Isolation of Chamigrene Sesquiterpenes and Absolute Configuration

Note Cite This: J. Nat. Prod. 2017, 80, 3049-3053

pubs.acs.org/jnp

Isolation of Chamigrene Sesquiterpenes and Absolute Configuration of Isoobtusadiene from the Brittle Star Ophionereis reticulata Genoveffa Nuzzo,*,‡,# Bruno A. Gomes,†,# Pietro Amodeo,‡ Helena Matthews-Cascon,§ Adele Cutignano,‡ Leticia V. Costa-Lotufo,⊥ Felipe A. C. Monteiro,∥ Otilia Deusdenia L. Pessoa,† and Angelo Fontana‡ †

Departamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, 60021-970, Fortaleza, CE, Brazil ‡ CNR, Istituto di Chimica Biomolecolare, Bio-Organic Chemistry Unit, 80078 Pozzuoli, Naples, Italy § Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará, 60021-970, 60455-760, Fortaleza, CE, Brazil ⊥ Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-010 São Paulo, SP, Brazil ∥ Instituto Federal de Educaçaõ , Ciência e Tecnologia, 63475-000, Jaguaribe, CE, Brazil S Supporting Information *

ABSTRACT: The chemical study of the Brazilian brittle star Ophionereis reticulata led to the isolation of three chamigrene sesquiterpenes, including the partially characterized isoobtusadiene (1), its unreported acetyl derivative (2), and the known (+)-elatol (3). The complete elucidation of the structures 1 and 2 was accomplished by 1D and 2D NMR spectroscopy. The first assignment of the absolute configuration of the isoobtusadiene skeleton is suggested as 6S,9R,10S on the basis of the NMR analysis of the Mosher’s ester derivatives of 1 and the ECD study of the acetyl derivative 2. Chamigrenes are typical constituents of Laurencia red algae. O. reticulata is a predator with a preference for algae. Thus, the origin of these metabolites can be likely ascribed to diet.

M

arine organisms are a prolific source of a large and diverse array of secondary metabolites.1 Among deuterostomian invertebrates, the echinoderms comprise about 6000 species that have been also screened for substances of biomedical interest.2,3 The brittle stars (Ophiuroidea) are the largest group of echinoderms based on number of species.4 These organisms are known to be a source of polyhydroxysterols and steroidal saponins,5−7 but their study has also revealed the presence of other classes of secondary metabolites, including carotenoid sulfates,8 gangliosides,9 brominated indoles,10 phenylpropanoids,11 and terpenes.11,12 As part of our ongoing effort to identify active natural products from diverse marine organisms, we have investigated the extracts of Ophionereis reticulate (Say, 1825), a brittle starfish widespread along the northeastern coast of South America. Herein, we describe the isolation and identification of three chamigrene sesquiterpenes, namely isoobtusadiene (1),13−16 its unreported acetyl derivative (2), and (+)-elatol (3).17,18 The study also gave us the opportunity to complete the NMR characterization of isoobtusadiene (1)13 and determine the absolute configuration of its spiro-sesquiterpene skeleton. O. reticulata was collected at Paracuru beach, on the coast of Ceará State, Brazil. The whole animal was extracted twice with MeOH, and after the removal of the organic solvent, the raw © 2017 American Chemical Society and American Society of Pharmacognosy

extract (75 mg) was subjected to two sequential steps of chromatographic purifications. Preliminary 1H NMR analysis of fractions obtained by Sephadex LH-20 chromatography eluted with MeOH indicated the presence of terpenoid compounds in fraction I (12.3 mg). Further purification on a silica gel column led to the isolation of isoobtusadiene (0.6 mg, 1), acetyl isoobtusadiene (0.4 mg, 2), and (+)-elatol (3). Isoobtusadiene (1) was first reported by Prof. Gerwick from the red alga Laurencia obtusa,13 but the full interpretation of the NMR data and the assignment of the absolute configuration of the spiro ring system have not been addressed. 14−16 Spectroscopic data (NMR, UV, and IR) of 1 and 2 differed Received: June 14, 2017 Published: November 7, 2017 3049

DOI: 10.1021/acs.jnatprod.7b00510 J. Nat. Prod. 2017, 80, 3049−3053

Journal of Natural Products

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only by the presence of signals related to the acetyl residue (δH 2.08; δC 22.9 and 170.2; IR 1738 cm−1). Accordingly, the 1H NMR spectra of both compounds showed two methyl groups at δH 1.03 and 1.22, two exomethylene appendages (δH 4.85− 5.01, H2-14 and 4.80−4.82, H2-15), and three independent spin systems. Acetylation at C-9 resulted in the deshielding of H-9 in 2 (δH 5.29), as well as slight differences of the protons H-8b (δH 2.58 in 1; δH 2.46 in 2) and H-10 (δH 4.65 in 1; δH 4.55 in 2). Heterocorrelation spectroscopy (HSQC and HMBC) allowed us to achieve the full assignments of protons and carbons in both products (Table 1). In particular, these

29.5) and C-4 (δC 134.2). The same correlations were also found in isoobtusadiene (1), thus leading to a reassignment of the exomethylene systems.13 A strong NOE between H-8a (δH 2.72) and H-10 (δH 4.55) of 2 suggested axial orientations of these two protons, supporting the equatorial position of the bromine atom in a pseudochair conformation of the corresponding cyclohexyl ring. Homodecoupling experiments showed the presence of three small coupling constants (J9,10 = 3.3 Hz; J9,8b = 2.0 Hz; J9,8a = 1.6 Hz) for H-9 that, in agreement with the axial orientation of H-10, implied an axial orientation of the acetoxy group (Figure 1). This analysis was further corroborated by the NOE correlation between the acetyl residue (CH3, δH 2.08) and the axial methyl group at δH 1.22 (H3-13) of 2, thus supporting the cis relative configuration of the substituents at C-9 and C10 as proposed by Gerwick and co-workers for isoobtusadiene (1).13 Finally, the depicted relative configuration of the spiro carbon was deduced on the basis of NOE correlations between H-1a (δH 1.91) with the axial protons H10 and H-8a (δH 2.72), as well as between H3-13 (δH 1.22) and H-5 (δH 5.84) (Figure 1). Conversion of 1 into 2 by acetylation confirmed the same relative configuration of both compounds. Because the specific rotation of compound 1 ([α]D = −25, c 0.007) was consistent with the value reported in the literature for isoobtusadiene from Laurencia ([α]D = −11, c 0.7),13 we concluded that the sesquiterpene of the brittle star is the same compound previously described in the alga. In order to establish the absolute configuration, we applied the modified Mosher’s method19 on the hydroxy group of isoobtusadiene (1). The (R)- and (S)-MTPA esters 1a and 1b were obtained by treating compound 1 with (S)- and (R)-MTPA chloride. Full assignment of the MTPA esters was achieved by 2D NMR experiments. In particular, the shielded methyl group at C-11 in both diastereoisomers was assigned to the β-oriented CH3-13 on the basis of a strong NOE correlation with H-5. The diagnostic Δδ(δSester−δRester) values are reported in Figure 1B and support the R absolute configuration of C-9. As underlined by the dashed lines in Figure 1B, the MTPA ester on 1a and 1b determined a negative effect only on the α-oriented H-10 and CH3-12, whereas all the other resonances showed a positive ΔδS,R value. The assignment of the absolute configuration of C9 allows us to suggest the 6S,9R,10S configuration for the stereocenters of the chamigrene skeleton of isoobtusadiene (1) and its acetyl derivative (2). This assignment was further supported by analysis of the electronic circular dichroism (ECD) spectrum of the acetyl derivative 2. The experimental spectrum of the natural product showed a positive band at 207 and a negative band at 248 nm, together with a shoulder at around 230 nm. As shown in Figure 2, according to the configuration suggested by NMR, these data

Table 1. NMR Spectroscopic Data (CDCl3, 1H NMR 600 MHz, 13C NMR 150 MHz) for Isoobtusadiene 1 and Acetyl Isoobtusadiene 2a 1 δC, type

δH, multi (J in Hz)

δC, type

δH, multi (J in Hz)

1a 1b

27.3, CH2

29.0, CH2

2a 2b 3 4 5 6 7 8a 8b 9

27.7, CH2

1.93, m 1.76, ddd (15.0, 13.0, 4.0) 2.27, dt (15.0, 3.6) 2.20, m

1.91, m 1.78, ddd (15.0, 13.0, 4.0) 2.28, dt (15.0, 4.0) 2.20, m

10 11 12 13 14a

71.6, CH 42.6, C 27.6, CH3 22.3, CH3 118.1, CH2

position

14b 15a 15b OAc a

2

142.4, C 132.5, CH 133.6, CH 51.1, C 143.5, C 38.9, CH2 73.1, CH

112.0, CH2

6.26, d (10.0) 5.84, d (10.0)

2.72, dd (15.5, 1.6) 2.58, dd (15.5, 2.0) 4.16, ddd (2.6, 2.0, 1.6) 4.65, d (2.6) 1.02, s 1.23, s 4.87, s 5.12, s 4.79, s

29.5, CH2 141.9, C 134.2, CH 135.3, CH 50.7, C 143.2, C 37.0, CH2 73.2, CH 63.5, CH 42.4, C 28.4, CH3 22.6, CH3 119.4, CH2 113.6, CH2

4.81, s

6.28, d (10.5) 5.84, d (10.5)

2.72, dd (15.5, 1.6) 2.46, dd (15.5, 2.0) 5.29, ddd (3.3, 2.0, 1.6) 4.55, d (3.3) 1.03, s 1.22, s 4.85, s 5.01, s 4.80, s 4.82, s

170.2, C 22.9, CH3

2.08, s

Numbering system is in agreement with Gerwick et al.13

experiments unambiguously identified the chemical shifts of both methylene groups in the cyclohexadiene and the exomethylene moieties that were not assigned in the original paper.13 In fact, analysis of 2 showed correlations of H2-14 (δH 4.85 and 5.01) with C-6 (δC 50.7) and C-8 (δC 37.0), whereas the protons at C-15 (δH 4.80 and 4.82) correlated with C-2 (δC

Figure 1. Proposed configuration of isoobtusadiene skeleton. (A) Diagnostic NOE correlations observed for acetyl isoobtusadiene 2. (B) Chemical shift differences [ΔδS,R] between the S- and R-MTPA derivatives of isoobtusadiene 1. 3050



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acquired by a Jasco V-650 spectrophotometer, and IR spectra by a Jasco FT-IR 4100 spectrophotometer. ECD spectra were acquired on a Jasco J-815 polarimeter. NMR spectra were recorded on a Bruker DRX 600 spectrometer equipped with an inverse TCI CryoProbe. Chemical shift values were referenced to internal signals of residual proton [CDCl3 1H (δH 7.26 ppm) and 13C (δC 77.0 ppm)]. Gas chromatography−mass spectrometry (GC-MS) analysis was carried out on a Focus GC Polaris Q (Thermo Scientific). High-resolution mass spectra were acquired on a Q-Exactive Hybrid QuadrupoleOrbitrap mass spectrometer (Thermo Scientific). Silica gel 60 chromatography was performed using precoated Merck F254 plates. All chemicals and solvents (Sigma-Aldrich) were of analytical reagent grade and were used without any further purification. Biological Material. Ophionereis reticulata was collected during low tide at Paracuru beach (3°24′0.22″ S and 39°0′48.60″ W), on the coast of Ceará State, Brazil. The marine organism (voucher number 400 CELIMCE) was taxonomically identified by Dr. Helena M. Cascon, and a voucher specimen (no. 400 CELIMCE) is deposited at the Laboratório de Invertebrados Marinhos do Ceará of Departamento de Biologia, Universidade Federal do Ceará. For morphological details of Ophionereis reticulata see Figure S29 (Supporting Information). Extraction and Isolation Procedure. Eleven animals, after their capture, were immediately extracted at room temperature two times with MeOH for 24 h (wet weight of the fresh animals was 876.2 mg). The organic solvent was evaporated under reduced pressure at 40 °C to yield 75 mg of extract. This material was fractionated on a Sephadex LH-20 column by MeOH to give 11 fractions (from A to M). Fraction I (12.3 mg) was further purified by silica gel column chromatography using a gradient of diethyl ether in petroleum ether as eluent to obtain, in order of increasing polarity, compounds 2 (0.4 mg), 1 (0.6 mg), and 3 (0.5 mg), respectively. Isoobtusadiene (1): colorless oil; [α]25D −25 (c 0.007, CH2Cl2) (lit.13 −11.7, c 0.7, CHCl3); Rf 0.7 (light petroleum−diethyl ether, 60:40); UV (MeOH) λmax (log ε) 230 (2.1); IR (KBr) νmax 3537, 2980, 1640, 1370, 1206, 1030 cm−1; 1H and 13C NMR data, Table 1; GC-EIMS (EI, 70 eV) m/z (%) [M] + m/z 295.99 (50) (C15H2179BrO), [M + 2]+ 297.99 (50) (C15H2181BrO). Acetyl Isoobtusadiene (2): colorless oil; [α]25D −27 (c 0.007, CH2Cl2); [α]25D −12 (c 0.007, CHCl3); Rf 0.85 (light petroleum− diethyl ether, 60:40); UV (MeOH) λmax (log ε) 230 (2.0); IR (KBr) νmax 2920, 1738, 1372, 1246, 1212, 1030 cm−1; 1H and 13C NMR data, Table 1; GC-EIMS (EI, 70 eV) m/z (%) [M − AcOH]+ m/z 279.02 (50), [M − AcOH+2]+ 281.06 (50); HRESIMS m/z (%) 361.07724 (50) [M + Na]+ (calcd for C17H2379BrNaO2, 361.07791), m/z 363.07507 (50) [M + Na]+ (calcd for C17H2381BrNaO2, 363.07587). (+)-Elatol (3): colorless oil; [α]25D +58 (c 0.02, CH2Cl2); [α]25D +57 (c 0.02, CHCl3) (lit.20 [α]25D +95.5); Rf 0.6 (light petroleum− diethyl ether, 60:40); 1H and 13C NMR data were identical to those reported in refs 17 and 21; GC-EIMS (EI, 70 eV) m/z (%) [M − Cl]+ 296.95 (50) (C 15 H 22 79 BrO), [M − Cl + 2] + 298.93 (50) (C15H2281BrO). Acetylation of Isoobtusadiene (1). A solution of isoobtusadiene (0.2 mg) in pyridine (0.5 mL) was treated with Ac2O (0.2 mL) and stirred at room temperature for 4 h. The reaction was quenched with H2O, and the mixture was extracted twice with CH2Cl2. The organic layer was concentrated and analyzed by 1H NMR and GC-EIMS. Preparation of MTPA Esters 1a and 1b of Isoobtusadiene. (S)- and (R)-MTPA-Cl (5 μL) and a catalytic amount of 4dimethylaminopyridine (DMAP) were separately added to two sample of 1 (0.1 mg each) in dry CH2Cl2 (0.5 mL). The resulting mixtures were allowed to stand at room temperature for 12 h. After evaporation of the solvent, the mixtures were extracted with diethyl ether, affording (R)- and (S)-MTPA esters 1a and 1b, respectively. (R)-MTPA ester of 1: selected 1H NMR values (CDCl3, 600 MHz) 6.21 (H-4), 5.69 (H5), 5.48 (H-9), 4.77/4.61 (H2-15), 4.58 (H-10), 2.69/2.38 (H2-8), 1.87/1.73 (H2-1), 2.23/2.13 (H2-2), 0.95 (H3-13), 0.98 (H3-12); ESIMS m/z (%) 535.1066 (50) [M + Na]+ (C25H2879BrF3NaO3), m/z 537.1048 (50) [M + Na]+ (C25H2881BrF3NaO3). (S)-MTPA ester of 1: selected 1H NMR values (CDCl3, 600 MHz) 6.27 (H-4), 5.78 (H-5), 5.51 (H-9), 5.01/4.89 (H2-14), 4.82/4.80 (H2-15), 4.55 (H-10), 2.81/

Figure 2. ECD spectroscopy of acetyl isoobtusadiene (2). Dotted line = experimental curve; black line = calculated spectrum of the 6S,9R,10S diastereomer; gray line = calculated spectrum of the 6R,9S,10R diastereomer.

were in good agreement with the calculated ECD spectrum of the 6S,9R,10S isomer. None of the other seven possible diastereomers of the chamigrene skeleton of 2 showed calculated ECD curves comparable to that of the natural product of O. reticulata (Supporting Information). This is the first complete assignment of the isoobtusadiene skeleton. The depicted 6S,9R,10S absolute configuration is in agreement with that indicated for the majority of the halogenated chamigrenes reported from Laurencia.20 The structure of compound 3 was inferred on the basis of the NMR data and polarimetric measurements. The 1H and 13C NMR chemical shifts were identical to those of (+)-elatol, as confirmed by the synthetic work of Stoltz and co-workers.21 Nevertheless, after one step of purification by HPLC, the specific rotation of 3 (+57) was different from that expected for (+)-elatol ([α]D = +95.5).21 The presence of impurities in the sample (Supporting Information) may explain the discrepancy of these data. Chamigrene sesquiterpenes including 1 and 3 are typical terpenes of algae, and this is the first report of these products in the brittle star O. reticulata. Marine algae are protected from herbivory by a variety of secondary metabolites, including terpenes, amino-acid-based and halogenated compounds, which can influence palatability.22 However, it is well-known that specialized feeders, such as several opisthobranch molluscs,23 can accumulate algal terpenes to deter predation. Many echinoderms of the class Echinoidea (e.g., sea urchins) are active herbivores and are protected from the negative effects of these molecules too. Lhullier and co-workers showed that Laurencia microcladia, containing elatol, is palatable to the sea urchin Echinometra lucunter.24 O. reticulata can be found in the Western Atlantic and Pacific Ocean from the intertidal zone up to 40 m deep, in reef environments and mangroves, and around algae.25,26 This animal is described as an unselective omnivore,27 although Hendler et al. reported that the diet of O. reticulate is based solely on green algae and diatoms.25 The identification of the chamigrene terpenes in the extract of the brittle star is a direct confirmation of this observation and suggests that this organism may be immune to or capable of detoxifying algal secondary metabolites. In consideration of the well-established role of herbivores on structuring algal populations,28 this finding opens intriguing questions about the possible ecological role of ophiuroids in the habitat of the northeast coast of Brazil.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Jasco P2000 digital polarimeter. UV spectra were 3051



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2.57 (H2-8), 1.90/1.77 (H2-1), 2.27/2.18 (H2-2), 1.00 (H3-13), 0.97 (H 3 -12); ESIMS m/z (%) 535.1062 (50) [M + Na] + ( C 2 5 H 2 8 7 9 B r F 3 N a O 3 ) , m / z 5 3 7. 1 0 4 3 ( 5 0 ) [ M + N a ] + (C25H2881BrF3NaO3). Computational Methods. Preliminary relaxed dihedral scans and unconstrained energy minimization (EM) were performed in implicit CHCl3 solvent (CPCM model, also used in all subsequent calculations)29 using resolution-of-identity (RI) approximation and a BP functional,30,31 combined with the def2TZVP(-f) basis set32 and the corresponding standard auxiliary basis set def2/J.33 Conformational scans on the Ac group were performed with full relaxation of all degrees of freedom except the scanned dihedral angles for each point and a resolution of 30°. The PBE0 functional34 and def2TZVP basis set32 in implicit CHCl3 were used for final unconstrained EM, using the RIJCOSX approximation35 and the corresponding standard auxiliary basis set def2/J.33 ECD spectra were calculated by applying the Tamm−Dancrof approximation to the time-dependent density functional approach,36 with the B3LYP functional,37,38 using the RIJCOSX approximation35 and ma-def2TZVP basis set32,39 with the corresponding automatically generated (AutoAux) auxiliary basis set40 and implicit CHCl3 solvent. Calculations utilize the atom-pairwise dispersion correction with the Becke−Johnson damping scheme (D3BJ).41,42 A total of 150 excited states were calculated, only including singlet excited states. Quantum chemical calculations were performed with ORCA version 4.0.1.43 ECD spectra were obtained using version 1.71 of the SpecDis software.44−46 Whenever used, peak broadening (expressed by the σ value in eV) and scaling (as a fraction of the experimental intensity) and UV shift parameters (in eV) to best match the resolution level of experimental data are reported in the text, tables, and figures. Similarity between calculated and experimental spectra is estimated with the similarity factor and Δ(enantiomeric similarity index),45 calculated with version 1.71 of the SpecDis software,44−46 allowing a UV shift range of ±30 nm and a range of σ from 0.1 to 0.26 eV for peak broadening in the predicted/experimental spectra best matching.

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for the technical support. A.F. acknowledges the support of MIUR-PRIN2015 “Top-down and bottom-up approach in the development of new bioactive chemical entities inspired on natural products scaffolds” (Project no. 2015MSCKCE_003).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00510. Table with the NMR data of elatol, 1D and 2D NMR spectra of compounds 1−3 and the derivatives 1a and 1b, ECD study of the acetyl derivative 2, pictures of O. reticulata with a morphological description of the brittle star (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: nuzzo.genoveff[email protected]. ORCID

Genoveffa Nuzzo: 0000-0001-7065-2379 Adele Cutignano: 0000-0003-3035-9252 Angelo Fontana: 0000-0002-5453-461X Author Contributions #

B. A. Gomes and G. Nuzzo contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work has been carried out in the frame of Italy-Brazil bilateral project (grant no. 490247/2011-3) founded by CNR and CNPq. The authors are gratefully to CNR, CNPq, and CAPES for the fellowships, financial support, and facilities. The authors also thank Mrs. D. Melck of NMR Service at ICB-NMR 3052



Note

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Isolation of Chamigrene Sesquiterpenes and Absolute Configuration

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