Non-halogenated new sesquiterpenes from Bornean Laurencia snackeyi

Abstract Two new non-halogenated sesquiterpenes, snakeol (1) and snakediol (2) were isolated together with 9 known sesquiterpenes such as (R,Z)-33-dimethyl-5-methylene-4-(3-methylpenta-24-dien-1-yl)cyclohex-1-ene (3), palisol (4), pacifigorgiol (5), palisadin D (6), palisadin A (7), palisadin B (8), 5-acetoxypalisadin B (9), debromolaurinterol (10) and α-bromocuparane (11) from the red algae Laurencia snackeyi. The structures of two new metabolites were determined from their spectroscopic data (IR, 1D and 2D NMR and MS). Compounds 1, 2, 10 and 11 showed strong antibacterial activity against selected human clinical bacterial pathogens.


Introduction
The red algae of genus Laurencia (Rhodomelaceae, Ceramiales) is the most studied genus of all marine algal genera. Nevertheless, new chemical studies of Laurencia species from different latitudes often result in the discovery of interesting secondary metabolites (Kamada & Vairappan 2012, 2015. It has been reported this genus is an extremely rich source of secondary metabolites predominantly in two major biosynthetic halogenated groups, terpenes and C 15 -acetogenins (Suzuki & Vairappan 2005) and the widespread occurrence of these compounds has implications for their potential ecological significance (Wang et al. 2013). However, the non-halogenated metabolites with diverse skeletons also occurred widely in Laurencia species all over the world (Ji & Wang 2014). Diversity of halogenated secondary metabolites is characteristic of Laurencia, they are considered as chemotaxonomical markers at the species level (Suzuki & Vairappan 2005). Besides, these secondary metabolites have been reported to possess diverse biological activities such as anti-inflammatory (Chatter et al. 2011) and cytotoxic activities (Juagdan et al. 1997;Alarif et al. 2016).

Results and discussion
Snakeol (1) was isolated as colorless oil, [ ] 28 D − 11.8 • (c 0.5, CHCl 3 ). Its molecular formula was established as C 15 H 24 o from the HR-eSIMS ion at m/z 243.1721 [M + Na] + (Calcd for C 15 H 24 oNa, 243.1719), implying 4° of unsaturation. The IR absorption at 3369 cm −1 indicated the presence of the hydroxyl functional group. The 1 H and 13 C NMR spectra of 1 are summarised in Table  S1. The 13 C NMR spectral data revealed the presence of 15 carbon atoms, which were attributed by HSQC spectra to 3 tertiary methyls, 5 methylenes (including an exomethylene and a methylene bearing oxygen atom), 4 methines and 3 quaternary carbons. The presence of three pairs of double bond in 1 suggested the remaining 1° of unsaturation was a monocyclic sesquiterpene.
Assignments were made with the aid of the 1 H-1 H CoSY and HMBC spectra data. The 1 H-1 H CoSY correlations and key HMBC correlations are shown in Figure S2. The 1 H-1 H CoSY correlations indicated the connectivity for the three spin systems C-1-C-2, C-4-C-5-C-6 and C-8-C-9-C-10. The three-bond correlations of H 3 -14 and H 3 -15 to the respective carbons C-14 and C-15, and the correlations of both to C-6, C-10 as well as C-11, permitted placement of the gem-dimethyl group at C-11. HMBC cross peaks between H 2 -13 and C-6/C-8 were important to confirm that the exomethylene moiety to be inserted between C-6 and C-8. Furthermore, HMBC correlations from H 3 -12 to C-2, C-3 and C-4 suggested the attachment of tertiary methyl group (C-12) to the quaternary carbon at C-3, as well as allowed connection of two partial structures of C-1-C-2 and C-4-C-5-C-6. The 1 H and 13 C chemical shift at position 1 (δ H 4.15; δ C 64.0) revealed a hydroxy methylene (liu et al. 2009). The relative stereochemistry of 1 was determined by NoeSY experiments. The H-6 exhibited NoeSY correlation to H 3 -15 suggested these protons were positioned on α-configuration. These configurations at C-6 and C-11 were similar to the analogues 2-4. The geometry of the C-2 double bond was determined to be E by the Noe correlation of H-2 to H-4 and H-5. To the best of our knowledge, most of the monocyclofarnesol sesquiterpenes found in Laurencia species have the Z configuration double bond at C-3/C-4 (Suzuki & Vairappan 2005;Wang et al. 2013), but this double bond has rearranged as E configuration double bond at C-2/C-3 in 1. This position of double bond at C-2/C-3 was considered as first record, this could be due to the diversity of Bornean Laurencia species. Thus, snakeol (1) having the unusual monocyclofarnesol structure would be represented by Figure 1. Snakediol (2) was isolated as colorless oil, [ ] 28 D − 10.8 • (c 0.4, CHCl 3 ). The molecular formula was established as C 15 H 24 o 2 from the HR-eSIMS ion at m/z 259.1669 [M + Na] + (Calcd for C 15 H 24 o 2 Na, 259.1669), implying 4° of unsaturation. Its IR absorption at 3290 cm −1 indicated the presence of the hydroxyl functional group. The 1 H and 13 C NMR spectra of 2 are summarised in Table S2. The 13 C NMR spectral data revealed the presence of 15 carbon atoms, which were attributed by HSQC spectra to 3 tertiary methyls, 4 methylenes (including an exomethylene and a methylene bearing oxygen), 5 methines (including a methine bearing oxygen) and 3 quaternary carbons. The presence of three pairs of double bond could be suggested a monocyclic structure similar to 4.
The 1 H-1 H CoSY correlations and key HMBC correlations are shown in Figure S2. The 1 H-1 H CoSY correlations indicated the connectivity for the three spin systems C-1-C-2, C-4-C-5-C-6 and C-8-C-9-C-10. The three-bond correlations of H 3 -14 and H 3 -15 to the respective carbons C-14 and C-15, and the correlations of both to C-6, C-10 as well as C-11, permitted placement of the gem-dimethyl group at C-11. HMBC cross peaks between H 2 -13 and C-6/C-8 allowed attachment of exomethylene moiety between C-6 and C-8. Furthermore, HMBC correlations from H 3 -12 to C-2, C-3 and C-4 suggested the attachment of tertiary methyl group (C-12) to the quaternary carbon at C-3, as well as permitted connection of two partial structures of C-1-C-2 and C-4-C-5-C-6. The chemical shifts at C-1 (δ C 65.7) and C-2 (δ C 71.8) indicated that the hydroxyl groups were attached to these two carbons. The relative stereochemistry of 2 was determined by NoeSY experiments. The Z geometry double bond was assigned at C-3/C-4 due to the Noe cross peak between H-4 and H 3 -12. The configuration of C-6 and C-11 were found to be similar to those of 1 based on NoeSY spectrum. Thus, snakediol (2) was very similar to known compound 4 which replaced the bromine atom to hydroxyl group shown in Figure 1.
Compound 6 named as palisadin D was previously isolated from okinawan L. snackeyi (Suzuki & Vairappan 2005;Makhanu et al. 2006). However, its stereochemistry was not determined. Judging from co-occurrence of 6 and 7 from the same alga and its biogenetic pathway, the relative stereochemistry of 6 is probably similar to those of 7 upon investigating NoeSY spectrum. The NoeSY correlations between H-5β/H 3 -14 and H 3 -14/H 3 -15 indicated these protons were located on β-orientation. In addition, lack of Noe correlation between H 3 -14 and H-6, further supported these configurations. These findings suggested the relative structure of 6 as shown in Figure 1.
Studies on natural products chemistry reveal that some compounds are only produced by certain species, therefore conferring upon them a chemical race. our earlier studies of L. snackeyi collected from the coast waters of Sabah revealed the presence of two chemical types for their component. First type contained snyderane-type compounds such as 7, 8 and 9 as their common metabolite (Vairappan et al. 2013). Second type produced chamigrene-type compounds together with snyderanes as their major metabolite (Tan et al. 2011). Here, we reported the third type which contained cyclolaurane-and cuparane-type compounds such as 10 and 11 together with snyderanes. The difference in the type of secondary metabolites produced by these populations could be due to geographic as well as genetic factors. There are also species that produce unrelated sets of compounds depending on geographical distribution (Suzuki & Vairappan 2005). Hence, our research suggests that three chemical types could be present in Bornean L. snackeyi.
The isolated compounds 1-11 were tested for their antimicrobial activity. The results are shown in Table S4. Compounds 1, 2, 10 and 11 showed strong antibacterial activity against Salmonella typhi with a MIC/MBC ratio of 2.79, 2.79, 2.79 and 2.72, respectively, indicating a bactericidal antibiosis. In addition, compounds 1 and 2 showed strong antibacterial activity against Escherichia coli with a MIC/MBC ratio of 3.02 and 2.76, respectively. These antibacterial results could be possibly due to the presence of the hydroxyl group at C-1 position in the monocyclofarnesol-type structures (1-4). In combination, both these values could reflect possible pharmacokinetic and pharmacodynamic pattern and help to predict drug's efficacy in vivo. The ratio of MBC to MIC less than four indicated the compounds to be of bactericidal mode of action (levison 2004). Therefore, new non-halogenated compounds 1 and 2 provided sufficient information to suggest an antibiosis mechanism of bactericidal in nature.

General experimental procedures
optical rotations were measured on an AuTopol IV automatic polarimeter (Rudolph Research Analytical). IR spectra were recorded on a Thermo Nicolet Nexus FT-IR spectrometer. 1 H NMR (600 MHz) and 13 C NMR (150 MHz) spectra were measured in CDCl 3 with TMS as the internal standard using a Jeol eCA 600 spectrometer. HR-eSI-ToFMS spectra were obtained with lCMS-IT-ToF (Shimadzu).

Biological material
Specimens of L. snackeyi were collected from kg Sikaudagan, Bum Bum Island, Semporna, Sabah (04°38.218′N, 118°59.535′e), on July, 2013. The voucher specimen (BoRH39002) was deposited in the BoRNeeNSIS Collection of Institute for Tropical Biology and Conservation, university of Malaysia Sabah.

Antibacterial activity
Antibacterial assay was performed using microdilution method against four strains of human pathogenic bacteria: E. coli (QeITBC1204), S. typhi (QeITBC1207), Staphylococcus aureus (QeITBC1218) and Vibrio cholera (QeITBC1202) (espinel-Ingroff et al. 2001). The bacterial suspensions were adjusted with sterile saline to a concentration of 1.0 × 10 5 CFu/ml and stored at 4°C. Inoculum was screened for contamination by culturing on a solid medium, and experiments were in triplicates and repeated three times. pure compounds were added (1 and 10 mg/ml) to 100 ml tryptic soy broth (TSB) with a bacteria inoculum (1.0 × 10 4 CFu per well), reaching the desired concentration in a microtitre plate to measure the MICs and MBCs. The mixtures in microplates were incubated for 24 h at 37°C. The MIC was defined as the lowest concentrations without visible growth, while the MBC was defined as the lowest concentration showing no visible growth, and was determined by serial subcultivations of 2 μl into microtitre plates which contained 100 μl of TSB for each well and incubated for 24 h using a microplate manager. The optical density was measured at 655 nm and compared to blank and positive control. positive control was Kanamycin (1 mg/ml), while 5% DMSo was used as the negative control.

Conclusion
In this paper, two new non-halogenated monocyclofarnesol-type sesquiterpenoids, namely snakeol (1) and snakediol (2) were isolated from L. snackeyi specimen collected from kg Sikaudagan, Sabah. This is the first record that monocyclofarnesol-type sesquiterpene 1 has the double bond at C-2/C-3. Compounds 1, 2, 10 and 11 showed strong antibacterial activity against selected clinical strains of bacteria. This is the first report on the presence of chemical types in L. snackeyi, which contained cyclolaurane-and cuparane-type compounds such as 10 and 11 together with snyderanes.