Vagiallene, a Rearranged C15 Acetogenin from Laurencia obtusa

Mar 12, 2019 - branched spin system. Even though the closure of the second ... while at the same time being in agreement with the 1H and 13C. NMR chem...
0 downloads 0 Views 955KB Size
Download PDF
Letter pubs.acs.org/OrgLett

Cite This: Org. Lett. XXXX, XXX, XXX−XXX

Vagiallene, a Rearranged C15 Acetogenin from Laurencia obtusa Stamatios Perdikaris,† Alfonso Mangoni,‡ Laura Grauso,§ Panagiota Papazafiri,⊥ Vassilios Roussis,† and Efstathia Ioannou*,†

Org. Lett. Downloaded from pubs.acs.org by IDAHO STATE UNIV on 04/19/19. For personal use only.

†

Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece ‡ Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Via D. Montesano 49, Napoli 80131, Italy § Dipartimento di Agraria, Università degli Studi di Napoli Federico II, Via Università 100, Portici, 80055 Città Metropolitana di Napoli, Italy ⊥ Section of Animal and Human Physiology, Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, Athens 15784, Greece S Supporting Information *

ABSTRACT: Vagiallene (1), a rearranged C15 acetogenin with a molecular formula and a carbon skeleton unprecedented in natural products, was isolated as a trace constituent from the organic extract of the red alga Laurencia obtusa from Lefkada island. The planar structure and the relative configuration of 1 were established on the basis of extensive analysis of its spectroscopic data, while its absolute configuration was determined by comparison of its experimental and quantummechanically predicted electronic circular dichroism spectra.

T

he genus Laurencia, comprised of 146 taxonomically accepted species, owes its reputation to the astonishing number of structurally diverse molecules that have been isolated from members of this genus during the last 60 years.1 This genus has attracted the interest of many scientists from multidisciplinary fields, pursuing investigations on the chemistry, ecology, and biology of Laurencia spp. and its secondary metabolites, as documented from the increasing number of scientific publications describing their isolation and bioactivity assessment as well as efforts toward their total synthesis.1 To date, more than 1050 secondary metabolites, mostly halogenated terpenes and acetogenins frequently displaying new carbocycles, have been reported from species of Laurencia as well as mollusks grazing on them.1,2 In the framework of our ongoing interest in the chemistry of Laurencia and the isolation of new bioactive natural products, in recent years we have developed an integrated metabolomic platform for the accelerated identification of known compounds and the detection of new metabolites at the early stages of phytochemical analysis.3 Application of this screening strategy on a large number of extracts and fractions derived from Laurencia populations collected from the Aegean and Ionian Seas revealed the presence of a trace constituent (1) (Figure 1) possessing the molecular formula C15H16Br2O5, never reported up to now for any marine or terrestrial natural product,4 in an extract of Laurencia obtusa collected from the island of Lefkada in the Ionian Sea. © XXXX American Chemical Society

Figure 1. Chemical structure of vagiallene (1).

Vagiallene (1),5 isolated through a series of chromatographic separations in minute amounts (∼230 μg),6 was obtained as a colorless oil. In its HR-ESI-MS analysis in negative-ion mode, compound 1 displayed an [M − H]− ion peak at m/z 432.9286 with isotopic ion peaks at m/z 434.9262 and 436.9241 at a relative intensity of 1:2:1, consistent with C15H15Br2O5. In the IR spectrum, the absorption band at 3423 cm−1 indicated the presence of at least one hydroxy group in the molecule, while the absorption band at 1768 cm−1 suggested the occurrence of an unsaturated five-membered lactone. The 13C NMR spectrum revealed 15 carbon signals, which as determined from the HSQC-DEPT spectrum corresponded to one methyl, one methylene, 10 methine, and three nonprotonated carbon atoms, whereas the 1H NMR spectrum included signals Received: March 12, 2019

A

DOI: 10.1021/acs.orglett.9b00897 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

H-14 with C-4 confirmed the closure of the first pyran ring through an ether bridge between C-4 and C-14. Additionally, the HMBC correlations of both H-7 and H-8 with C-6 and C-9 indicated the formation of an unsaturated γ-lactone ring. On the basis of the HMBC correlation of H-5 with the ketal carbon C-6, as also indicated by its 13C NMR chemical shift (δC 107.1), the latter ring was connected on C-5 of the branched spin system. Even though the closure of the second pyran ring through an ether bridge between C-6 and C-10 was not experimentally verified by an HMBC correlation between the hemiacetal proton H-10 (δH 5.44) and C-6 as it might have been expected,7 the spiro system is the only possible planar structure that can justify the tricyclic carbon skeleton of 1 while at the same time being in agreement with the 1H and 13C NMR chemical shifts as well as the 3J coupling of H-10 to the OH proton at C-10 observed in the COSY spectrum. The relative configuration of vagiallene (1) was determined on the basis of the key correlations displayed in the NOESY spectrum (Figure 3). The cis junction of the dioxadecaline

reminiscent of C15 acetogenins, commonly isolated from species of the genus Laurencia. The structural elements displayed in the 1H and 13C NMR spectra of 1 (Table 1) included one methyl group on a tertiary Table 1. 1H and 13C NMR Data of Vagiallene (1) (δ in ppm, J in Hz) in chloroform-d no.

δC

δH

a

1 2 3 4 5 6 7 8 9 10 11

74.6 204.0 100.4 70.1 49.4 107.1 152.7 122.5 169.0e 95.3 28.4

12

36.3

13 14 15 −OH

55.7 72.0 20.6

b

6.17, d (5.7) 5.39, dd (8.6, 5.7) 4.49, dd (10.4, 8.6) 2.09, dd (10.4, 3.9) 7.30, d (5.7) 6.15, d (5.7) 5.44, m α 1.85, ddd (13.7, 13.5, 10.2) β 2.48, ddd (13.7, 2.9, 2.8) 3.04, m 3.97, 3.91, 1.36, 3.27,

dd (10.4, 4.6) dq (10.4, 5.7) d (5.7) d (5.4)

in benzene-d6 δC

c

73.9 203.8 100.8 70.2 49.3 106.9 152.8 122.3 168.6 95.2 28.3

36.4 56.7 71.7 20.5

δH d 5.43, d (5.7) 4.71, dd (8.9, 5.7) 3.96, dd (10.5, 8.9) 1.27, dd (10.5, 3.9) 6.72, d (5.8) 5.60, d (5.8) 4.80, m α 1.46, ddd (13.7, 13.2, 10.1) β 2.13, ddd (13.7, 2.9, 2.6) 2.56, dddd (13.2, 4.8, 3.9, 2.9) 3.34, dd (10.5, 4.8) 3.20, dq (10.5, 5.9) 1.11, d (5.9) 2.24, brs

a

Measured at 50.3 MHz. bMeasured at 400 MHz. cBased on HMBC correlations. dMeasured at 600 MHz. eBased on HMBC correlations. Figure 3. Key NOE correlations of vagiallene (1).

carbon (δH/C 1.36/20.6), four oxygenated or halogenated methines (δH/C 3.91/72.0, 3.97/55.7, 4.49/70.1, and 5.44/ 95.3), one conjugated 1,2-disubstituted double bond (δH/C 6.15/122.5 and 7.30/152.7), and two deshielded methines (δH/C 5.39/100.4 and 6.17/74.6) which together with a carbon resonating at δC 204.0 were characteristic for a bromoallene functionality as well as one ester carbonyl at δC 169.0 and a nonprotonated carbon at δC 107.1. Since the carbon−carbon double bond, the carbonyl group, and the bromoallene moiety accounted for four of the seven double-bond equivalents dictated by the molecular formula, the planar structure of 1 was determined as tricyclic. The homonuclear and heteronuclear correlations observed in the COSY and HMBC spectra of 1 established its planar structure (Figure 2). The cross-peaks observed in the COSY spectrum revealed the presence of an extended branched spin system, starting from H-3 of the bromoallene functionality, which in turn exhibited a long-range (4J) coupling to H-1 as well as an isolated spin system for the two olefinic methines of the 1,2-disubstituted double bond. The HMBC correlation of

system was deduced by the strong NOE correlation between H-5 and H-12 and further confirmed by the correlation between H-4 and H-11α, which also established the configuration at C-4. The intense 1,3-diaxial correlations of H-4 with H-14, H-5 with H-13, and H-10 with H-12 determined the configurations at C-14, C-13, and C-10, respectively. Furthermore, the configuration of the spiro carbon atom C-6 was indicated by the NOE correlation of H-4 with H-7. Finally, the strong NOE correlation between H3 and H-5, fixing the predominant rotamer about the C-3/C-4 bond, in combination with a weaker but clear NOE correlation of H-1 with H-7 determined the axial chirality of the allene system. The absolute configuration of 1 was determined by comparison between the experimental and the quantummechanically predicted electronic circular dichroism (ECD) spectra of the 2R,4S,5S,6S,10R,12R,13R,14S enantiomer of 1. It is well-known8 that theoretical ECD spectra are strongly dependent on the conformation of the molecule, so a conformational search was initially performed using molecular dynamics (MD) (see the Supporting Information). Vagiallene (1) is a rigid molecule and low conformational mobility is expected for it. Indeed, the MD calculation revealed only two low-energy conformers (1a and 1b, Figure 4), differing in the dihedral angle around the C-3/C-4 bond. These structures were used as starting structures for the quantum-mechanical calculations, performed using the program Gaussian 09.9 The geometry of the conformers 1a and 1b was optimized using the ωB97X-D functional10 and the TZVP basis set, and

Figure 2. COSY and key HMBC correlations of vagiallene (1). B

DOI: 10.1021/acs.orglett.9b00897 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

of vagiallene (1) is indeed 2R,4S,5S,6S,10R,12R,13R,14S. These results are in accordance with the empirical rule proposed by Lowe about the absolute configuration of chiral allenes.12 According to Lowe’s rule, the strongly negative optical rotation measured for 1 (−378°) is indicative of the 2R configuration of the bromoallene moiety. The optical rotation predicted by quantum-mechanical calculations also shows a negative sign (−323°, see the Supporting Information), along with a magnitude close to the experimental one. Final validation of the structure of vagiallene (1) was obtained by quantum-mechanical prediction of its 13C NMR chemical shifts13 and 1H−1H coupling constants,14 using the same level of theory as for ECD prediction. The predicted chemical shifts were in excellent agreement with the experimental values (see the Supporting Information), with an average absolute error of 1.34 ppm and a maximum error of 3.28 ppm. Proton−proton coupling constants were also in good agreement (average absolute error 0.80 Hz, maximum error 1.60 Hz) with the experimentally determined values (see the Supporting Information). Through a series of experiments, Murai has shown that electrophilic bromoetherification, by the action of bromoperoxidase, on the straight-chain C15 fatty acid laurediol results in the formation of the obtusallene carbocycle.15 As subsequently proposed by Braddock,16 obtusallenes I, III, and V−IX can be derived through multiple electrophilic events on obtusallene II. In the case of vagiallene (1), a plausible scheme for its biogenesis can be envisaged starting from obtusallene I (2),17 which was isolated as one of the main constituents of the investigated algal extract. In particular, vagiallene (1) could arise from the carbon skeleton of obtusallene I (2) through the cleavage of Δ9 and the formation of a carbon−carbon bond between C-5 and C-12 (Scheme 1). Specifically, oxidative cleavage of Δ9 could result in the formation of an aldehyde group at C-10 and a 10-membered lactone ring. The formation of an intermediate bromonium ion, the opening of the 10membered lactone ring, and a nucleophilic attack of Δ5 at C-12 could result in the shift of the bromine atom from C-12 to C13 and the formation of the first pyran ring of vagiallene. Subsequent nucleophilic attack of the C-9 hydroxy group at C6 carbocation could give rise to the formation of the γ-lactone ring. Dehydrohalogenation would lead to the formation of the Δ7 bond. Allylic oxidation at C-6 would form the carbinol that through nucleophilic attack at the electron-deficient C-10

Figure 4. Minimum energy conformers of vagiallene (1) as determined by quantum-mechanical calculations.

then time-dependent density functional theory (TD-DFT) was used to predict their theoretical CD spectra. The Boltzmannweighted mean of the two spectra was calculated and a CD curve was obtained from the Gaussian output using the program SpecDis v. 1.70 (see the Supporting Information).11 The two empirical parameters used by SpecDis to optimize the fit between the experimental and predicted spectra were adjusted as follows: the half-width of the CD bands, σ, was set to 0.48 eV and the UV shift was set to 15 nm. The calculated spectrum was in excellent agreement with the theoretical spectrum (Figure 5), showing that the absolute configuration

Figure 5. Predicted (dashed line) and experimental (solid line) ECD spectra of (2R,4S,5S,6S,10R,12R,13R,14S)-1.

Scheme 1. Plausible Biosynthetic Pathway for Vagiallene (1)

C

DOI: 10.1021/acs.orglett.9b00897 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

(4) ReaxysElsevier Life Sciences IP Ltd.: Oxford. https://www. reaxys.com/#/search/quick (accessed Mar 01, 2019). (5) Vagiallene (1) was named after the late Professor Constantinos Vagias, colleague and friend, who focused his research efforts on the study of marine organisms from the Greek coastlines. (6) The weight of 1 was determined using the NMR-based procedure reported in: Dalisay, D. S.; Molinski, T. F. NMR Quantitation of Natural Products at the Nanomole Scale. J. Nat. Prod. 2009, 72, 739−744. (7) HMBC experiments were measured using different J values (1.5, 3, 5, 8, 10, 12, and 15 Hz). Nonetheless, the correlation of H-10 to C6 was not observed under these experimental conditions. (8) Esposito, G.; Bourguet-Kondracki, M.-L.; Mai, L. H.; Longeon, A.; Teta, R.; Meijer, L.; Van Soest, R.; Mangoni, A.; Costantino, V. Chloromethylhalicyclamine B, a Marine-Derived Protein Kinase CK1δ/ε Inhibitor. J. Nat. Prod. 2016, 79, 2953−2960. (9) Gaussian 09, Revision E.01. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian, Inc.: Wallingford, CT, 2009. (10) Chai, J.-D.; Head-Gordon, M. Long-Range Corrected Hybrid Density Functionals with Damped Atom−Atom Dispersion Corrections. Phys. Chem. Chem. Phys. 2008, 10, 6615−6620. (11) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDis: Quantifying the Comparison of Calculated and Experimental Electronic Circular Dichroism Spectra. Chirality 2013, 25, 243−249. (12) Lowe, G. The Absolute Configuration of Allenes. Chem. Commun. 1965, 411−413. (13) Moosmann, P.; Ueoka, R.; Grauso, L.; Mangoni, A.; Morinaka, B. I.; Gugger, M.; Piel, J. Cyanobacterial Ent-Sterol-Like Natural Products from a Deviated Ubiquinone Pathway. Angew. Chem., Int. Ed. 2017, 56, 4987−4990. (14) Costantino, V.; Fattorusso, E.; Imperatore, C.; Mangoni, A. Glycolipids from Sponges. 20. J-Coupling Analysis for Stereochemical Assignments in Furanosides: Structure Elucidation of Vesparioside B, a Glycosphingolipid from the Marine Sponge Spheciospongia vesparia. J. Org. Chem. 2008, 73, 6158−6165. (15) Murai, K. In Comprehensive Natural Product Chemistry; Barton, D. H. R., Meth-Cohn, O., Nakanishi, K., Eds.; Pergamon: Elmsford, NY, 1999; Vol. 1, pp 303−324. (16) Braddock, D. C. A Hypothesis Concerning the Biosynthesis of the Obtusallene Family of Marine Natural Products via Electrophilic Bromination. Org. Lett. 2006, 8, 6055−6058. (17) (a) Cox, P. J.; Imre, S.; Islimyeli, S.; Thomson, R. H. Obtusallene I, a New Halogenated Allene from Laurencia obtusa. Tetrahedron Lett. 1982, 23, 579−580. (b) Cox, P. J.; Howie, R. A. Xray Structure Analysis of Obtusallene. Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. 1982, 38, 1386−1387.

could give rise to the spiro-hemiacetal ring system of vagiallene (1). It is noteworthy that the relative configuration of the stereogenic centers C-4 and C-14 as well as the absolute configuration of the bromoallene functionality are identical in both vagiallene (1) and obtusallene I (2). Vagiallene (1) was evaluated for its in vitro cytotoxic activity against four human tumor cell lines (A549, A431, HT29, and MCF7). Compound 1 was proven inactive against A549, HT29, and MCF7 cells (IC50 > 50 μM) but exhibited weak activity (IC50 42.6 ± 5.7 μM) against A431 cells, albeit slightly higher than that of miltefosine used as a positive control (see the Supporting Information).

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00897. Details of experimental procedures, additional references, additional figures and tables, and NMR and MS spectra of 1 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Alfonso Mangoni: 0000-0003-3910-6518 Efstathia Ioannou: 0000-0003-3103-4951 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Dr. K. Tsiamis (Hellenic Centre for Marine Research) for his help with the taxonomic identification of the alga. This research has been cofinanced by the European Union (European Social Fund − ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) − Research Funding Program: Heracleitus II. Investing in knowledge society through the ESF (Grant No. 346942-ΠΕ1.93), as well as through the Operational Program “Competitiveness and Entrepreneurship” of the National Strategic Reference Framework (NSRF) − Research Funding Program: Cooperation 2011. Regions at the Centre of Development were supported through the ESF (Grant No. 11ΣΥΝ-3-770).

■

REFERENCES

(1) Harizani, M.; Ioannou, E.; Roussis, V. The Laurencia Paradox: An Endless Source of Chemodiversity. In Progress in the Chemistry of Organic Natural Products; Kinghorn, A. D., Falk, H., Gibbons, S., Kobayashi, J., Eds.; Springer International Publishing, 2016; Vol.102, pp 91−252. (2) MarinLit: A Database of the Marine Natural Products Literature; Royal Society of Chemistry: London. http://pubs.rsc.org/marinlit/ (accessed Aug 30, 2018). (3) Kokkotou, Κ.; Ioannou, Ε.; Nomikou, Μ.; Pitterl, F.; Vonaparti, A.; Siapi, E.; Zervou, M.; Roussis, V. An Integrated Approach Using UHPLC-PDA-HRMS and 2D HSQC NMR for the Metabolic Profiling of the Red Alga Laurencia: Dereplication and Tracing of Natural Products. Phytochemistry 2014, 108, 208−219. D

DOI: 10.1021/acs.orglett.9b00897 Org. Lett. XXXX, XXX, XXX−XXX