Secondary Metabolites with Anti-Inflammatory Activity from Laurencia majuscula Collected in the Red Sea

The chemical investigation of the organic extract of the red alga Laurencia majuscula collected from Hurghada reef in the Red Sea resulted in the isolation of five C15 acetogenins, including four tricyclic ones of the maneonene type (1–4) and a 5-membered one (5), 15 sesquiterpenes, including seven lauranes (6–12), one cuparane (13), one seco-laurane (14), one snyderane (15), two chamigranes (16, 17), two rearranged chamigranes (18, 19) and one aristolane (20), as well as a tricyclic diterpene (21) and a chlorinated fatty acid derivative (22). Among them, compounds 1–3, 5, 7, 8, 10, 11 and 14 are new natural products. The structures and the relative configurations of the isolated natural products have been established based on extensive analysis of their NMR and MS data, while the absolute configuration of maneonenes F (1) and G (2) was determined on the basis of single-crystal X-ray diffraction analysis. The anti-inflammatory activity of compounds 1, 2, 4–8, 10, 12–16, 18 and 20–22 was evaluated by measuring suppression of nitric oxide (NO) release in TLR4-activated RAW 264.7 macrophages in culture. All compounds, except 6, exhibited significant anti-inflammatory activity. Among them, metabolites 1, 4 and 18 did not exhibit any cytostatic activity at the tested concentrations. The most prominent anti-inflammatory activity, accompanied by absence of cytostatic activity at the same concentration, was exerted by compounds 5 and 18, with IC50 values of 3.69 μM and 3.55 μΜ, respectively.


Introduction
The marine environment is considered to be one of the richest sources of diverse natural products with a wide array of biological activities [1]. The Red Sea, lying between Africa and Asia, is the world's northernmost tropical sea, with its seawater inlet at the Indian Ocean. The more than 2000 km-long stretch of coral reef system of the Red Sea, hosting a rich biodiversity with a high number of endemic species, is among the five most extended reefs in the world. Nonetheless, the biota of the Red Sea, in comparison to that of other tropical areas, has not been as extensively investigated as a source of bioactive marine natural products [2].
Among red algae, the genus Laurencia (order Ceramiales, family Rhodomelaceae), including approx. 140 accepted species distributed in tropical, subtropical and temperate coastal waters, is one of the most prolific sources of new secondary metabolites in the marine environment, even though its members have been the subject of intense studies during the last 60 years [1, [3][4][5]. To date, more than 1100 secondary metabolites, mostly halogenated acetogenins and terpenes often displaying unprecedented carbocycles, have been reported from Laurencia species and mollusks feeding on them, among which a significant number has displayed antibacterial, antifungal, antiviral, anti-inflammatory, antiproliferative, cytotoxic, antifouling, antifeedant, ichthyotoxic and insecticidal activity [1, 4,5].
In the framework of our interests, aiming for the isolation of new bioactive marine metabolites, we investigated the chemical profile of Laurencia majuscula collected from Hurghada reef. Herein, we report the isolation and structure elucidation of 22 metabolites (1-22, Figure 1), among which nine (1-3, 5, 7, 8, 10, 11 and 14) are new natural products, as well as the evaluation of their anti-inflammatory activity.
of other tropical areas, has not been as extensively investigated as a source of bioactive marine natural products [2].
Among red algae, the genus Laurencia (order Ceramiales, family Rhodomelaceae), including approx. 140 accepted species distributed in tropical, subtropical and temperate coastal waters, is one of the most prolific sources of new secondary metabolites in the marine environment, even though its members have been the subject of intense studies during the last 60 years [1, [3][4][5]. To date, more than 1100 secondary metabolites, mostly halogenated acetogenins and terpenes often displaying unprecedented carbocycles, have been reported from Laurencia species and mollusks feeding on them, among which a significant number has displayed antibacterial, antifungal, antiviral, anti-inflammatory, antiproliferative, cytotoxic, antifouling, antifeedant, ichthyotoxic and insecticidal activity [1,4,5].
In the framework of our interests, aiming for the isolation of new bioactive marine metabolites, we investigated the chemical profile of Laurencia majuscula collected from Hurghada reef. Herein, we report the isolation and structure elucidation of 22 metabolites (1-22, Figure 1), among which nine (1-3, 5, 7, 8, 10, 11 and 14) are new natural products, as well as the evaluation of their anti-inflammatory activity.
Maneonene F (1) was obtained as a white amorphous solid, possessing the molecular formula C 16 H 20 BrClO 3 as deduced from the HR-APCIMS measurements. The presence of one bromine and one chlorine in the molecule was also evident in the LR-EIMS spectrum, as indicated by the fragment ion [M − OCH 3 ] + cluster at m/z 343/345/347 with relative intensities 3:4:1. The spectroscopic data of metabolite 1 (Table 1), in conjunction with the correlations observed in its HSQC, HMBC and COSY spectra, suggested a tricyclic C 15 acetogenin of the maneonene type. Specifically, the HSQC-DEPT and HMBC spectra revealed the presence of one methyl on a secondary carbon atom and an oxymethyl, two methylenes, ten methines and two non-protonated carbons. In the COSY spectrum, the presence of one extended spin system spanning from H-1 to H-11 and a shorter one from H-13 to H 3 -15 ( Figure 2a) were evident. The HMBC correlations of H-10 (δ H 5.30) with C-7 (δ C 77.5), as well as of H-9 (δ H 4.40) with C-12 (δ C 108.2), confirmed the presence of two ether bridges between C-7 and C-10 and between C-9 and C-12, while the correlations of C-12 with H-6 (δ H 2.87), H-13 (δ H 4.04) and H 3 -16 (δ H 3.27) connected the two spin systems concluding the tricyclic skeleton and secured the position of the methoxy group at C-12. In addition, the HMBC correlations of C-2 (δ C 78.3) with H-3 (δ H 5.60) and H-4 (δ H 5.90), as well as of C-4 (δ C 141.6) with H-1 (δ H 3.24), confirmed the presence of the terminal -enyne moiety, which is frequently encountered in C 15 acetogenins [4]. The relative configuration of metabolite 1 was proposed on the basis of the cross-peaks observed in its NOESY spectrum. Specifically, the correlations of H 3 -16 with H-13 and of H-11 with H-14b (δ H 1.48), as well as that of H-11 with H-10 and of H-10 with H-9, secured the relative configuration at C-9, C-10, C-11 and C-12. Furthermore, the absence of measurable coupling of H-6 with both H-7 and H-11, indicating an almost 90 • dihedral angle between H-6-C-6-C-7-H-7, as well as between H-6-C-6-C-11-H-11, in conjunction with the NOE enhancement of H-5 with H-11, secured the relative configuration at C-6 and C-7. The Z geometry of the double bond in the -enyne moiety was deduced from the coupling constant of H-3 with H-4 (J = 10.6 Hz) and the chemical shift of the acetylenic proton (δ H 3.24). Single crystal X-ray diffraction analysis of a crystal of 1 (Figure 2b) allowed for the verification of its proposed structure, including the unambiguous assignment of the relative configuration at C-5 and C-13, which could not be determined by analysis of the NMR spectroscopic data. The absolute stereochemistry of maneonene F (1) was determined as 5R,6S,7R,9R,10R,11S,12S,13R.
The spectroscopic data of metabolites 2 and 3 (Table 1), possessing the same molecular formulae as compound 1, closely resembled those of maneonene F (1), suggesting the same planar structure for 2 and 3, as was further confirmed from the correlations observed in their respective HSQC-DEPT, HMBC and COSY spectra. The observed correlations in the NOESY spectrum of metabolite 2 were similar to those observed for metabolite 1, suggesting the same relative configuration in the rigid tricyclic scaffold of compound 2. However, the fact that H-10, H-13 and H-14a were shielded (δ H 4.88, 3.96 and 1.79, respectively, for 2 vs. δ H 5.30, 4.04 and 1.93, respectively, for (1) suggested a change in the relative configuration at C-13. Indeed, single-crystal X-ray diffraction analysis of a crystal of 2 ( Figure 2c) verified that maneonene G (2) was the 13-epimer of 1 and established its absolute stereochemistry as 5R,6S,7R,9R,10R,11S,12S,13S. In the NOESY spectrum of maneonene H (3), the correlations of H-9 with H-10 and of H-10 with H-11, in combination with that of H-13 with H-6, secured the relative configuration at C-9, C-10 and C-11 and suggested the inversion of relative configuration at C-12. The fact that H-6 resonates in higher fields in 3 in comparison to 1 and 2 (δ H 2.43 in 3 vs. δ H 2.87 and 2.97 in 1 and 2, respectively) also corroborates the inversion of the orientation of the methoxy group at C-12. The relative configuration at C-6 and C-7 was determined, as in the case of 1 and 2, on the basis of the absence of coupling of H-6 with both H-7 and H-11 (J 6,7 ≈ 0 Hz and J 6,11 ≈ 0 Hz) and the NOE cross-peak of H-5 with H-11, whereas the relative configuration at C-5 and C-13 could not be unambiguously assigned on the basis of analysis of the NMR spectroscopic data. Table 1. 13 C and 1 H NMR data (δ in ppm, J in Hz) of compounds 1, 2, 3 and 5.
Position    Surprisingly, in comparison to 1 and 2, maneonene H (3) proved unstable and rapidly converted upon standing to a mixture of compounds 1, 2 and 4, thus securing the absolute configuration for the asymmetric centers C-5, C-6, C-7, C-9, C-10 and C-11 in 3 and 4, as in the cases of 1 and 2. It can be hypothesized that 3, having a methoxy substituent at C-12 with an R configuration, is less stable and, through demethoxylation and formation of 4 as an intermediate, results in the production of the more stable stereoisomers 1 and 2. Interestingly, when compound 4 was subjected to reversed-phase HPLC, it afforded a mixture of the hydroxylated at C-12 derivatives 23-26 ( Figure 3). Attempts to purify the four derivatives were proven unsuccessful, since it seems that these derivatives exist in a dynamic equilibrium. Specifically, even though four distinct peaks could be observed in normal-phase HPLC that were separately collected, the 1 H NMR spectra of the individual peaks revealed their interconversion. Therefore, structure elucidation of these derivatives was based on 2D NMR and MS data of the mixture of 23-26 (at a 1:2:1:2 ratio), as well as of the mixture of 23 and 24 (at a 1:1 ratio) ( Table S1). The S configuration at C-12 for 23 and 25 was assigned on the basis of NOE interactions between H-10 and H-13, while the absence of the particular NOE cross-peak for 24 and 26 indicated an R configuration at C-12. Based on the above observations, it cannot be excluded that compounds 1-3 might not be the actual natural products present in the fresh algal tissues of the red alga, since the acetals 1-3 could be produced during the extraction process upon addition of MeOH on the enol ether 4.
3 in comparison to 1 and 2 (δΗ 2.43 in 3 vs. δΗ 2.87 and 2.97 in 1 and 2, respectively) also corroborates the inversion of the orientation of the methoxy group at C-12. The relative configuration at C-6 and C-7 was determined, as in the case of 1 and 2, on the basis of the absence of coupling of H-6 with both H-7 and H-11 (J6,7 ≈ 0 Hz and J6,11 ≈ 0 Hz) and the NOE cross-peak of H-5 with H-11, whereas the relative configuration at C-5 and C-13 could not be unambiguously assigned on the basis of analysis of the NMR spectroscopic data.
Surprisingly, in comparison to 1 and 2, maneonene H (3) proved unstable and rapidly converted upon standing to a mixture of compounds 1, 2 and 4, thus securing the absolute configuration for the asymmetric centers C-5, C-6, C-7, C-9, C-10 and C-11 in 3 and 4, as in the cases of 1 and 2. It can be hypothesized that 3, having a methoxy substituent at C-12 with an R configuration, is less stable and, through demethoxylation and formation of 4 as an intermediate, results in the production of the more stable stereoisomers 1 and 2. Interestingly, when compound 4 was subjected to reversed-phase HPLC, it afforded a mixture of the hydroxylated at C-12 derivatives 23-26 ( Figure 3). Attempts to purify the four derivatives were proven unsuccessful, since it seems that these derivatives exist in a dynamic equilibrium. Specifically, even though four distinct peaks could be observed in normal-phase HPLC that were separately collected, the 1 H NMR spectra of the individual peaks revealed their interconversion. Therefore, structure elucidation of these derivatives was based on 2D NMR and MS data of the mixture of 23-26 (at a 1:2:1:2 ratio), as well as of the mixture of 23 and 24 (at a 1:1 ratio) ( Table S1). The S configuration at C-12 for 23 and 25 was assigned on the basis of NOE interactions between H-10 and H-13, while the absence of the particular NOE cross-peak for 24 and 26 indicated an R configuration at C-12. Based on the above observations, it cannot be excluded that compounds 1-3 might not be the actual natural products present in the fresh algal tissues of the red alga, since the acetals 1-3 could be produced during the extraction process upon addition of MeOH on the enol ether 4.   (Table 1), indicated a monocyclic carbocycle. The HSQC-DEPT and HMBC spectra revealed the presence of one methyl on a secondary carbon atom and an acetyl methyl, six methylenes, seven methines, among which one was halogenated and three were oxygenated, as well as two non-protonated carbon atoms. The correlations in the COSY spectrum revealed the presence of a single extended spin system spanning from H-1 to H 3 -15, positioning the bromine atom at C-6 and confirming the presence of the -enyne functionality. The HMBC correlation of H-7 (δ H 3.69) with C-10 (δ C 81.8) confirmed the ether bridge between C-7 and C-10. The acetoxy group was placed at C-9 on the basis of the HMBC correlation between H-9 (δ H 5.08) and the carbonyl carbon C-16 (δ C 169.2). The relative configurations of the asymmetric centers of metabolite 5 were proposed on the basis of the NOE correlations between H-7 and H-10 indicating their cis orientation, while the lack of NOE correlation between H-7 and H-9 indicated their trans orientation. The Z geometry of the double bond in the -enyne moiety was dictated from the coupling constant of H-3 with H-4 (J = 10.6 Hz) and the chemical shift of the acetylenic proton (δ H 2.75). Thus, metabolite 5 was identified as (3Z,7S*,9R*,10S*)-9-acetoxy-6-bromo-7,10-epoxypentadec-3-en-1-yne.
Metabolite 7, obtained as colorless oil, possessed the molecular formula C 15 H 18 O, as indicated by the HR-ESIMS and NMR spectroscopic data. The presence of a substituted benzene ring was suggested from the absorbances at 1648 and 1508 cm −1 in the IR spectrum and the two doublets resonating at δ H 7.04 and 7.10 and integrating for two protons each, indicative for a para-substituted aromatic ring. In addition, the intense absorption band at 1703 cm −1 dictated the presence of a carbonyl group in the molecule. The HSQC-DEPT and HMBC spectra revealed the presence of four methyls, one methylene, four methines and six non-protonated carbon atoms. The spectroscopic features of metabolite 7 (Table 2), in conjunction with the correlations observed in the HMBC and COSY spectra, suggested a laurane skeleton for compound 7. Specifically, the position of H 3 -14 on C-1 was confirmed by the HMBC correlations of H 3 -14 (δ H 1.56) with C-1 (δ C 47.8), C-2 (δ C 176.2), C-5 (δ C 53.3) and the aromatic carbon C-6 (δ C 142.0). The correlations of H 3 -12 (δ H 1.71) with C-1, C-2 and C-3 (δ C 135.5) and those of H 3 -13 (δ H 1.76) with C-2, C-3 and C-4 (δ C 208.6) secured the positions of H 3 -12 on C-2 and H 3 -13 on C-3, thus allowing for the identification of 7 as 4-oxoisolaurene.   Table 2). The remaining signals included three singlet methyls, one on an aliphatic quaternary carbon (δ H 1.47) and two on oxygenated quaternary carbons (δ H 1.10 and 1.49), as well as an oxygenated aromatic methylene resonating at δ H 4.66 (H 2 -15). The six degrees of unsaturation and the presence of an aromatic ring dictated a tricyclic skeleton. The presence of a 1,2,3-trimethylcyclopentanyl moiety was deduced from the COSY correlations between H 2 -4 and H 2 -5 and the correlations in the HMBC spectrum from H 3 -12 to C-1, C-2 and C-3, from H 3 -13 to C-2, C-3 and C-4, and from H 3 -14 to C-1, C-2, C-5 and C-6, suggesting a laurane carbocycle. Furthermore, according to the molecular formula of compound 10, the remaining oxygen atom in the molecule was assigned to an oxirane ring between C-2 and C-3, as supported from the chemical shifts of C-2 (δ C 72.6) and C-3 (δ C 70.4) and the observed HMBC correlations. The relative configuration of the asymmetric centers of compound 10 was determined on the basis of the correlations observed in the NOESY spectrum. Specifically, the NOE correlations of the aromatic protons at δ H 7.15 (H-7/H-11) with H 3 -12, H 3 -13 and H-4b (δ H 1.83) suggested the anticoplanar orientation of H 3 -14 in relation to H 3 -12 and H 3 -13, thus allowing for the identification of 10 as (1S*,2S*,3R*)-2,3-epoxy-15-hydroxydihydroisolaurene.
Metabolite 11, isolated as colorless oil, possessed the molecular formula C 15 H 20 O, as suggested by its LR-EIMS and NMR data. The spectroscopic data of compound 11 were rather similar to those of compound 10, with the main differences being the absence of the singlet at δ H 4.66 attributed to the hydroxymethylene at C-9 of the aromatic ring and the presence of a singlet resonating at δ H 2.31 attributed to an aromatic methyl ( Table 2). The COSY and HMBC correlations verified the planar structure of 11. Compound 11 was proven quite unstable and degraded prior to the acquisition of a NOESY spectrum. Nevertheless, the high structural similarity of 11 with metabolite 10 rendered safe the assumption that both 10 and 11 share the same relative configuration. Therefore, metabolite 11 was identified as (1S*,2S*,3R*)-2,3-epoxydihydroisolaurene.
Compound 14 was isolated as colorless oil. The pseudomolecular ion [M + H] + at m/z 249.1487 observed in its HR-ESIMS was consistent with the molecular formula C 15 H 20 O 3 . The absorption bands at 3428 and 1703 cm −1 in the IR spectrum, in conjunction with the observed 13 C signals at δ C 210.0, 208.1 and 64.8 (Table 2), suggested the presence of two carbonyl moieties and one hydroxy group. The presence of a 1,4-disubstituted benzene ring was indicated by the two doublets at δ H 7.20 and 7.34 integrating for two protons each. Moreover, the presence of an aromatic hydroxymethylene at position C-9 was verified by the HMBC correlations of H 2 -15 (δ H 4.69) with C-8/C-10 (δ C 127.7) and C-9 (δ C 139.2). The aliphatic methyl H 3 -14 (δ H 1.45) was fixed at C-1 due to its correlations with C-1 (δ C 54.8), C-2 (δ C 210.0), C-5 (δ C 31.2) and C-6 (δ C 141.5) as observed in the HMBC spectrum. In addition, the HMBC correlations of H 3 -12 (δ H 1.90) with the carbonyl carbon C-2 and of H 3 -13 (δ H 2.07) with the carbonyl carbon C-3 (δ C 208.1) supported the cleavage of the C-2/C-3 bond of the cyclopentane ring. Thus, compound 14 was identified as 2,3-dioxo-15hydroxy-seco-laurene.
The anti-inflammatory activity of metabolites 1, 2, 4-8, 10, 12-16, 18 and 20-22 was evaluated using the RAW 264.7 macrophage cell line, which has been proven to be a powerful tool for the detection of bioactivity of natural products [18,19]. The bioactivity of compounds 3, 9, 11, 17 and 19 was not evaluated since they were either proven unstable or isolated in insufficient amounts. RAW 264.7 macrophages were stimulated with the TLR4 ligand LPS, which triggers a pro-inflammatory signal that induces nitric oxide (NO) production, and simultaneously treated with increasing concentrations of the tested metabolites. The detection of NO was achieved using Griess reaction 48 h following cell activation and was used to determine the IC 50 values. Inhibition of NO production was determined by comparing metabolite-treated cells with cells exposed to the vehicle solvent only (0.1% v/v Carbowax TM 400 and 0.01% v/v ethanol). All tested compounds revealed significant anti-inflammatory activity in the concentration range used (Table 3, Figure 4).  In order to verify that the anti-inflammatory activity observed was not due to a potential cytostatic effect of the metabolites, an MTT assay was performed in cells exposed to increasing concentrations of metabolites 1, 2, 4-8, 10, 12-16, 18 and 20-22 for 24, 48 and 72 h (Table 3, Figure 5). The time point that the cytostatic activity was observed reflected its potency since cytotoxicity is a cumulative process. In order to verify that the anti-inflammatory activity observed was not due to a potential cytostatic effect of the metabolites, an MTT assay was performed in cells exposed to increasing concentrations of metabolites 1, 2, 4-8, 10, 12-16, 18 and 20-22 for 24, 48 and 72 h (Table 3, Figure 5). The time point that the cytostatic activity was observed reflected its potency since cytotoxicity is a cumulative process. Compounds 1 and 2 exhibited IC50 values of 10.17 and 12.66 μΜ, respectively, and although they differ only in the configuration at C-13, compound 1 did not show any cytostatic effect, in contrast to compound 2 exhibiting cytostatic activity above 25 μΜ. Compound 4 displayed an IC50 value of 8.91 μΜ with no significant cytostatic activity at the tested concentration. Interestingly, compound 5 exhibited significant anti-inflammatory activity with an IC50 value of 3.69 μΜ and cytostatic activity above 50 μΜ, further supporting its strong anti-inflammatory action. Although displaying structural similarities, compound 6 exhibited significant NO reduction only at 62.5 μΜ, which could be attributed to its cytostatic effect, whereas compounds 7 and 8 showed IC50 values of 25.27 and 6.92 μΜ, respectively, as well as cytostatic activity above 25 μΜ. Compounds 10 and Compounds 1 and 2 exhibited IC 50 values of 10.17 and 12.66 µM, respectively, and although they differ only in the configuration at C-13, compound 1 did not show any cytostatic effect, in contrast to compound 2 exhibiting cytostatic activity above 25 µM. Compound 4 displayed an IC 50 value of 8.91 µM with no significant cytostatic activity at the tested concentration. Interestingly, compound 5 exhibited significant anti-inflammatory activity with an IC 50 value of 3.69 µM and cytostatic activity above 50 µM, further supporting its strong anti-inflammatory action. Although displaying structural similarities, compound 6 exhibited significant NO reduction only at 62.5 µM, which could be attributed to its cytostatic effect, whereas compounds 7 and 8 showed IC 50 values of 25.27 and 6.92 µM, respectively, as well as cytostatic activity above 25 µM. Compounds 10 and 12-15 exhibited significant anti-inflammatory activity, but in rather high concentrations, with IC 50 values of 20.46, 45.24, 23.81, 22.73 and 33.09 µM, respectively, and cytostatic activity above 50 µM for compounds 10, 12 and 13, and above 25 µM for compounds 14 and 15. Importantly, compound 16 displayed substantial anti-inflammatory activity with an IC 50 value of 4.97 µM and cytostatic effect at concentrations over 25 µM. Compound 18 exhibited the most potent anti-inflammatory activity with an IC 50 value of 3.55 µM, and it is noteworthy that no significant cytostatic activity was observed at any of the tested concentrations. Compounds 20-22 showed anti-inflammatory action with IC 50 values of 10.51, 6.66 and 13.19 µM, respectively, and cytostatic activity above 50, 25 and 3.125 µM, respectively. It can be inferred that the anti-inflammatory activity of compound 22 is mainly due to its strong cytostatic activity. Overall, compounds that displayed minimal or no cytostatic activity and had the capacity to inhibit NO production have the potential to serve as lead molecules for novel anti-inflammatory compounds. NO is a central mediator of inflammation and its inhibition is a hallmark of anti-inflammatory activity; yet, further studies are required to determine the mechanisms of action, including evaluating their action on inflammatory cytokine production in macrophages and, furthermore, in in vivo models of inflammatory diseases.

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 recorded on a Shimadzu UV-1900i UV-Vis spectrophotometer (Shimadzu Europa GmbH, Duisburg, Germany). IR spectra were recorded on a Bruker Alpha II FTIR spectrometer (Bruker Optik GmbH, Ettlingen, Germany). NMR spectra were recorded on Bruker DRX 400, Avance NEO 950 (Bruker BioSpin GmbH, Rheinstetten, Germany) and Varian 600 (Varian, Inc., Palo Alto, CA, USA) spectrometers. Chemical shifts are provided on the δ (ppm) scale with reference to the solvent signals. The 2D NMR experiments (HSQC, HMBC, COSY, NOESY) were performed using standard Bruker or Varian pulse sequences. Low-resolution EI mass spectra were measured on an Agilent Technologies 5977B mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) or a Thermo Electron Corporation DSQ mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). High-resolution APCI or ESI mass spectra were measured on a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Column chromatography separations were performed with Kieselgel 60 (Merck, Darmstadt, Germany). HPLC separations were conducted on a Waters 600 liquid chromatography pump equipped with a Waters 410 differential refractometer (Waters Corporation, Milford, MA, USA) or an Agilent 1100 series liquid chromatography pump equipped with an Agilent 1100 series refractive index detector, using (a) an Econosphere Silica 10µ (Grace, 25 cm × 10 mm i.d) column or (b) a Supelcosil Si (Supelco, 25 cm × 10 mm i.d). TLC were performed with Kieselgel 60 F 254 aluminum plates (Merck, Darmstadt, Germany) and spots were detected after spraying with a 25% H 2 SO 4 in MeOH reagent and heating at 100 • C for 1 min.

Biological Material
The biomass of L. majuscula was hand-picked by SCUBA diving at a depth of 10 m from the reefs near the National Institute of Oceanography and Fisheries (NOIF), Hurghada, Egypt (GPS coordinates 27 • 17 06 N, 33 • 46 24 E), in July 2016, and transported to the laboratory in ice chests, where they were stored at −20 • C until analyzed. A voucher specimen has been deposited at the Herbarium of NOIF in Hurghada and the Herbarium of the Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens (ATPH/MP0548).

Single-Crystal X-ray Diffraction Analysis of Compounds 1 and 2
Compounds 1 (maneonene F) and 2 (maneonene G) were crystallized by slow evaporation of saturated solutions of MeOH, in both cases as colorless plates. Single crystal X-ray diffraction data were collected using a dual source Bruker D8-Venture diffractometer equipped with four-circle kappa goniometer, performing ϕ and ω scans to fill the Ewald sphere, and a Photon-III CMOS area detector at 100 K using an ImS Diamond Mo/Kα radiation source. Control of data collection, data processing and reduction were performed using the APEX 4 software suite. Data for both 1 and 2 were collected to a resolution of 0.7 Å. A multi-scan absorption correction was applied in both cases [20]. Data solution and model refinement were performed using Olex2-1.5 and all software packages within [21]. Collection and refinement details for compounds 1 and 2 are provided in Table S2. cytostatic activity up to concentrations of 50 µM. In addition, the strong cytostatic potential of compound 22 warrants further studies in the context of anti-cancer research.

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