Key words
Laurencia majuscula
- Rhodomelaceae - red alga - halogenated - chamigrane sesquiterpene
Introduction
Red algae of the genus Laurencia are known to produce an array of diverse halogenated secondary metabolites represented as terpenoid, alkaloid and C15 -acetogenin chemical skeletons [1 ]
[2 ]
[3 ]
[4 ]. Presence and quantity of the major halogenated metabolites within Laurencia populations often vary with their species and geographical distribution [5 ]
[6 ]
[7 ]
[8 ]
[9 ]. Halogenated compounds have also been shown to exhibit various biological activities with pharmaceutical importance, such as antibacterial [7 ], cytotoxic [10 ], and insect repellent activities [11 ]. As part of our ongoing effort to document the diversity of halogenated secondary metabolites, we collected a population of Laurencia majuscula from the coastal waters of North Borneo (Mantanani Island), which led to the isolation of two new sesquiterpenoids, lauremantanones A (1 ) and B (2 ), along with seven known compounds, dendroidiol (3 ) [12 ], (+)-elatol (4 ) [13 ], cartilagineol (5 ) [14 ]
[15 ]
[16 ], obtusol (6 ) [15 ]
[16 ]
[17 ], (+)-laurencenone B (7 ) [18 ]
[19 ], 2-chloro-3-hydroxy-α -chamigren-9-one (8 ) [13 ]
[20 ], and puertitol A (9 ) [21 ] ([Fig. 1 ]). Herein, we describe the isolation, structural elucidation, cytotoxicity, and antibacterial activities of these compounds.
Fig. 1 Structures of sesquiterpenoids 1–9 from L. majuscula .
Results and Discussion
Compound 1 was isolated as colorless oil, [α ]D
28 +22.3 (c 1.0, CHCl3 ). The molecular formula was established as C15 H21 ClO2 by the HRESIMS [M + H]+ ion at m /z 269.1305 (calcd. for C15 H22 ClO2 , 269.1303) and it accounted for 5 degrees of unsaturation. Its IR absorption was seen at 3 420 and 1 650 cm−1 , indicating the presence of hydroxyl (-OH) and α ,β unsaturated carbonyl (C=O) functionalities. The 13 C NMR revealed the presence of 15 signals whose multiplicities were attributed by DEPT-135 and HSQC spectra to three methyls, five methylenes, including an oxygenated methylene, a trisubstituted, and a tetrasubstituted olefins, two quaternary carbons, and a carbonyl carbon. These signals accounted for three degrees of unsaturation, implying 1 possesses a bicyclic system. The NMR data ([Table 1 ]) of 1 closely resembled that of 7 except for the replacement of a vinyl methyl at C-14 in 7 by a vinyl carbinol moiety in 1 [[18 ]
[19 ]
[22 ]. The presence of a carbinol unit was further confirmed by its deshielded chemical shifts at C-14 (δ
C 64.0; δ
H 4.24) [23 ], IR absorption at 3 420 cm−1 , and HRESIMS spectrum.
Table 1
13 C and 1 H NMR (150 and 600 MHz) data of 1 and 2 (CDCl3 , δ in ppm, J in Hz).
1
2
No.
δ
C
δ
H
δ
C
δ
H
1
36.2
2.55 d (17.9)
35.4
2.24 d (6.2)
2.26 d (17.9)
2.23 d (11.0)
2
126.4
68.4
4.28 dd (11.0, 6.2)
3
130.4
70.6
4
30.8
2.17 dd (17.2, 5.5)
36.9
1.93–1.95 m
2.00–203 m
1.78–1.81 m
5
31.2
1.92 td (12.4, 5.5)
23.8
1.97–1.99 m
1.76 ddt (12.4, 4.8, 2.1)
1.78–1.81 m
6
46.2
47.5
7
171.1
168.7
8
124.1
6.32 s
124.5
6.18 s
9
199.3
199.1
10
49.8
2.64 d (17.2)
50.6
2.41 d (19.3)
2.10 d (17.2)
2.36 d (19.3)
11
41.3
41.6
12*
25.2
1.06 s
28.1
1.16 s
13*
24.6
0.96 s
27.9
1.13 s
14
64.0
4.24 s
63.9
4.46 d (16.5)
4.42 d (16.5)
15
20.4
1.79 s
29.3
1.36 s
* Assignments may be reversed
The 1 H-1 H COSY (Fig. 1S , Supporting Information) correlation between H2 –4 and H2 –5, together with the HMBC (Fig. 1S , Supporting Information) correlations from H3 –12, 13 to C-6, 10, 11, from H3 –15 to C-2, 3, 4, from H2 –14 to C-6, 7 , 8, from H2 –1 to C-2, 5, 6, from H2 –10 to C-9, and from H-8 to C-10 were enough to establish the planar structure of an α -chamigrane-type sesquiterpenoid for 1 . The relative configuration at C-6 was assigned S *, identical to that of (+)-7 , based on a chemical shift (δ
C 46.4) and the optical rotation [α ]D
25 +39.4 (c 0.2, CHCl3 ) as well as a biogenetic pathway given the fact that both compounds were isolated from the same specimen. (Fig. 2S–7S )
Compound 2 was isolated as colorless oil, [α ]D
28–35.0 (c 0.5, CHCl3 ). The molecular formula was determined as C15 H23 ClO3 through the HRESIMS [M + H]+ ion at m /z 287.1412 (calcd. for C15 H24 ClO3 , 287.1409). Both hydroxyl and carbonyl functionalities were detected by IR absorption at 3 444 and 1 651 cm−1 , respectively. Upon careful comparison, NMR data of 2 ([Table 1 ]) were almost identical to those of 8 except for the replacement of an olefinic methyl at C-14 in 8 by a vinyl carbinol unit in 2
[13 ]. Detailed assignment of 1 H-1 H COSY and HMBC correlations (Fig. 1S , Supporting Information) revealed an α -chamigrane framework for 2 . (Fig. 8S–13S )
The relative stereochemistry of 2 was determined by NOESY experiments. The NOESY correlations between H-2/H2 –4 α , H-2/H3 –15, and H2 –4 α /H3 –15 demonstrated that these protons were located on the same orientation, while both 2-Cl and 3-OH were located on the opposite orientation of the ring B. The relative configuration at C-6 was identical to that of (-)-8 based the NOE correlation between H2 –1/H3 –12, chemical shifts, and the optical rotation [α ]D
25–46.0 (c 0.22) [13 ]. The methylene H2 –14 (δ
H 4.46 and 4.42) of 2 experienced a downfield shift of 0.2 ppm and was presented as an individual signal instead of superimposed as compared to that of 1 (δ
H 4.24, H2 –14). This could be due to the additional hydroxyl moiety in 2 at C-3, which results in a restricted rotation of the sigma bond between C-7 and C-14. Finally, judging from the co-occurrence of 2 and 8 in the same alga, the relative stereochemistry of the B ring in 2 is the same as that of 8 . Therefore, 2S *, 3R *, and 6S * were assigned similarly to those of (-)-8 . The natural product designated as laurencenone B [22 ] was incorrectly assigned due to discrepancies that existed between published 1 H NMR data of the natural product and that of a synthetic compound [19 ]. Since then, no report of complete NMR and specific rotation measurements for the natural product laurencenone B (7 ) was found, therefore, herein, we reported its specific rotation and 1 H and 13 C NMR data. The specific rotation and 1 H and 13 C NMR data of 7 were consistent to semisynthetic and synthetic materials [13 ]
[19 ].(Fig. 14S–15S )
The α -chamigrane-type sesquiterpenoids 1 and 2 showed strong cytotoxic activities against HeLa and P-388 cell lines with an IC50 ≤ 5.0 μg/mL ([Table 2 ]), whereas β -chamigrane-type sesquiterpenoids 3 –6 displayed a much stronger cytotoxicity against cell lines HeLa, MCF-7, and P-388 with an IC50 ≤ 1.0 μg/mL. On the other hand, compounds 7 –9 were found inactive against all cell lines. Compounds 1 and 2 exhibited weak activity against Bacillus cereus with MIC and MBC values of 250 and 1 000–1 250 μg/mL, respectively [Table 3 ]). In a previous literature, compound 3 was reported as having no activity against NO and TNF-α production in LPS-induced RAW 264.7 macrophages, and no antibacterial property on Mycobacterium bovis
[12 ]. Compounds 4 and 6 were reported to show antileishmanial activity against Leishmania amazonensis on promastigotes (IC50 of 9.7 and 6.2 μg/mL, respectively) and amastigotes (IC50 of 4.5 and 3.9 μg/mL, respectively), but were not active against NO production by macrophages [15 ]. In addition, compounds 4 and 6 were also reported to exhibit cytotoxic activities against gastric carcinoma (IC50 of < 1.0 and 7.0 μg/mL, respectively), liver carcinoma (IC50 of < 1.0 μg/mL), and breast carcinoma (IC50 of < 1.0 and 1.5 μg/mL, respectively), while 5 showed an IC50 of 1.0, 0.25, and 1.0 μg/mL on non-small lung cancer, human colon carcinoma, and melanoma cells, respectively. Compounds 4 –6 , however, showed negligible antibacterial activity [24 ]
Table 2 Cytotoxicity of compounds 1 –9 against cancer cell lines HeLa, MCF-7, and P-388.
Cells
IC50 (μg/mL)
1
2
3
4
5
6
7
8
9
HeLa
2.50
2.50
0.75
0.75
1.00
1.00
-
-
-
MCF-7
2.50
5.00
1.00
0.75
0.75
1.00
-
-
-
P-388
5.00
2.50
0.75
1.00
1.00
1.00
-
-
-
Table 3 Antibacterial activities of compounds 1 and 2 against clinical strains.
Strains
MIC (MBC) in μg/mL
1
2
Bacillus cereus
250 (1000)
250 (1250)
Escherichia coli
-
-
Salmonella typhi
> 500
> 500
Vibrio cholera
> 500
> 500
Positive control = kanamycin, with an MIC and MBC of 2.0 μg/mL
Materials and Methods
General experimental procedures
The 1 H NMR (600 MHz) and 13 C NMR (150 MHz) spectra were recorded on an NMR spectrometer (Jeol). The HRESIMS was acquired via LCMS-ESI-IT-TOF (Shimadzu). An AUTOPOL IV automatic polarimeter was used to measure the optical rotation (Rudolph Research Analytical). Infrared spectra were recorded on an FTIR (Thermo Nicolet). For preparative TLC (Kieselgel 60 F254 , Merck), the spots were visualized by UV light (254 and 365 nm) and spraying with a 5% phosphomolybdic acid-ethanol solution. Column chromatography was performed with silica gel (Kieselgel 60, 70–230 mesh; Merck). All organic solvents for extraction and isolation were of analytical grade (Fisher Scientific). The HPLC solvent was HPLC grade (Fisher Scientific). The deuterated CDCl3 was purchased from Merck.
Plant material
Specimens of L. majuscula (Harvey) Lucas were collected from Mantanani Island, Sabah (06º59.728’N, 116º34.830’E) in November 2017. A voucher specimen (BORH39093) was deposited in the BORNEENSIS Collection of the Institute for Tropical Biology and Conservation, University of Malaysia, Sabah. Field identifications were done by Prof. Dr. Charles S. Vairappan, who is the corresponding author of this paper. Voucher specimens were examined by Assoc. Prof. Dr. Abe Tsuyoshi, Hokkaido University Museum, Sapporo, Japan.
Extraction and isolation
After air drying for 3 days, the alga (110 g) was extracted with methanol (MeOH). The resulting MeOH extract was concentrated in vacuo and partitioned between ethyl acetate (EtOAc) and H2 O. The EtOAc fraction was washed with water, dried over anhydrous Na2 SO4 , and evaporated to leave a dark green oil (3.0 g). The EtOAc extract (1.5 g) was then fractionated by Si gel column chromatography with a step gradient (hexane and EtOAc). The fraction eluted with hexane-EtOAc (8:2) was subjected to preparative TLC with toluene to give compounds 4 (160 mg: 10.7%), 5 (99 mg: 6.6%), 6 (90 mg: 6.0%), 7 (101 mg: 6.7%), and 9 (41 mg: 2.7%). The fraction eluted with hexane-EtOAc (7:3) was subjected to preparative TLC with chloroform (CHCl3 ) to give 3 (41 mg: 2.7%). The fraction eluted with hexane-EtOAc (1:1) was subjected to preparative TLC with CHCl3 to give 8 (82 mg: 5.5%). The fraction eluted with EtOAc (100%) was subjected to preparative TLC with CHCl3 -MeOH (95:5) to give 1 (16 mg: 1.1%) and 2 (15 mg: 1.0%). Compounds that were isolated using the preparative TLC technique were further purified using reverse phase HPLC with an ODS-3 column under acetonitrile, MeCN-H2 O (50–100% MeCN), at 210 nm UV detection. Yields were calculated as a percentage of the EtOAc crude extract.
Lauremantanone A (1 ): colorless oil; [α ]28
D +22.3 (c 1.0, CHCl3 ); IR (neat) ν
max 3420, 2930, 1650, 1455, 1318, 1078, and 979 cm−1 ; 1 H and 13 C NMR (CDCl3 ) spectral data: see [Table 1 ]; HRESIMS m/z 269.1305 [M + H]+ (calcd. for C15 H22 ClO2 , 269.1303).
Lauremantanone B (2 ): colorless oil; [α ]28
D -35.0 (c 0.5, CHCl3 ); IR (neat) ν
max 3444, 2925, 1651, 1455, and 1118 cm−1 ; 1 H and 13 C NMR (CDCl3 ); spectral data: see [Table 1 ]; HRESIMS m/z 287.1412 [M + H]+ (calcd. for C15 H24 ClO3 , 287.1409).
(+)-Laurencenone B (7 ): white powder; [α ]25
D +39.4 (c 0.2, CHCl3 ); 1 H NMR (CDCl3 , 600 MHz) δ : 5.89 (1H, s), 2.54–2.62 (2H, br m), 2.27 (1H, d, J = 17.2 Hz), 2.16–2.20 (1H, br m), 2.02–2.09 (2H, br m), 1.97 (3H, s), 1.93 (1H, td, J = 12.7, 5.6 Hz), 1.81 (3H, s), 1.75–1.77 (1H, br m), 1.06 (3H, s), 0.97 (3H, s); 13 C NMR (CDCl3 , 150 MHz) δ : 198.1, 168.5, 129.7, 127.5, 126.3, 48.9, 46.4, 40.5, 36.3, 30.4, 30.2, 24.8, 23.8 (overlapped two signals), 19.7.
Cytotoxic assay
The in vitro cytotoxicity assay was conducted against the cell lines human cervical epithelioid carcinoma (HeLa), human breast carcinoma (MCF-7), and murine lymphocytic leukemia (P-388). The tested concentrations were 100, 10, 1, 0.1, 0.01, and 0.001 µg/mL. The assay was carried out using a previous procedure [25 ]. Positive control experiments were conducted using H2 O2 as the test chemical.
Antibacterial assay
The antibacterial assay was carried out using 96-well plates with a known microdilution method against the strains Bacillus cereus (QEB2018–01), Escherichia coli (QEB2018–02), Salmonella typhi (QEB2018–03), and Vibrio cholera (QEB2018–04) obtained from Queen Elizabeth General Hospital, Kota Kinabalu, Sabah, Malaysia. The tested concentrations were 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, and 2.0 µg/mL. The assay was carried out using a previous procedure [6 ]. The positive control kanamycin (contained ≥ 98% kanamycin A) was purchased from Merck.
Supporting Information
NMR spectra of the new compounds are available as Supporting Information.
