New Marine Antifouling Compounds from the Red Alga Laurencia sp.

Abstract

Six new compounds, omaezol, intricatriol, hachijojimallenes A and B, debromoaplysinal, and 11,12-dihydro-3-hydroxyretinol have been isolated from four collections of Laurencia sp. These structures were determined by MS and NMR analyses. Their antifouling activities were evaluated together with eight previously known compounds isolated from the same samples. In particular, omaezol and hachijojimallene A showed potent activities (EC₅₀ = 0.15–0.23 µg/mL) against larvae of the barnacle Amphibalanus amphitrite.

1. Introduction

Biofouling is a major cause of increased fuel consumption of ships and facilitates the spread of invasive marine organisms [1]. After many years of utilizing toxic antifoulants to control biofouling, the IMO (International Maritime Organization) banned the use of organotin compounds in 2008, paving the way for the development of environmentally friendly fouling-resistant coatings [2]. In addition, effective and new fouling treatment is needed to control and curb the introduction of non-indigenous species (NIS), that are introduced into local waters partly via ballast water uptake and discharge in vessels. The ballast water management convention will enter into force in September 2017. However, to effectively control NIS introduction, an effective antifouling treatment approach is still needed [3]. Search for natural antifouling compounds to solve biofouling has been “a work in progress” for almost four decades. Lately, a number of natural antifouling products have been reported and some of them have shown potent activities [4,5]. Furthermore, recent efforts to identify molecular mechanisms of antifouling compounds improve the possibility of their industrial importance [6]. As expected, industrial utilization of natural products as antifouling agents would warrant systematic synthetic efforts, and these have been implemented [5]. The total synthesis of 10-isocyano-4-cadinene [7] and dolastatin 16 [8] were achieved by our group [9,10]. The red alga Laurencia sp. is a rich source of biologically active secondary metabolites [11,12] and produces antifouling compounds such as elatol [13], omaezallene [14], and 2,10-dibromo-3-chloro-7-chamigrene [15]. As a continuous effort, our group has been searching for novel and potent antifouling compounds from Laurencia sp. by using barnacle larvae and epiphytic diatom assays. Hence, here we report the structures (Figure 1) and activities of new antifouling compounds from Laurencia sp.

2. Results

2.1. Omaezol (1) and Intricatriol (2)

Laurencia sp., which was collected in Omaezaki, Japan, was extracted with MeOH. The extract yielded omaezol (1), intricatriol (2), in addition to the previously isolated omaezallenes and intricatetraol [14]. The molecular formula of 1 was determined to be C₂₀H₃₅BrO₂ (m/z 368.1707, calcd. for C₂₀H₃₃BrO, 368.1709 [M − H₂O]⁺) by HR-EIMS (High Resolution-Electron Ionization Mass Spectrometry), suggesting three degrees of unsaturation. The existence of a hydroxy group was revealed by an IR absorption at 3386 cm⁻¹. ¹³C NMR data (

Table 1. ¹H and ¹³C NMR spectroscopic data for compounds 1 and 2 in CDCl₃.

Carbon Number	Compound 1		Compound 2		Carbon Number	Compound 2
	13C	1H	13C	1H		13C	1H
1	67.6	3.91	27.8	1.689	24	27.5	1.695
2	30.2	2.44, 2.01	72.0		23	72.0
3	41.9	1.55, 1.70	67.1	4.07	22	67.1	4.08
4	71.4		28.7	1.85, 2.61	21	28.9	1.75, 2.45
5	48.2	1.10	36.8	1.72, 1.89	20	36.9	1.47, 1.72
6	32.1	1.48, 2.00	72.2		19	73.3
7	74.6		113.4		18	84.3	3.85
8	31.8	1.49, 1.90	27.9	1.60, 1.70	17	26.4	1.93
9	38.7	1.05, 1.76	32.8	1.91	16	32.7	1.57, 2.19
10	39.4		86.7		15	85.6
11	33.6	1.67	84.7	3.61	14	76.0	3.57
12	30.2	1.52	29.6	1.55, 2.40	13	29.4	1.74
13	26.5	1.12			25	33.1	1.785
14	124.3	5.10			30	32.9	1.793
15	131.7				26	21.2	1.28
16	25.5	1.70			29	24.5	1.26
17	17.6	1.62			27	17.6	1.45
18	12.3	0.90			28	23.0	1.19
19	14.4	1.26
20	29.7	1.18

) showed the presence of one double bond, suggesting a bicyclic structure. COSY correlations connected C-1 to C-3. HMBC peaks from H-19 to C-1, 5, 9, 10 and from H-20 to C-3, 4, 5 concluded a cyclohexane ring (Figure S1). Based on ¹³C NMR chemical shifts, a bromine atom is attached to C-1 (δ 67.6) and a hydroxy group is connected to C-4 (δ71.4). A side chain was determined by COSY correlation (H-11/H-18 and H-12/H-13) and HMBC peaks (H-18/C-7, 12 and H-14/C-13, 16, 17). Another hydroxy group is attached to C-7 (δ 74.6). Overlapping of NMR chemical shifts (C-6; δ 32.1, C-8; δ 31.8, C-9; δ 38.7, C-10; δ 39.4, H-6; δ 1.48, H-8; δ 1.49) hampered the positioning of the two remaining methylenes. However, HSQC-TOCSY peaks from H-8 to C-9 and H-6 to C-5 concluded the planar structure of 1. NOESY correlations determined relative configurations of the trans-decalin (Figure 2).

The molecular formula of 2 was determined to be C₃₀H₅₂Br₂Cl₂O₆ (m/z 757.1584, calcd. for C₃₀H₅₃Br₂Cl₂O₆, 757.1581 [M + H]⁺) by HR-FABMS (High Resolution-Fast Atom Bombardment Mass Spectrometry). The ¹H and ¹³C NMR spectra of 2 (Table 1) were similar to intricatetraol [16,17], but were not symmetric. The 2D NMR interpretations (Figure S2) showed that both compounds had the same carbon skeleton. However, ¹³C chemical shift changes were observed (C-7: δ 84.1 in intricatetraol, δ 113.4 in 2, and C-11: δ 77.5 in intricatetraol, δ 84.7 in 2). C-7 in 2 was proposed to be an acetal carbon and C-11 was suggested to be a part of an ether instead of a secondary alcohol to make another five-membered ring. In conclusion, the planar structure of 2 was determined and its configuration was assumed to be the same as intricatetraol [16,17], which was extracted from the same sample.

2.2. Hachijojimallenes A (3) and B (4)

Hachijojimallenes A (3) and B (4) were isolated together with N-methyl-2,3,6-tribromoindole [18] and pinnaterpene C [19] from a red alga, Laurencia sp., which was collected in Hachijojima Island, Japan. Based on HR-EIMS data, the molecular formula of 3 was determined to be C₁₅H₁₉Br₃O₃ (m/z 466.8851, calcd. for C₁₅H₁₈Br₃O₂, 466.8852 [M − OH]⁺). The existence of a bromoallene moiety was suggested by an IR absorption at 1957 cm⁻¹, ¹³C NMR chemical shifts (δ 74.1, 99.4, and 201.9), and ¹H NMR chemical shifts (δ 5.45 and 6.08) (

Table 2. ¹H and ¹³C NMR spectroscopic data for compounds 3–5 in CDCl₃.

Carbon Number	Compound 3		Compound 4		Compound 5
	13C	1H	13C	1H	13C	1H
1	74.1	6.08	74.0	3.09	58.7
2	201.9		201.9		103.4
3	99.4	5.45	100.0	5.48	42.4	2.59
4	73.8	4.73	74.1	4.72	31.7	1.26, 1.74
5	39.3	1.81, 2.26	39.6	1.82, 2.35	43.0	1.74, 1.92
6	79.4	4.56	88.6	5.11	131.6
7	81.4	4.61	82.8	4.77	159.4
8	50.5	2.77	51.0	3.19	110.0	6.72
9	102.1		119.7		138.8
10	53.6	4.07	42.8	4.13	122.1	6.74
11	44.3	2.50, 2.76	40.6	1.74, 2.70	122.4	6.92
12	51.3	3.79	75.6	4.03	21.5	2.33
13	44.7	1.97	50.2	2.13	24.2	1.31
14	24.7	1.47, 2.11	23.3	1.44, 1.60	203.5	9.78
15	11.6	1.03	12.5	0.99	13.1	1.04
9-OH		3.65

). COSY correlations established a carbon skeleton C-3—C-8—C-13—C-10 and an ethyl group. ¹³C chemical shifts revealed that C-4, 6, and 7 were oxymethines and C-10 and 12 were brominated. These carbons and an acetal carbon (δ 102.1) were assembled by HMBC data to conclude the planar structure of 3 (Figure S3). Relative configurations were determined by NOEs (H-6/H-10, H-10/H-11 α, H-11 α/H-12, H-7/H-12, and H-8/H-13).

The molecular formula of 4 was determined to be C₁₅H₁₈Br₂O₃ (m/z 403.9627, calcd. for C₁₅H₁₈Br₂O₃, 403.9617 [M]⁺) by HR-EIMS, suggesting the existence of another ring. The striking difference in its ¹³C NMR (Table 2) was observed in C-9 (3; δ 102.1, 4; δ 119.7) and C-12 (3; δ 51.3, 4; δ 75.6). In addition, HMBC was observed from H-12 to C-9. In conclusion, the hemiacetal in 3 was replaced by acetal in 4 to form an ether between C-9 and C-12, which was debrominated at C-12. NOEs of 4 supported its relative configurations, similar to those of 3.

2.3. Debromoaplysinal (5)

Re-investigation of the red alga L. okamurae, collected at Oshoro Bay, Hokkaido, Japan, yielded debromoaplysinal (5). The molecular formula of 5 was determined to be C₁₅H₁₈O₂ (m/z 231.1379, calcd. for C₁₅H₁₉O₂, 231.1380 [M + H]⁺) by ESI-TOFMS. Its 1D NMR spectra (Table 2) revealed the existence of an aldehyde and a trisubstituted benzene. COSY and HMBC correlations (Figure S4) clarified its planar structure, which is similar to aplysinal and debromoaplysinol. In fact, chemical shifts in the trisubstituted benzene of 5 are similar to those of debromoaplysinol, and those in the cyclopentane of 5 are similar to those of aplysinal. NOESY peaks between H-13 and H-14 suggested cis configuration of Me-13 and the aldehyde. Irradiation of H-3 by DPFGSE 1D NOE showed the enhancement of H-13. In conclusion, H-3, Me-13, and the aldehyde are located on the same face.

2.4. 11,12-Dihydro-3-hydroxyretinol (6)

Re-investigation of the red alga L. nipponica collected at Muroran, Hokkaido, Japan, yielded 11,12-dihydro-3-hydroxyretinol (6), along with three known halogenated chamigrene-type sesquiterpenoids [20,21]. The molecular formula of 6 was deduced to be C₂₀H₃₂O₂ (m/z 304.2400, calcd. for C₂₀H₃₂O₂, 304.2397 [M]⁺) by HR-EIMS, accounting for five degrees of unsaturation. The ¹H- and ¹³C-NMR spectroscopic data (

Table 3. ¹H and ¹³C NMR spectroscopic data for compound 6 in CDCl₃.

Carbon Number	13C	1H	Carbon Number	13C	1H
1	37.9		11	27.5	2.29
2	49.1	1.47, 1.76	12	40.0	2.12
3	65.9	4.00	13	140.0
4	43.2	2.02, 2.37	14	124.2	5.44
5	125.9		15	60.1	4.18
6	138.2		16	22.4	1.71
7	124.0	5.92	17	31.0	1.06
8	139.0	6.00	18	29.5	1.06
9	134.6		19	13.2	1.79
10	131.4	5.40	20	17.2	1.71

) as well as the HSQC experiment of 6 showed the presence of eight sp² carbons, three vinyl methyls, a pair of geminal methyls, an oxymethylene, an oxymethine, four methylenes, and a quaternary carbon. These signals explained four degrees of unsaturation, implying that one ring was present in 6. COSY and HMBC data (Figure S5) clearly indicated the gross structure of 6. The E configurations of double bonds were deduced from the ¹H-¹H coupling constants (³J_7–8 15.9 Hz) and ¹³C-NMR chemical shifts of vinyl methyls (C-19; δ 13.2, and C-20; δ 17.2). Unfortunately, the configuration at C-3 was not determined due to its limited amount.

2.5. Antifouling Activity

Most of the compounds obtained in this study were tested for antifouling activity against larvae of the barnacle Amphibalanus amphitrite (

Table 4. Antifouling activities (µM) against barnacle larvae.

Compound	EC50	LC50
1	0.59	9.6
2	—	—
3	0.31	20
4	0.76	17
5	4.3	—
7	1.6	—
8	2.2	20
9	1.2	—
10	2.3	—
11	—	—
12	1.5	—
13	5.5	—
14	6.1	—
CuSO4	0.72	1.4

—: EC₅₀ or LC₅₀ is more than 10 µg/mL.

). The antifouling activity of 1 was potent (EC₅₀ = 0.23 µg/mL) and its toxicity was low (LC₅₀ = 3.7 µg/mL), while 2 was not potent (EC₅₀ > 10 µg/mL). The antifouling activity of 3 was potent (EC₅₀ = 0.15 µg/mL) and its toxicity was low (LC₅₀ = 9.8 µg/mL), while 4 was a little less potent (EC₅₀ = 0.31 µg/mL, LC₅₀ = 6.8 µg/mL). The antifouling activity of the known compound, pinnaterpene C (7, EC₅₀ = 0.82 µg/mL, LC₅₀ > 10 µg/mL), was similar to that of 4, while another known compound, N-methyl-2,3,6-tribromoindole, showed weak activity (EC₅₀ = 4.3 µg/mL, not toxic at 10 µg/mL). Compound 5 also showed moderate antifouling activity (EC₅₀ = 1.0 µg/mL) and did not kill any larvae at 10 µg/mL. Some known compounds (Figure 3) isolated from L. okamurae were tested. Three aromatic sesquiterpenoids [22] showed antifouling activities (laurinterol 8: EC₅₀ = 0.65 µg/mL, LC₅₀ = 5.8 µg/mL; isolaurinterol 9: EC₅₀ = 0.34 µg/mL, LC₅₀ > 10 µg/mL; debromolaurinterol 10: EC₅₀ = 0.5 µg/mL, LC₅₀ > 10 µg/mL), while α-bromocuparene (11) was inactive. Three sesquiterpenoids (Figure 3) isolated from L. nipponica collected in Muroran showed antifouling activities (prepacifenol 12: EC₅₀ = 0.63 µg/mL, LC₅₀ > 10 µg/mL; pacifenol 13: EC₅₀ = 2.36 µg/mL, LC₅₀ > 10 µg/mL; 2,10-dibromo-3-chloro-9-hydroxy-α-chamigrene 14 EC₅₀ = 2.51 µg/mL, LC₅₀ > 10 µg/mL). In addition, anti-diatom activities against the epiphytic marine diatoms, Nitzschia sp. and Cylindrotheca closterium, were tested for three chamigrenes and 6. Compounds 6, 12, and 14 showed inhibition against both diatoms at 5–10 µg/cm².

3. Discussion

From a chemical point of view, 1 is a rare halo-diterpenoid, prenylated selinene-type compound like anhydroaplysiadiol [23]. Oxasqualenoids such as 2 have been proposed to be synthesized by the epoxide-opening cascades. These reactions could be catalyzed by vanadium-dependent bromoperoxidases [24]. However, we do not know how different cyclization patterns are controlled to produce 2 and the similar compound intricatetraol [16,17]. Although most of C₁₅ acetogenins from Laurencia contain only ether rings, 3 and 4 contain a carbocyclic ring, similar to lembynes [25]. Compound 5 is one of common laurane-type sesquiterpenes. Retinols occur in nature only in animals and in the limited green algae Caulerpa sp. [26] This is the first record of a retinane-type diterpene (6) isolated from Laurencia. Although the genus of Laurencia is one of the most studied organisms in marine natural product chemistry [11,12], recent isolation techniques yielded six new compounds in this study. Twelve compounds showed antifouling activities against the barnacle larvae. Notably, the activities of 1 and 3 were equivalent to CuSO₄ (EC₅₀ = 0.18 µg/mL). Three compounds showed antifouling activities against diatoms. However, most of compounds isolated from Laurencia sp. so far have not been tested for antifouling activity. Our results suggest that Laurencia is a potential source of antifouling compounds.

4. Materials and Methods

4.1. General Procedures

IR spectra were measured on a JASCO IR-700 spectrophotometer (JASCO, Tokyo, Japan). ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃ by using JEOL JNM-ECA600 (JEOL, Tokyo, Japan), JEOL JNM-EX400 (JEOL, Tokyo, Japan), or BRUKER ASX300 spectrometer (Bruker BioSpin, Faellanden, Switzerland), unless otherwise stated. EI-MS were obtained on a JEOL JMS-FABmate spectrometer (JEOL, Tokyo, Japan). FAB-MS were obtained on a JEOL JMS-HX110 spectrometer (JEOL, Tokyo, Japan). ESI-MS were obtained on a JEOL JMS-700TZ (JEOL, Tokyo, Japan) or BRUKER DALTONICS micro TOF-HS focus spectrometer (Bruker Daltonics, Bremen, Germany). Optical rotations were recorded on a HORIBA SEPA-300 polarimeter (Horiba, Kyoto, Japan).

4.2. Plant Material

Algal samples of Laurencia sp. were collected at Omaezaki, Shizuoka Prefecture, and Hachijojima Island, Tokyo, Japan. L. okamurae was collected at Oshoro Bay and L. nipponica was collected at Muroran, Hokkaido, Japan. The voucher specimens are deposited in the Herbarium of Graduate School of Science, Hokkaido University.

4.3. Omaezol and Intricatriol

The dried algal sample (250 g) was extracted and separated as described in a previous paper [12]. Omaezol (1, 3.2 mg) and intricatriol (2, 17.0 mg) were isolated by HPLC (YMC-Pack Pro C18 (YMC, Kyoto, Japan) with CH₃CN and H₂O) from the omaezallene containing silica-gel fraction.

1: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −67.4 (c 0.17, CHCl₃); IR (neat), ν_max 3428, 1259, 1185, 1088, 1065, 954, 801 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1.

2: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −45.2 (c 0.39, CHCl₃) ; IR (neat), ν_max 3414, 2972, 1451, 1369, 1214, 1101, 756 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1.

4.4. Hachijojimallenes A and B

The air-dried algae (80 g) were soaked in MeOH (500 mL) for seven days. The MeOH solution was concentrated in vacuo, and the residue was partitioned between EtOAc and H₂O. The EtOAc layer was then washed with water, dried over anhydrous Na₂SO₄, and evaporated in vacuo to leave a dark green oily substance (1.23 g). The extract (700 mg) was fractionated by Si gel CC with a step gradient (hexane and EtOAc). The fraction (41 mg) eluted with hexane-EtOAc (9:1) was further subjected to PTLC (preparative thin-layer chromatography) with hexane to give 1-methyl-2,3,6-tribromoindole (4.0 mg), which was determined on the basis of ¹H NMR and MS data. The fraction (411 mg) eluted with hexane–EtOAc (7:3) was further chromatographed on a Si gel column with hexane–EtOAc (3:1) (each eluate, 10 mL) to give 10 fractions. The fourth fraction (79.1 mg) was then separated by PTLC with hexane-EtOAc (3:1) to afford hachijojimallene B (4) (3.5 mg). The combined fifth and sixth fractions (74.9 mg) were further separated by repeated PTLC with hexane–EtOAc (1:1) to give hachijojimallene A (3) (40.0 mg). The seventh fraction (43.8 mg) was subjected to PTLC with hexane–EtOAc (3:1) followed by HPLC (Develosil-ODS-T-5 with CH₃CN and H₂O) to give pinnaterpene C (7, 15.0 mg).

3: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{24}$ −88.2 (c 0.13, CHCl₃); IR (neat), ν_max 3490, 1957, 1258, 1184, 1165, 1143, 1120, 1038, 959, 788, 756 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1.

4: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{24}$ −151 (c 0.71, CHCl₃); IR (neat), ν_max 1950, 1243, 1181, 1127, 1080, 1045, 1022, 970, 855, 821 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1.

4.5. Debromoaplysinal

The air-dried algae (100 g) were soaked in MeOH (3 L) for seven days. The MeOH solution was concentrated in vacuo, and the residue was partitioned between EtOAc and H₂O. The EtOAc layer was then washed with water, dried over anhydrous Na₂SO₄, and evaporated in vacuo to obtain the crude extract (4.3 g). The extract (2.1 g) was fractionated by Si gel column chromatography with a step gradient (hexane and EtOAc). The fraction (1.4 g) eluted with hexane-EtOAc (3:1) was further subjected to two sets of Si gel column chromatography and PTLC with toluene to give debromoaplysinal (5) (0.9 mg). The other known compounds (laurinterol 8, 30.6 mg; isolaurinterol 9, 12.4 mg; debromolaurinterol 10, 11.0 mg; α-bromocuparene 11, 1.1 mg) were isolated from the same fraction (hexane–EtOAc 3:1) in a similar way.

5: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{24}$ −91.9 (c 0.08, CHCl₃); IR (neat), ν_max 2927, 2856, 1731, 1593, 1499, 1456, 1377, 1266, 1146, 1117, 971, 805 cm⁻¹; ¹H NMR and ¹³C NMR, see Table 1.

4.6. 11,12-Dihydro-3-hydroxyretinol

Fresh algal sample (400 g wet weight) was extracted in MeOH at room temperature for three days. The resulting MeOH extract was concentrated in vacuo and partitioned between diethyl ether and H₂O. The diethyl ether fraction (340 mg) was subjected to Si gel CC eluting with a gradient of hexane and EtOAc in an increasing polarity. Fraction 4, eluted with hexane-EtOAc (3:1), was subjected to PTLC to yield pacifenol (12, 4.1 mg) and prepacifenol (13, 6.3 mg). Fraction 5, eluted with hexane-EtOAc (1:1), was subjected to PTLC with CHCl₃-MeOH (97:3) to yield 2,10-dibromo-3-chloro-9-hydroxy-α-chamigrene (14, 7.3 mg). Fraction 7, eluted with hexane-EtOAc (1:3), gave 6 (1.3 mg) after purification with PTLC using CHCl₃-MeOH (95:5).

6: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{27}$ −61.9 (c 0.07, CHCl₃); ¹H NMR and ¹³C NMR, see Table 1.

4.7. Antifouling Assay

An antifouling assay against larvae of the barnacle Amphibalanus amphitrite was conducted according to the previous literature [9]. The antifouling assay against diatoms Nitzschia sp. and Cylindrotheca closterium was conducted as follows. The diatoms were maintained in test tubes under 16 h light and 8 h dark cycle conditions at 25 °C in modified Jorgensen’s medium. Pure compounds were applied to a sample zone (16 mm in diameter) of cellulose TLC aluminum sheet (58 × 58 mm), and then each treated sheet was placed in Petri dish (15 × 90 mm in diameter). After that, 25-mL aliquots of modified Jorgensen’s medium were introduced into Petri dishes with treated sheets, and inoculated with 1 mL of cultivated diatom suspension in equivalent cell densities. Petri dishes were sealed with Parafilm to ensure a closed system, and were incubated under the same conditions. Cell growth was estimated daily, and the adhesive condition was evaluated and compared to those of the control after seven days of incubation.