Belizentrin, a Highly Bioactive Macrocycle from the Dinoflagellate

Letter pubs.acs.org/OrgLett

Belizentrin, a Highly Bioactive Macrocycle from the Dinoflagellate Prorocentrum belizeanum Humberto J. Domínguez,† José G. Napolitano,† M. Teresa Fernández-Sánchez,‡ David Cabrera-García,‡ Antonello Novelli,§ Manuel Norte,† José J. Fernández,*,† and Antonio Hernández Daranas*,†,∥ †

Institute for Bio-Organic Chemistry “Antonio González”, Center for Biomedical Research of the Canary Islands, and ∥Department of Chemical Engineering and Pharmaceutical Technology, Faculty of Pharmacy, University of La Laguna, 38206 La Laguna, Tenerife, Spain ‡ Department of Biochemistry and Molecular Biology, and §Department of Psychology, Institute of Biotechnology of Asturias, Campus “El Cristo”, University of Oviedo, Oviedo 33006, Spain S Supporting Information *

ABSTRACT: Belizentrin (1), a novel 25-membered polyketide-derived macrocycle, was isolated from cultures of the marine dinoflagellate Prorocentrum belizeanum. This metabolite is the first member of an unprecedented class of polyunsaturated and polyhydroxylated macrolactams. The structure of 1 was primarily determined by NMR and computational methods. Pharmacological assays with cerebellar cells showed that 1 produces important changes in neuronal network integrity at nanomolar concentrations.

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958.4794). The UV spectrum of 1 showed an absorption maximum at 270 nm, which is consistent with the presence of a polyconjugated carbonyl group. Analysis of the HSQC experiment showed the presence of 9 olefinic carbons (accounting for 10 olefinic protons as one carbon is an exotype methylene), 4 methyl groups, 13 aliphatic methylenes, and 14 aliphatic methines (including 13 oxymethynes). In addition, 8 quaternary carbons were identified in the HMBC experiment, including 2 carboxamides, 1 carboxy carbon, 3 olefinic carbons, and 2 aliphatic carbons directly attached to oxygen atoms as deduced by their chemical shifts (72.2 and 97.9 ppm). A comprehensive analysis of COSY, TOCSY, HSQC, and HSQC-TOCSY experiments led to the identification of six 1 H−1H spin systems (Figure 1). These fragments were connected through the analysis of long-range 1 H, 13 C correlations extracted from the HMBC experiment (Figure 1). An interesting structural feature of 1 is the presence of both an ester and an amide group on its macrocycle backbone. The location of the amide group was determined by the distinctive chemical shifts of C11′ (41.3 ppm) and the methylene protons H11′ (3.41/3.84 ppm) as measured in CD3OD. On the other hand, considering the downfield shifts of both C19 and H19 (73.0 and 5.36 ppm, respectively), the ester functionality connected this position with C1′, closing a 25-membered macrocycle. Further analysis of the HMBC experiment enabled the identification of three ether linkages (Figure 1), thereby

arine organisms are an important source of bioactive molecules, with a significant number of them currently in clinical trials. In fact, due to their novel structural features, many of these marine-derived products are considered as “firstin-class” drugs.1 Marine dinoflagellates, in particular the genus Prorocentrum, produce some of the most active and complex secondary metabolites found in nature.2−7 Many of these compounds exhibited unparalleled antiproliferative or immunosuppressive bioactivities, making them potential drug candidates or valuable pharmacological tools in drug discovery and development.8 In this report, we describe the isolation and structure elucidation of belizentrin (1), a chemically unique and highly bioactive macrocycle obtained from large-scale cultures of Prorocentrum belizeanum. This new polyketide-derived compound has a highly oxidized side chain and is, to the best of our knowledge, the first macrolactam isolated from a marine dinoflagellate.9 Belizentrin was isolated from the methanol extract of a cell pellet obtained from a 1000 L culture of P. belizeanum (strain PBMA01). Chromatographic fractionation of the crude extract was carried out using gel permeation and reversed-phase chromatography, which led to the isolation of 3.1 mg of 1 as a white amorphous solid. After a preliminary purity assessment was performed by LC−MS (obtaining a single-peak total ion current chromatogram), samples of 1 were subjected to both NMR analysis and biological testing. The molecular formula of 1, C48H73NO17 (with a total of 13 degrees of unsaturation), was established by HRMS analysis ([M + Na]+ C48H73NO17Na, calcd m/z 958.4776, obsd m/z © XXXX American Chemical Society

Received: July 17, 2014

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dx.doi.org/10.1021/ol502102f | Org. Lett. XXXX, XXX, XXX−XXX

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Figure 1. Planar structure of belizentrin (1), including key NMR correlations. The 1H,1H spin systems are numbered I−VI. Figure 2. Selected dipolar correlations observed in the five- and sixmembered rings of belizentrin (1).

forming one oxolane (B) and two oxane rings (A/C), and accounting for the remaining unsaturations. Once the planar structure of 1 was established, the determination of key stereochemical features of this complex molecule was undertaken. The configuration of the double bonds Δ17,18, Δ2′,3′, and Δ9′,10′ was assigned as E on the basis of the observed 3JH,H values (>15 Hz in all cases) and ROE crosscorrelations (Figure 1). Because the remaining double bonds included quaternary carbons, the determination of their geometry relied on the analysis of dipolar correlations. As a result, the configuration of the Δ4′,5′ olefin was assigned as E based on the ROE cross-peaks between Me12′ and one of the H6′ methylene protons, as well as by the ROE between H3′ and H5′. Similarly, the Z configuration of the Δ21,22 double bond was assigned by the observation of an intense ROE correlation between Me34 and H22. The relative configuration of all the stereogenic centers in the six-membered ring A was unambiguously determined using a combination of scalar coupling measurements and a thorough analysis of the ROESY experiment. A trans-diaxial relationship between protons H3, H4, H5, and H6 was established on the basis of their large vicinal coupling constants (3JH3,H4 = 8.9 Hz, 3 JH4,H5 = 8.9 Hz, and 3JH5,H6 = 10.5 Hz). The smaller J-coupling value between H6 and H7 (3JH6,H7 = 4.2 Hz) was diagnostic of a gauche relationship between these neighboring protons. The cross-peak network observed in the ROESY experiment between H3, H5, and H8 confirmed that these protons were located on the same ring face, whereas the dipolar correlations between H2, H4, H6, and H7 positioned these protons on the opposite side (Figure 2A). Even though the configurational analysis of saturated fivemembered rings represents a substantial challenge due to their inherent conformational flexibility,10 the relative configuration of the oxolane ring B could be established through the analysis of dipolar correlations between cis-oriented protons in 1,3relative positions.11 The ROE cross-peak between H12 and H14, as well as the one between H14 and one of the H16 methylene protons, indicated that these nuclei were located on the same ring face. Additional dipolar correlations between one of the H13 methylene protons and H11, Me33, and H15 were observed, thereby confirming that these protons were positioned on the opposite ring face (Figure 2B). In the case of ring C, the relative configuration of all four chiral centers was established by ROE analysis due to the existence of two quaternary carbons (Figure 2C). Thus, the

dipolar correlation between H25 and Me35 indicated that both groups are located on the same ring face, whereas the dipolar correlation between H26ax and H28 positioned these nuclei on the opposite ring face. In addition, the ROE cross-peaks between H28, and both H30 and H31 were crucial to establish the relative configuration of the quaternary carbon C29 (Figure 2C). Next, we approached the relative configuration within the 25membered macrocycle, where four out of the five chiral carbons within this moiety are located in the oxane ring C. Consequently, the difficulty was to connect their relative configurations with the remote position C19. Taking into account that all of the involved stereocenters are confined within a conformationally restricted macrocycle, this task should be accessible from ROE and 3JH−H measurements followed by computational simulations.12−14 Therefore, in order to analyze the NMR data, we fixed a 25R*,27S*,28R*,29S* relative configuration for ring C, and molecular modeling simulations were undertaken using a C1− C15 truncated model of the two possible C19 epimers (Figure 3 and Figure S15, Supporting Information).15 For this task, an

Figure 3. Selected dipolar correlations observed for the macrocyclic moiety of belizentrin (1). Interatomic distances are shown in angstroms. B


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unrestricted hybrid search method was used.16 First, 5000 cycles of molecular dynamics simulated annealing at 1000 K were carried out, followed by 5000 cycles of large-scale low mode search steps. These cycles resulted in 24 and 8 stable conformers for the 19S* and 19R* epimers within a 10 kJ/mol threshold of the global minimum. Afterward, geometrical optimizations at the DFT level using the B3LYP functional with the 6-31G**(+) basis set were undertaken. Once the resulting Boltzmann populations were calculated, four relevant conformers (70, 20, 5, and 4%) were found for the 19S* epimer, while only one major conformer (98%) resulted for the 19R* epimer (Figure S15, Supporting Information). The calculated structures were examined looking for concordances with the available NMR data (ROE and 3JH−H). Thus, it turned out that the calculated structures for the 19S*,25R*,27S*,28R*,29S* stereoisomer explained the dipolar correlations observed between the pairs H2′−H312′, H312′− H6′, H6′−H8′, and H8′−H10′ as well as the transannular ROE between H2′ and H24 (Figure 3). Moreover, on the other face of the macrocycle an analogous NOE network could be observed, reinforcing the previous observations. Particularly diagnostic were the correlations between H19 and both Me34 and H17 (Figure 3 and Table S2, Supporting Information). Moreover, the measured 3JH,H values for H19 (ddd, 9.2, 8.9, 4.8 Hz) perfectly matched the selected structure. On the other hand, the structure of the 19R* epimer showed discrepancies with the experimental data. We also used quantum mechanical calculations of chemical shifts as an alternative approach of analyzing the NMR data. This methodology has proved to be an excellent tool for addressing the stereochemistry of complex natural products.17,18 Thus, we calculated 1H and 13C chemical shifts for both C19 epimers of the macrocyclic portion of 1 and estimated their DP4 probabilities.19 Particularly informative were the calculated chemical shifts for both H20 (Figure S14, Supporting Information). Using the DP4 parameter, it is possible to assign stereochemical relationships with quantifiable confidence.20 The result was that, the 19S*,25R*,27S*,28R*,29S* configuration (also selected using the NOE and 3JH,H data) was predicted with >99% probability, reinforcing our previous conclusions. The C1−C18 side chain of 1 is stereochemically more complex than the macrocyclic portion as includes 11 asymmetric carbons. Nevertheless, considering that the relative configuration within the C3−C7 and C12−C15 cyclic stereoclusters was previously determined, the remaining task was to connect both rings via a four-carbon acyclic tether (C8− C11) that included three stereogenic centers. In the absence of long-range heteronuclear coupling constants (decomposition of 1 occurred before we could do such measurements, Figure 11, Supporting Information), we made use of Kishi’s universal NMR database concept.21 Thus, we measured 1H−1H coupling constants within the C9−C12 portion of 1 (3JH9,H10 = 3.9 Hz, 3 JH10,H11 = 2.8 Hz, and 3JH11,H12 = 5.8 Hz) from 1H and COSYDQF experiments (Table S1, Supporting Information). The observed 3JH,H profile corresponds either to an all-anti or all-syn relationship between the oxygen substituents of this portion but discards all other possibilities.21 In order to solve this uncertainty, we also made use of chemical shift calculations. Thus, we built models of the 16 possible diasteroisomers of the C1−C18 fragment and performed conformational searches for each one (Table S5, Supporting Information). Afterward, all conformers within a 10 kJ/mol threshold of the global

minimum found for each stereoisomer were geometrically optimized at the HF/3-21G level. Finally, single-point energy and NMR shielding constants calculations were calculated using DFT at the mpw1pw91/6-31G**(+) level for all those conformers (201 structures) using a Poisson−Boltzmann methanol solvation model. Subsequently, for each diasteroisomer, a Boltzmann weighted average of their NMR chemical shifts and the corresponding DP4 probabilities were estimated. This calculation resulted in the prediction of a single stereoisomer of relative configuration, 3R*,4S*,5R*,6R*,7R*,9S*,10R*,11S*,12S*,14S*,15R*, with a 90.1% probability when both 1H and 13C chemical shifts were taken into account (Tables S6 and S7, Supporting Information). Remarkably, this stereoisomer shows an all syn relationship between the oxygen substituents of the C9−C12 portion in correspondence with Kishi’s NMR database results. In summary, the relative configurations within the macrolactam and the polyhydroxilated side chain had been assigned, although the relationship between their configurations is arbitrary as they are connected by a double bond at C17−C18. From our point of view, belizentrin shows structural similarities with macrolide immunosuppressants.22 Recently, several lines of evidence have demonstrated unexpected activities of immunossuppresant drug targets in the central nervous system including the modulation of tubulin polymerization and τ protein function,23 axon regeneration and sprouting,24 neurite outgrowth,25 and neuronal apoptosis.26 Consequently, to study the possible biological actions of 1 on neuronal survival and function we used primary cultures of cerebellar cells.26,27 Exposure of cultured neurons to 1 resulted in strong changes in neuronal network integrity followed by cell death. Neurite weakness and fragmentation was evident starting at concentrations of 100 nM, while exposure to concentrations of 1 over 300 nM caused complete degeneration of neuronal somas (Figure 4). These effects required 24 h exposure to 1, while no visible morphological signs of toxicity could be

Figure 4. Effect of various concentrations of belizentrin (1) on cultures of cerebellar cells. (Top) Fluorescence photomicrographs of neurons before and after exposure to 1 for 24 h. Live neurons showed a bright green color in the cell body, whereas neurites and dead neurons did not retain any fluorescein and their nuclei appeared stained in red by ethidium bromide. Complete disintegration of neurites was observed in cells exposed to 200 nM of 1. (Bottom) Dose−response curve (mean ± SD). C


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(10) Taha, H. A.; Richards, M. R.; Lowary, T. L. Chem. Rev. 2013, 113, 1851−1876. (11) Napolitano, J.; Gavín, J.; García, C.; Norte, M.; Fernández, J. J.; Daranas, A. H. Chem.Eur. J. 2011, 17, 6338−6347. (12) Steinmetz, H.; Irschik, H.; Kunze, B.; Reichenbach, H.; Höfle, G.; Jansen, R. Chem.Eur. J. 2007, 13, 5822−5832. (13) Bock, M.; Buntin, K.; Müller, R.; Kirschining, A. Angew. Chem., Int. Ed. 2008, 47, 2308−2311. (14) Menche, D. Nat. Prod. Rep. 2008, 25, 905−918. (15) Janssen, D.; Albert, D.; Jansen, R.; Müller, R.; Kalesse, M. Angew. Chem., Int. Ed. 2007, 46, 4898−4901. (16) Macromodel, version 10.0 and Jaguar, version 8.0; Schrödinger, LLC: New York, NY, 2013. (17) Cen-Pacheco, F.; Rodriguez, J.; Norte, M.; Fernandez, J. J.; Daranas, A. H. Chem.Eur. J. 2013, 19, 8525−8532. (18) Lodewyk, M. W.; Siebert, M. R.; Tantillo, D. R. Chem. Rev. 2012, 112, 1839−1862. (19) Smith, S. G.; Goodman, J. M. J. Am. Chem. Soc. 2010, 132, 12946. (20) Domínguez, H. J.; Crespín, G. D.; Santiago-Benítez, A. J.; Gavín, J. A.; Norte, M.; Fernández, J. J.; Daranas, A. H. Mar. Drugs 2014, 12, 176−192. (21) Higashibayashi, S.; Czechtizky, W.; Kobayashi, Y.; Kishi, Y. J. Am. Chem. Soc. 2003, 125, 14379−14393. (22) Schreiber, S. L. Science 1991, 251, 283−28. (23) Chambraud, B.; E. Sardin, E.; Giustiniani, J.; Dounane, O.; Schumacher, M.; Goedert, M.; Baulieu, E. E. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 2658−2663. (24) Buchmaster, P. S.; Wen, X. Epilepsia 2011, 52, 2057−2064. (25) Chen, H.; Xiong, T.; Qu, Y.; Zhao, F.; Ferriero, D.; Mu, D. Neurosci. Lett. 2012, 507, 118−123. (26) Fernández-Sánchez, M. T.; Cabrera-García, D.; FerreroGutierrez, A.; Pérez-Gómez, A.; Cruz, P. G.; Daranas, A. H.; Fernández, J. J.; Norte, M.; Novelli, A. Toxicol. Sci. 2013, 132, 409− 418. (27) D’Mello, S. R.; Galli, C.; Ciotti, T.; Calissano, P. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10989−1099.

observed with shorter exposures. Dose−response experiments were performed taking advantage of the capability of viable cells to retain fluorescein. The concentration of 1 that produced a 50% reduction in maximum neuronal survival after 24 h (EC5024) was estimated at approximately 193 ± 7 nM. These biological effects on neuronal cultures were consistently observed over time. This bioactivity is therefore a stable characteristic of the compound, suggesting the involvement of structural elements not affected by the observed decomposition of the molecule. In summary, this study described the structural characterization of belizentrin (1), a new and structurally unique macrolide that exhibited potent bioactivity in neuronal survival assays in vitro. Interestingly, 1 affected the strength and integrity of neurites long before any reduction in neuronal viability could be observed. From a structural point of view, the relatively low stability of 1 precluded the acquisition of a complete set of spectroscopic data, in particular heteronuclear n JC,H measurements, thus adding a new degree of difficulty to an already challenging elucidation task. Nevertheless, a thorough analysis of the available NMR data, combined with molecular modeling simulations and quantum mechanical calculations, enabled the evaluation of potential diastereomeric structures and ultimately made possible a full proposal for the relative configuration of this complex natural product.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures, spectral data, and computational results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research work was funded by EU Grant Nos. FP7-KBBE3-245137-MAREX and FP7-REGPOT-2012-CT2012-31637IMBRAIN as well as by SAF2011-28883-C03-01 and 03 from MINECO and CEI10/00018 from MECD, Spain. H.D. acknowledges MINECO for a Ph.D. scholarship.

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REFERENCES

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Belizentrin, a Highly Bioactive Macrocycle from the Dinoflagellate

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