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Abstract

A new manzamenone derivative (4) and four known manzamenones were isolated from the tropical marine sponge Plakortis sp. These compounds exhibited an improved inhibitory effect against the copper-induced low-density lipoprotein oxidation. To identify their structural properties for antioxidant effects, 43-O-methylmanzamenone A (2) has been exposed oxidative conditions such as hydrogen peroxide, mCPBA and primary alkyl amines to give various derivatives (6)-(9). The structures of the compounds were determined by spectroscopic analyses and comparison with literature values.

The marine sponges of the genus Plakortis have been recognized as a rich source of oxygenated fatty acids such as plakortin,¹ cyclic peroxides,^2,3 untenone,⁴ plakoridine A,⁵ untenolide A,⁶ and manzamenones.^7

-11 Manzamenones constitute a family of natural dimeric^7

-10 and trimeric¹¹ fatty acid derivatives. Especially, it has been proposed that 3,6-dioxo-4-docosenoic acid might be a universal key intermediate of either the manzamenones or untenone A, and furthermore, all of these molecules are likely to be biosynthetically related to one another.⁸ As a consequence, the manzamenones feature saturated long alkyl chains and ester groups around a cyclopentenone core. From the biological activity viewpoint, several Plakortis-derived manzamenones exhibit a broad spectrum of bioactivity.^10
-12

As part of our ongoing efforts to identify bioactive metabolites from marine sponges,¹³ we collected the sponge Plakortis from Weno Island, Chuuk State, Federated States of Micronesia. The crude organic extract of this marine sponge exhibited considerable antioxidant activity. Stimulated by the fact, we initiated an investigation on this organism. Fractions of interest were selected on the basis of the ¹H nuclear magnetic resonance (NMR) analysis of the crude extracts obtained from solvent-partitioning. Subsequently, an integrated separation process for isolation and purification of compounds was executed by different chromatographic methods including vacuum flash chromatography on silica and normal-phase high-performance liquid chromatography (HPLC). In consequence, the separation process yielded 5 compounds, which were later identified by combined spectroscopic analyses to be manzamenone A (1),⁷ 43-O-methylmanzamenone A (2),⁷ manzamenone F (3),⁷ compound 4, and manzamenone L (5)¹⁰ (Figure 1), respectively.

Figure 1

Structures of compounds 1- 5.

Compound 4 was obtained as a colorless gem. Its molecular formula of C₆₂H₁₁₀O₇was supported by the presence of molecular ion peak in the high-resolution fast atom bombardment mass spectra (HRFABMS) ion at m/z 967.8320 (M + H)⁺ (calculated for C₆₂H₁₁₁O₇, 967.8324) (Supplemental figure S3). The ¹H NMR spectrum of compound 4 is very similar to that of manzamenone F (3) except for the integral value from the difference in the number of methylene moieties, and a triplet methyl signal of ¹H δ 0.92 from buthoxy group in 3 (Supplemental figure S1). And the ¹³C NMR spectra of compounds 4 and 3 are also similar to each other except for the signals from buthoxy group in 3 (Supplemental figure S2). The number of methylene moieties was revealed by mass spectrometry experiment including the comparison of fragmentation patterns of manzamenones and the structure of compound 4 was elucidated as shown. The inspection of the ¹H–¹H correlation spectroscopy (COSY) spectrum of 4 revealed connection of C-1 to C-2, C-4 to C-5, C-26 to C-27, and C-47 to C-48 (Figure 2). And the connectivity of 2-ring system was indicated by heteronuclear multiple bond correlations (HMBCs) (Figure 2). The COSY and HMBC spectra of 4 are also similar to those of 3.

Figure 2

Selected 2-dimensional correlations of compound 4.

The isolation of natural products from the sponge Plakortis yielded a considerable quantity of 43-O-methylmanzamenone A (2), which is likely to be used for further derivatization in order to identify their structural properties for antioxidant effects. Compound 2 was exposed to hydrogen peroxide, m-chloroperbenzoic acid, and primary alkyl amines, oxidized products were obtained from each reaction.

As shown in Scheme 1, the reaction of compound 2 with 30% hydrogen peroxide gave epoxide 6 in 53% yield. The spectral data of 6 were highly compatible with those obtained for 2 (Supplemental figure S4) while the molecular formula of C₄₇H₈₀O₈was assigned by combined HRFABMS. Additionally, the ¹³C NMR spectrum showed that 2 alkene signals at δ 133.7 (C-8) and 187.7 (C-9) of 2 were replaced by epoxide carbon signals at δ 65.7(C-8) and 75.1(C-9). A similar difference was also observed in the ¹H NMR spectrum in which the signal of the methine proton, H-1, at δ 3.17 of 2 was upfield shifted at δ 2.77 (Supplemental figure S5). It is assumed that these NMR shifts are the results of the transformation of a double bond to an epoxide. The result indicates that epoxide is formed by the reactions of H₂O₂, a nucleophilic reagent, with electron-deficient double bond in 2. The relative stereochemistry of the epoxide moiety in 6 was deduced from nuclear Overhauser effect spectrum as shown Figure 3 (Supplemental figure S6).

Scheme 1

Oxidations of compound 2. (a) H₂O₂, methanol, 40°C, 53%; (b) mCPBA, CH₂Cl₂, rt, 34%; (c) RNH₂, THF, 50°C, 35% for 8 and 32% for 9.

Figure 3

Key nuclear Overhauser effect spectroscopy correlation for compound 6.

Interestingly, oxidation of 2 using m-chloroperbenzoic acid (mCPBA) gave 7 as a major product, which was produced by an oxidation reaction on the isolated double bond in 2. The spectral data for 7 were similar to those observed for 2 and 6, whereas the most notable change in the NMR data was the disappearance of a trisubstituted double bond in 2. The signals of ¹³C δ 136.8, 123.1; ¹H δ 5.76 in 2 were upfield shifted to ¹³C δ 60.1 (C-3), 58.2 (C-4); ¹H δ 3.50 (H-4), respectively. These changes could be explained by an epoxidation of the double bond between C-3 and C-4 of 2 by mCPBA. The formation of epoxide in this position can be rationalized by the electrophilic epoxidation of isolated double bonds in 2. The relative stereochemistry of the epoxide moiety was determined by the measurement of the vicinal ¹H-¹H coupling constants. Probably, the relationship between H-4 and H-5 might be cis as a small coupling constant (J < 1.0 Hz) in 6-membered ring (Supplemental figure S7). A similar phenomenon was observed with the authentic epoxides.¹⁴

The treatment of compound 2 with primary alkylamines in THF gave amides 8 and 9. The structures of these derivatives were defined by combined spectroscopic analysis. The carbomethoxy group at C-5 in compound 2 was converted into a carboxamide group by an alkyamine.

Based on our preliminary data on antioxidant activity of the crude MeOH extract, natural manzamenones 1-5 and synthetic derivatives 6-9 were evaluated for the inhibitory effect on the copper-induced low-density lipoprotein (LDL) oxidation. Most of the natural compounds 1-5 exhibited better inhibitory effect against the copper-induced LDL oxidation compared to oxidized derivatives 6-9. Particularly, the manzamenone 5 showed better effect than others (Figure 4). The results of inhibitory effect showed that oxidized manzamenone derivatives reduced the antioxidant effects. These observations suggest that the 2 double bonds in natural manzamenone are involved in the antioxidant effect on copper-mediated LDL oxidation.

Figure 4

Inhibitory effects of manzamenone derivatives 1-9 on copper-induced low-density lipoprotein oxidation.

In conclusion, a series of manzamenones have been prepared by the isolation of secondary metabolites from the marine sponge Plakortis sp. and the subsequent chemical modifications of the manzamenone 2 using oxidizing agents and alkylamines. The inhibitory effect on the copper-induced LDL oxidation of these compounds was evaluated. In consequence, natural products 1-5 were found to be more active than other compounds. These observations suggest that the 2 double bonds in natural manzamenone are involved in the antioxidant effect. By modification reaction of the manzamenone substituent, we were able to derive several compounds that showed structural properties, antioxidant effects, and improved potency profiles in our bioassay.

Experimental

NMR spectra were recorded on a Varian Unity 500 instrument at 500 MHz for ¹H and 125 MHz for ¹³C. All chemical shifts were recorded with respect to the residual solvent signals as an internal standard (CDCl₃ δ _H 7.26 ppm, δ _C 77.0 ppm). The infrared spectra were recorded using a JASCO FT/IR-4100 spectrophotometer as thin film. Optical rotations were measured from a JASCO DIP-370 automatic polarimeter. Mass spectral data were obtained at the Korean Basic Science Institute, Seoul, Korea.

Collection and Identification

Specimens (Registry No. 102-CH-231) of Plakortis sp. were collected by hand with scuba equipment at a depth of 20-25 m off the shore of Weno Island, Chuuk State, Federated States of Micronesia, in February 2010. The sample was identified by Dr Y. A. Kim, and a voucher specimen has been deposited at the Marine Biotechnology Research Center, Korea Institute of Ocean Science & Technology.

Extraction and Isolation

The specimens were lyophilized (wet wt. 153 g) and repeatedly extracted with MeOH (500 mL × 2) and CH₂Cl₂ (500 mL × 1). The extract was filtered and concentrated under reduced pressure to afford 13.4 g of crude extract. The residue was partitioned between H₂O and n-BuOH to yield 12.4 g of organic-soluble material. The n-BuOH layer was repartitioned between 15% aqueous MeOH and n-hexane (9.3 g). The residue of the n-hexane layer was subjected to silica vacuum flash chromatography using gradient mixtures of n-hexane and EtOAc as eluents (elution order: 10%, 20%, 30%, 40%, 50% EtOAc in n-hexane, and 100% EtOAc). The fraction eluted with 20% EtOAc in n-hexane (2.71 g) was dried and separated by silica HPLC (YMC silica column, 250 × 10 mm; 10% EtOAc in n-hexane) to afford in order of elution, 53.0 and 2.1 mg of 3 and 4, respectively. The fraction eluted with 30% EtOAc in n-hexane (1.01 g) was dried and separated by silica HPLC (15% EtOAc in n-hexane) to yield 25.4 and 411 mg of 5 and 2, respectively. The fraction eluted with 40% EtOAc in n-hexane (623 mg) was dried and separated by silica HPLC (20% EtOAc in n-hexane) to afford 13.2 mg of 1. The purity of these compounds was checked by HPLC.

Compound 4 : [α]_D −12.1 (c 0.1, CHCl₃); IR 2943, 2832, 2360, 1026 cm^-1 ^{; 1}H NMR (500 MHz, CDCl₃) and ¹³C NMR (125 MHz, CDCl₃), see Table 1; HRFABMS: m/z 967.8320 [calculated for C₆₂H₁₁₁O₇ (M + H)⁺; 967.8324].

Table 1

¹H and ¹³C nuclear magnetic resonance data of compound 4.

Position	δ _H (J in Hz)	δ _Cb
Position	δ _H (J in Hz)	δ _Cb	1	3.15 (1H, t, 6.0)	43.1
2	3.41 (1H, d, 6.0)	47.0
3	-	136.9
4	5.73 (1H, br s),	123.5
5	3.63 (1H, m, H-5)	42.1
6	3.30 (1H, t, 6.5)	46.0
7	-	202.4
8	-	133.0
9	-	183.1
10	2.10 (2H, m)	37.1
10	1.66 (2H, t, 7.0)	37.1
11-24	1.2-1.6 (br s)	30.4-22.9
25	0.86 (3H, t, 7.2)	14.3
26	3.04 (1H, m)	32.1
26	2.37 (1H, m)	32.1
27-40	1.2-1.6 (br s)	30.4-22.9
41	0.86 (3H, t, 7.2)	14.3
42	-	170.9
43	-	174.1
44	-	164.9
45	3.80 (3H, s)	52.0
46	3.46 (3H, s),	52.1
47	4.16 (2H, t, 6.7)	65.9
48-61	1.2-1.6 (br s)	30.4-22.9
62	0.86 (3H, t, 7.2)	14.3

Reaction of 2 with H₂O₂

43-O-Methylmanzamenone A (2, 10.0 mg) in methanol (0.4 mL) was treated with 30% aqueous H₂O₂ solution (30 µL) at 40°C for 5 hours. The resulting mixture was evaporated under reduced pressure. The residue was purified by HPLC using normal-phase column with 85% hexane in EtOAc to give 8,9-epoxy form (6, 5.4 mg, 53 %): [α]_D +4.3 (c 0.1, CHCl₃); IR 2925, 2853, 2360, 1742 cm^-1 ^{; 1}H NMR (CDCl₃) δ_H 5.73 (1H, d, J = 1.5 Hz, H-4), 3.84 (3H, s, 44-OMe), 3.75 (3H, s, 43-OMe), 3.59 (3H, s, 42-OMe), 3.42 (1H, t; J = 3.0 Hz, H-6), 3.39 (1H, d, J = 6.0 Hz, H-5), 3.21 (1H, d, J = 5.0 Hz, H-2), 2.77 (1H, dd, J = 5.5 and 5.0 Hz, H-1), 2.14 (2H, m, H-10), 1.93 (2H, m, H-26), 1.4-1.2 (56H, m, H-11-H-24 and H-27-H-40), 0.86 (6H, t, J = 7.30 Hz, H-25 and H-41); ¹³C NMR (CDCl₃) δ_C 203.5 (C-7), 173.9 (C-43), 170.9 (C-42), 164.4 (C-44), 137.4 (C-3), 122.8 (C-4), 75.1 (C-8), 65.7 (C-9), 53.0 (44-OMe), 52.9 (43-OMe), 52.5 (42-OMe), 44.3 (C-2), 41.8 (C-5), 41.0 (C-6), 38.1 (C-1), 36.6 (C-10), 32.1-29.4 (24 carbons), 27.2 (C-26), 27.4, 25.4 and 22.9 (C-11-C-24 and C-27-C-40), 14.3 (C-25 and C-41); HRFABMS: m/z 773.5933 [calculated for C₄₇H₈₁O₈ (M + H)⁺; 773.5931].

Reaction of 2 with mCPBA

43-O-Methylmanzamenone A (2, 9.9 mg) in CH₂Cl₂ (1 mL) was treated with mCPBA (8.3 mg) at room temperature for 18 hours. The resulting mixture was filtered, and then the filtrate was washed with Na₂SO₃ solution, NaHCO₃ solution, water, and brine. The dichloromethane layer was evaporated under reduced pressure. The residue was purified by HPLC to give 3,4-epoxy form (7, 3.4 mg, 34 %): [α]_D +5.6 (c 0.1, CHCl₃); IR 2924, 2853, 2360, 1742 cm^-1 ^{; 1}H NMR (CDCl₃) δ_H 3.80 (3H, s, 43-OMe), 3.78 (3H, s, 44-OMe), 3.56 (1H, d, J = 6.5 Hz, H-5), 3.52 (3H, s, 42-OMe), 3.50 (1H, br s, H-4), 3.39 (2H, s, H-1 and H-6), 3.17 (1H, m, H-2), 3.05 (1H, m, H-26a), 2.34 (1H, m, H-26b), 1.69 (1H, m, H-10a), 1.59 (1H, m, H-10b), 1.4-1.2 (56H, m, H-11-H-24 and H-27-H-40), 0.85 (6H, t, J = 7.30 Hz; H-25 and H-41); ¹³C NMR (CDCl₃) δ_C 201.7 (C-7), 183.9 (C-9), 173.0 (C-43), 170.8 (C-42), 163.6 (C-44), 132.2 (C-8), 60.1 (C-3), 58.2 (C-4), 53.0 (43-OMe), 52.4 (42-OMe), 52.1 (44-OMe), 46.8 (C-1), 43.5 (C-2), 41.3 (C-6), 41.0 (C-5), 34.2 (C-10), 32.1 (C-26), 30.3-29.5 (24 carbons), 27.9, 23.8 and 22.9 (C-11-C-24 and C-27-C-40), 14.3 (C-25 and C-41); HRFABMS: m/z 773.5930 [calculated for C₄₇H₈₁O₈ (M + H)⁺; 773.5931].

Reaction of 2 with Alkylamines

Compound 2 (5.1 mg) in THF (1 mL) was treated with ethylamine (5 µL) at 50°C for 6 hours. The resulting mixture was evaporated under reduced pressure. The residue was purified by HPLC to give compound 8 (1.8 mg, 35 %): [α]_D −6.8 (c 0.1, CHCl₃); IR 2924, 2853, 1735, 1568 cm^-1 ^{; 1}H NMR (CDCl₃) δ_H 9.79 (1H, s, NH), 8.28 (1H, s, H-4), 3.97 (1H, d, J = 7.0 Hz, H-6), 3.94 (3H, s, 44-OMe), 3.76 (3H, s, 42-OMe), 3.49 (2H, m, ethylamine H-1`), 3.49 (1H, m, H-1), 2.86 (1H, m, H-10a), 2.68 (1H, m, H-10b), 1.69 (2H, m, H-26), 1.26 (3H, m, ethylamine H,-2`), 1.4-1.2 (56H, m, H-11-H-24 and H-27-H-40), 0.85 (6H, t, J = 7.3 Hz, H-25 and H-41); ¹³C NMR (CDCl₃) δ_C 201.5 (C-7), 169.2 (C-42), 168.0 (C-44), 163.6 (C-43), 158.9 (C-9), 150.9 (C-3), 135.2 (C-8), 134.4 (C-4), 132.7 (C-5), 129.8 (C-2), 60.2 (C-1), 53.3 (42-OMe), 52.7 (44-OMe), 42.2 (C-6), 35.9 (C-26), 35.4 (ethylamine C-1`), 34.6 (C-10), 32.1-29.5 (25 carbons), 27.4 and 22.9 (C-11-C-24 and C-27-C-40), 14.3 (C-25 and C-41), 14.5 (ethylamine C-2`); HRFABMS: m/z 768.6143 [calculated for C₄₈H₈₂NO₆ (M + H)⁺; 768.6142]. Compound 9 was also prepared analogously to compound 8 in 32% yield as yellow solid: HRFABMS: m/z 782.6301 [calculated for C₄₉H₈₄NO₆ (M + H)⁺; 782.6299].

Inhibition of LDL Oxidation

The inhibitory effects against copper-induced LDL oxidation were measured by the reported method.¹⁵ LDL (100 µg/mL) was incubated in a phosphate-buffered saline (pH 7.4, 1 mL) containing 25 µM CuSO₄, in the absence (control) or presence of manzamenone derivatives (5 µM) at 37°C for 6 hours. The reaction mixture (1 mL) (15% trichloroacetic acid, 0.375% thiobarbituric acid, and 0.25 N hydrochloric acid) was added to the LDL mixture prepared as mentioned earlier. The resultant mixture was heated at 95°C for 30 minutes. After cooling, the absorbance of the pink chromophore was measured at 515 nm. A similar amount of 2,6-di-tertbutyl-4-methylphenol (BHT) was used as a reference compound. When comparing with the control, the inhibition rates against LDL oxidation for the compounds 1-5 were found to be more active than derivatives 6-9. Among these active compounds, 5 was found to be more active (58 %) than other compounds 1 - 4.

Supplemental Material

Supplementary material - Supplemental material for Antioxidant Properties of the Manzamenones from the Tropical Marine Sponge Plakortis sp.

Supplemental material, Supplementary material, for Antioxidant Properties of the Manzamenones from the Tropical Marine Sponge Plakortis sp. by MyoungLae Cho, Yeon-Ju Lee, Jong Seok Lee, Hee-Jae Shin and Hyi-Seung Lee in Natural Product Communications

Footnotes

Acknowledgments

We are grateful to the Department of Marine Resources, State of Chuuk, Federated States of Micronesia, for supporting marine organism research. This research was partially supported by the KIOST (PE99721), and the Ministry of Oceans and Fisheries, Republic of Korea (PM61620). We would like to thank the Basic Science Research Institute in Seoul, Korea, for providing mass spectrometric data.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID ID

Hyi-Seung Lee

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Supplementary Material

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Antioxidant Properties of the Manzamenones from the Tropical Marine Sponge Plakortis sp.

Abstract

Experimental

Collection and Identification

Extraction and Isolation

Reaction of 2 with H2O2

Reaction of 2 with mCPBA

Reaction of 2 with Alkylamines

Inhibition of LDL Oxidation

Supplemental Material

Supplementary material - Supplemental material for Antioxidant Properties of the Manzamenones from the Tropical Marine Sponge Plakortis sp.

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

ORCID ID

References

Supplementary Material

Reaction of 2 with H₂O₂