure 6 being the only plausible one ( Figure 5 and Figure S16). A suitable crystal allowed us finally to confirm this structure including the absolute configuration unambiguously by X-ray diffraction ( Figure 6). DFT calculations afforded the same absolute configuration, so that the validity of the computational methods applied here were additionally confirmed (see Supporting Information). We named compound 6 as 5-epi-maneolactone, as it looks like an oxidative cleavage product of the cis-maneonenes. It should be mentioned, however, that C-5 is (R)-configured in these compounds, oppositely to the (5S)-configured 6. Figure 6. Crystal structure and absolute configuration of 5-epi-maneolactone (6) by X-ray diffraction.

Collection and Taxonomy of the Marine Alga
The red alga L. papillosa (Forsk., Grev) was collected in summer 2009 at Ras Abu-Bakr, 65 km north of Ras Gharib on the Suez-Gulf, Red Sea, Egypt. The identification was carried out by G. S. Abou-El Wafa according to Nasr's method [30,31]. A reference specimen of the alga is kept at the Department of Botany, Faculty of Science, Mansoura University, Egypt. Samples of L. papillosa were separated from epiphytes and the algal material rinsed in tap water and distilled water. The sample was then spread on string nets, allowed to dry in air, ground, and stored in closed bottles at room temperature.

Antimicrobial Assay
Antimicrobial assays using the agar diffusion test [32] were performed as described previously [33]. M. miehei Tü 284 and S. viridochromogenes Tü 57 were obtained from the collection of H. Zähner (University of Tübingen, Germany), and Chlorella vulgaris was provided by the Algal Collection Göttingen. B. subtilis ATCC 6051 was obtained from the American Type Culture Collection, while S. aureus, E. coli and C. albicans are clinical isolates from Göttingen hospitals. Strains are kept in the strain collection of H. Laatsch, Institute of Organic and Biomolecular Chemistry, Georg-August University, Göttingen, Germany.

Ab Initio Calculations
DFT calculations were performed as described previously [34].