Lissodendoric Acids A and B, Manzamine-Related Alkaloids from the

Letter pubs.acs.org/OrgLett

Lissodendoric Acids A and B, Manzamine-Related Alkaloids from the Far Eastern Sponge Lissodendoryx florida Ekaterina G. Lyakhova,*,†,§ Sophia A. Kolesnikova,†,§ Anatoly I. Kalinovsky,† Dmitrii V. Berdyshev,† Evgeny A. Pislyagin,† Aleksandra S. Kuzmich,† Roman S. Popov,† Pavel S. Dmitrenok,† Tatyana N. Makarieva,† and Valentin A. Stonik*,†,‡ †

G. B. Elyakov Pacific Institute of Bioorganic Chemistry, The Far East Branch of the Russian Academy of Sciences, Prospect 100-let Vladivostoku 159, Vladivostok-22, Russia ‡ Far Eastern Federal University, Sukhanova Street 8, Vladivostok-91, Russia S Supporting Information *

ABSTRACT: The first representatives of a new group of manzaminerelated alkaloids with a previously unknown skeletal systems, namely, lissodendoric acids A (1) and B (2), were isolated from the sponge Lissodendoryx florida collected from the Sea of Okhotsk. The structures and absolute configurations have been elucidated by extensive spectroscopic analysis together with chemical transformations and quantum-chemical modeling. The lissodendoric acids show a potent capability to decrease the production of reactive oxygen species in neuroblastoma Neuro 2a and somewhat increase the survival of these cells upon treatment with 6hydroxydopamine (an in vitro antiparkinson biotest).

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anzamines and so-called manzamine-related compounds are a group of bioactive polycyclic alkaloids from marine sponges. Since the report on the antitumor metabolite manzamine A from Haliclona sp. in 1986,1 many alkaloids of this diverse group have been chemically elucidated.2 Most of them have been found to possess different biological activities, including antitumor, antimalarial, antimicrobial, insecticidal, anti-inflammatory, and immune-suppressant properties.3,4 Herein we describe the isolation, chemical structure, and bioactivity of two new manzamine-related metabolites 1 and 2 from the marine sponge Lissodendoryx florida, which have novel skeletal systems that are significantly different in comparison with known alkaloids. Their unique structural features include a conjugated carboxydiene moiety, a longer saturated intramolecular hydrocarbon chain, and an unprecedented (CH2)10NH2 substituent instead of the canonical manzaminebridged system (see S4 in the Supporting Information (SI)). The frozen sponge L. florida (dry weight 18 g), which was collected in the cold waters of the Sea of Okhotsk in August 2015, was finely chopped and extracted with EtOH. The extract was concentrated under reduced pressure, and an adsorbed aqueous residue was freed of inorganic salts by washing with water using polychrome (powdered Teflon) column chromatography. The organic layer, eluted with EtOH (from 50 to 100%), was concentrated and separated on a silica column using a stepwise gradient (CHCl3−EtOH−H2O/TFA 0.1%). Both compounds 1 and 2 (Figure 1) were isolated as trifluoroacetate salts from the polar fractions by reversedphase HPLC (see the SI for the details). Compound 1 was isolated as an optically active amorphous solid with [α]D −37.5 (c 0.48, MeOH). The chemical formula © 2017 American Chemical Society

Figure 1. Structures of lissodendoric acids A (1) and B (2) and chemical transformations of 1.

of 1, C29H50N2O2, was determined from the HR-ESI-MS data (m/z 459.3944 [M + H]+, calcd for C29H51N2O2 459.3945). The ESI-MS data for 1 in CD3OD disclosed that the molecule has four exchangeable protons (m/z 463.4, calcd for C29H47D4N2O2 463.4) (SI S6−S8). The 1H and 13C NMR spectra of 1 revealed two trisubstituted double bonds (δH 5.88, 6.94; δC 134.5, 139.5, 132.2, 135.5) conjugated to a carboxyl group (δC 169.4). In support of the latter, the UV spectrum (CH3OH) showed λmax Received: August 22, 2017 Published: September 21, 2017 5320

DOI: 10.1021/acs.orglett.7b02608 Org. Lett. 2017, 19, 5320−5323

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Organic Letters 298 nm (ε 2840). The analysis of the HSQC spectrum along with 13C NMR, DEPT, and MS data indicated that compound 1 contains four quaternary carbons, 22 sp3 methylenes, and two sp2 and one sp3 methines. The 1H and 13C chemical shifts of CH2-1 (δH 3.27, d, J = 13.5 and 3.54, dd, J = 13.5, 1.4; δC 57.3), CH2-3 (δH 3.25, td, J = 13.5, 2.5 and 3.40, br dd, J = 13.5, 4.8; δC 49.0), CH2-10 (δH 3.37, m and 3.42, m; δC 52.6), and CH210′ (δH 2.89, br t, J = 7.6; δC 40.8) allowed the assignment of these four methylenes to positions α- to amine N atoms (Table S3 in the SI). Further inspection of 1D and 2D NMR spectra (COSY, HSQC, HMBC, and 1H−15N GHMBC) as well as evaluation of the 1H−1H coupling constants revealed the presence of a hexahydroisoquinoline fragment in 1 (Figure 2). Moreover, the

Scheme 1. Characteristic HR-ESI-MS/MS Fragmentations for Compounds 1 and 2

aliphatic chain at N-2. Consequently, the 4aS*,8aS* relative stereochemistry and chair conformation of the piperidine ring with axial positions of Hβ-1, Hβ-3, H-4a, and aliphatic substituents at C-8a and N-2 were deduced (Figure 3).

Figure 2. Key COSY (bold bonds), 1H−13C HMBC (black ↶), and 1 H−15N GHMBC (red ↶) correlations of lissodendoric acid A (1).

chemical shifts of the protons on C-1, C-3, and C-10 supported the protonated form of the tertiary N-2 in 1 isolated in the presence of TFA.5,6 This was also in agreement with the intense ion peak at m/z 459 [M + H]+ in the MALDI MS spectrum of 1 registered in positive ion mode without a matrix (SI S7). With the absence of methyl group signals in the spectra, the remaining 19 methylenes were attributed to the two aliphatic chains based on 1D and 2D TOCSY data. One of them is attached to the isoquinoline core between N-2 and C-7, and another one with an amino group at the end is connected to C8a. The key long-range correlations of H-4a (δH 2.80, dd, J = 12.1, 4.5) to C-1′ (δC 39.5); H2-1 (δH 3.54, dd, J = 13.5, 1.4 and 3.27, d, J = 13.5) to C-10 (δC 52.6) and C-3 (δC 49.0); H2-10 (δH 3.37, m and 3.42, m) to C-3 (δC 49.0); H2-18 (δH 2.24, dt, J = 15.0, 5.2 and 2.39, m) to C-6 (δC 135.5), C-7 (δC 139.5), and C-8 (δC 134.5); H2-10′ (δH 2.89, br t, J = 7.6) to C-8′ (δC 27.5) and C-9′ (δC 28.6); H-11 (δH 1.61, m) to N-2 (δN 32.4); and H2-9′ (δH 1.63, br qv, J = 7.6) and H2-10′ (δH 2.89, br t, J = 7.6) to NH2-11′ (δN 31.5) confirmed the linkages (Figure 2). The lengths of the chains were determined by HR-ESI-MS/ MS. Specifically, the fragment ion at m/z 302.2113 (calcd 302.2115) was observed in the correspondence with the loss of a CH3−(CH2)9−NH2 fragment from the molecular ion (Scheme 1 and SI S6). The relative configuration of 1 was established by a ROESY experiment and analysis of the coupling constants (SI S13). NOE correlations were observed between H-4a/H2-1′ and H4a/Hβ-1, Hβ-3, and the value of 4JH1α−H3α (1.4 Hz) as well as the Hα-1/Hα-3 COSY correlation supported a coplanar arrangement along a W path of Hα-1 and Hα-3. The Hα-4/H10 and H-8/H-11 correlations suggested the α-oriented

Figure 3. Key NOE correlations (dashed arrows) of lissodendoric acid A (1).

Treatment of 1 with Ac2O in pyridine gave substituted acetamide 1a, as confirmed by HR-ESI-MS data (m/z 501.4053 [M + H]+, calcd for C29H51N2O2 501.4051) and the HMBC correlations H3 (δH 1.76, s, Ac)/CO (δC 168.8, Ac) and H210′ (δH 2.97, td, J = 6.9, 5.9)/CO (δC 168.8, Ac). Detailed analysis of the 1D and 2D NMR spectra demonstrated the free tertiary amine form of 1a because the 1H NMR signals due to the N-methylene groups were approximately 1 ppm lower than those for protonated 1.5 Moreover, the unshared electron pair of N-2 is anti to C−H1β (δH 1.76, d, J = 11.0) and C−H3β (δH 1.64) and induces an upfield shift of the latter.7 Consequently, the aliphatic substituent at N-2 is equatorial (SI S21−S30). Theoretical calculation of the electronic circular dichroism (ECD) spectra of the two enantiomers of 1a with the relative stereochemistry suggested by the NMR data was performed using TDDFT with the nonlocal exchange−correlation functional B3LYP,8 as implemented in the Gaussian 03 suite.9 The 6-311G(d) basis set was used. Solvent effects were taken into account using the polarizable continuum model (PCM).10 The ECD spectrum was derived by population-weighted superposition of the individual ECD spectra calculated for the conformers, where the population weighting factors were obtained by the B3LYP/6-311G(d)_PCM method. Each of these individual ECD spectra was modeled as a superposition of 5321

DOI: 10.1021/acs.orglett.7b02608 Org. Lett. 2017, 19, 5320−5323

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C−N bond with −(CH2)10−NH2 side chain formation (g) and further oxidation to afford 1 or 2 (Scheme 2).

Gaussian-type functions with bandwidth ΔE = 0.44 eV taken at the 1/e peak heights. As shown in Figures 4 and S1, good agreement was observed between the experimental and theoretical spectra with the 4aS,8aS absolute configuration for 1a and, consequently, for 1 (SI S31−S33).

Scheme 2. Possible Biogenetic Pathway from Corresponding Cyclostellatamine to 1 (n = 5, m = 4) or 2 (n = 5, m = 5)

Figure 4. Experimental ECD spectrum of 1a and theoretical spectra of two possible enantiomers. The UV shifts Δλ = 14 nm and Δλ = −26 nm were used for the regions λ ≤ 270 nm and λ > 270 nm, respectively, to reproduce the peak positions of the absorption bands in the experimental UV spectrum of 1a.

The presence of the acetamide in 1a instead of the primary amine in 1 allowed us to avoid difficulty in theoretical modeling of its ECD spectrum caused by the possible formation of additional H-bonded conformations. The reactive capacity of functional groups in 1 was shown in its reaction with EDC and DMAP. The cyclic product 1b was obtained and characterized by MS and NMR. The HR-ESI-MS peak at m/z 441.3838 [M + H]+ (calcd for C29H49N2O 441.3839), the low-field-shifted NMR signals of CH2-10′ (δH 3.61, m and 3.06, m), the characteristic signals of an amide bond11 NH-11′ (δH 8.11, m; δN 116.9), and the COSY correlation from CH2-10′ to NH-11′ confirmed the structure of 1b (SI S34−S38). Compound 2 was isolated as a minor component, and its molecular formula, C30H52N2O2, was determined from the HRESI-MS data in MeOH (m/z 473.4104 [M + H]+, calcd for C30H53N2O2 473.4102), indicating that it has one more methylene than 1. The NMR (Table S3) and ECD data as well as the optical rotation ([α]D −18.0, c 0.2, MeOH), were almost identical and support the same nature as 1. Analysis of the MS/MS spectrum demonstrated that the ion peak at m/z 316.2271 (calcd 316.2271) corresponds to a daughter ion with a (CH2)10 aliphatic bridge between N-2 and C-7 (Scheme 1 and SI S40−S47). The well-known scheme proposed by Baldwin and Whitehead12 and enhanced by Tsuda et al.13 discloses the biosynthesis of two possible types of antipodal manzaminerelated compounds. For lissodendoric acids, the Diels−Alder cyclization of cyclostellatamine precursors14 (a) seems to be realized and leads to the formation of polycyclic product d with a β-cis-fused hexahydroisoquinoline core. Further transformations include general steps of dismutation and enamine (e) hydrolysis to give hypothetical intermediate f. Typically, oxidation of f leads to the tetra- or pentacyclic framework of the majority of manzamines and manzamine-related metabolites with a hydroxyl at C-7. Alternatively, in case of the lissodendoric acids, conversions of f cause the cleavage of the

Although an α-cis-fused configuration is characteristic of the bicycle in most of the known manzamines and related compounds,1−4,15 the absolute configurations of several of them, including (−)-keramaphidin B,13 ircinols,16 and entmanzamines,17 were assigned as antipodal. The latter compounds share levorotatory properties with 1 and 2. Remarkably, lissodendoric acids A and B are the first representatives of a new structural series that have only three fused rings as well as saturated and elongated hydrocarbon chains in their structures. Only ingenamines18 have the combination of a 3-en-C8 chain and an elongated linear carbon bridge. The lissodendoric acids A and B did not demonstrate significant cytotoxicity (IC50 > 28.5 μM) against HeLa and SKMEL-5 cell lines. Then we examined the effect of sublethal concentrations of 1 and 2 on the generation of reactive oxygen species (ROS) in Neuro 2a cells treated with 6-hydroxydopamine (6-OHDA). The neuroblastoma cell damage induced by 6-OHDA is known as an in vitro model of Parkinson’s disease.19 The compounds show a significant (around 50%) reduction in ROS levels at concentrations of 0.1 and 10 μM for 1 and 0.1 μM for 2 (SI S49). The demonstrated activities of 1 and 2 are similar to the effects of some compounds demonstrating antiparkinson activity,20 including the antioxidant astaxanthin, pretreatment with which at a concentration of 0.1 μM significantly inhibited apoptosis, mitochondrial abnormalities, and intracellular ROS generation in 6-OHDAtreated cells.21,22 Also, the neuroprotective in vivo effect of astaxanthin in a model of Parkinson’s disease has been described.23

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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.orglett.7b02608. 5322

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(12) Baldwin, J. E.; Whitehead, R. C. Tetrahedron Lett. 1992, 33, 2059−2062. (13) Tsuda, M.; Inaba, K.; Kawasaki, N.; Honma, K.; Kobayashi. Tetrahedron 1996, 52, 2319−2324. (14) Lee, Y.; Jang, K. H.; Jeon, J.; Yang, W.-Y.; Sim, C. J.; Oh, K.-B.; Shin, J. Mar. Drugs 2012, 10, 2126−2137. (15) Furusato, A.; Kato, H.; Nehira, T.; Eguchi, K.; Kawabata, T.; Fujiwara, Y.; Losung, F.; Mangindaan, R. E. P.; de Voogd, N. J.; Takeya, M.; Yokosawa, H.; Tsukamoto, S. Org. Lett. 2014, 16, 3888− 3891. (16) Tsuda, M.; Kawasaki, N.; Kobayashi, J. Tetrahedron 1994, 50, 7957−7960. (17) El Sayed, K. A.; Kelly, M.; Kara, U. A. K.; Ang, K. K. H.; Katsuyama, I.; Dunbar, D. C.; Khan, A. A.; Hamann, M. T. J. Am. Chem. Soc. 2001, 123, 1804−1808. (18) Kong, F.; Andersen, R. J. Tetrahedron 1995, 51, 2895−2906. (19) Pasban-Aliabadi, H.; Esmaeili-Mahani, S.; Abbasnejad, M. Rejuvenation Res. 2017, 20, 125−133. (20) Bernstein, A. I.; Garrison, S. P.; Zambetti, G. P.; O’Malley, K. L. Mol. Neurodegener. 2011, 6, 2. (21) Ikeda, Y.; Tsuji, S.; Satoh, A.; Ishikura, M.; Shirasawa, T.; Shimizu, T. J. Neurochem. 2008, 107, 1730−1740. (22) Liu, X.; Shibata, T.; Hisaka, S.; Osawa, T. Brain Res. 2009, 1254, 18−27. (23) Grimmig, B.; Daly, L.; Hudson, C.; Nash, K. R.; Bickford, P. C. Funct. Foods Health Dis. 2017, 7, 562−576.

Experimental details; spectral data for 1, 1a, 1b, and 2; computational method and bioassay (PDF)

AUTHOR INFORMATION

Corresponding Authors

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

Ekaterina G. Lyakhova: 0000-0001-9487-1496 Valentin A. Stonik: 0000-0002-8213-8411 Author Contributions §

E.G.L. and S.A.K. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant 17-14-01065 from the Russian Science Foundation, and the study was carried out on the equipment of the Collective Facilities Center of The Far Eastern Center for Structural Molecular Research (NMR/MS) of PIBOC FEB RAS.

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DOI: 10.1021/acs.orglett.7b02608 Org. Lett. 2017, 19, 5320−5323