Elucidation of Spirodactylone, a Polycyclic Alkaloid from the Sponge

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Elucidation of Spirodactylone, a Polycyclic Alkaloid from the Sponge Dactylia sp., and Nonenzymatic Generation from the Co-metabolite Denigrin B Unwoo Kang,† Donald R. Caldwell,‡ Laura K. Cartner,†,§ Dongdong Wang,† Chang-Kwon Kim,† Xiangrong Tian,†,⊥ Heidi R. Bokesch,†,§ Curtis J. Henrich,†,§ Girma M. Woldemichael,†,§ Martin J. Schnermann,*,‡ and Kirk R. Gustafson*,†

Downloaded by UNIV OF SOUTHERN INDIANA at 15:51:05:602 on May 31, 2019 from https://pubs.acs.org/doi/10.1021/acs.orglett.9b01636.

†

Molecular Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States ‡ Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States § Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland 21702-1201, United States ⊥ Research & Development Center of Biorational Pesticide, College of Plant Protection, Northwest A&F University, Yangling 712100, P. R. China S Supporting Information *

ABSTRACT: Spirodactylone (1), a hexacyclic indolizidone alkaloid possessing a novel spiro ring system, was isolated from the marine sponge Dactylia sp. The structure was elucidated by extensive spectroscopic methods including application of the LR-HSQMBC NMR pulse sequence. Oxidative cyclization of denigrin B (2), an aryl-substituted 2-oxo-pyrroline derivative that was also isolated from the sponge extract, provided material identical to spirodactylone (1). This confirmed the assigned structure and provides insight into the probable biogenesis of 1.

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the oncogenic PAX3-FOXO1 transcription factor,7 provided an inactive indolizidone alkaloid with a novel natural product skeleton. Although 18 species have been described for the genus Dactylia,8 the only previous report in the chemical literature from this genus detailed the isolation of ircinamine B, a fatty acid conjugated thioether alkaloid.9 Sequential chromatographic fractionation of the Dactylia sp. extract on diol and C18 solid supports provided spirodactylone (1, Figure 1) and the tetrasubstituted pyrrolinone denigrin B (2).10 The structural elucidation and semisynthetic preparation of 1 are reported herein. Spirodactylone (1) was isolated as a yellow amorphous solid, and its molecular formula of C31H23NO5, determined by HRESIMS measurements, required 21 degrees of unsaturation. A UV absorption maximum at 332 nm (MeOH) revealed extended conjugation in 1, and IR bands at 3266, 1656, and 1605 cm−1 suggested the presence of hydroxy and carbonyl groups. The 1H NMR spectrum showed signals indicative of two mutually coupled methylene groups at δH 4.00 (2H, t, J = 6.1 Hz, H2-6), 2.28 (2H, t, J = 6.1 Hz, H2-7), two pairs of

he marine environment has provided many unprecedented secondary metabolites comprised of unique structural scaffolds and unusual functional group arrays.1 These natural products often occur as a series of structurally related analogues that can arise from genetic processes involving insertion, deletion, or mutation events, as well as biosynthetic gene cluster duplication followed by retailoring or rearrangement.2 Substrate promiscuity of the associated biosynthetic enzymes also provides a rich source of secondary metabolite chemical diversity.3 However, there is growing evidence that important diversity and chemical complexity in various alkaloid families can also arise via nonenzymatic processes involving reactive biosynthetic intermediates. Examples where chemically diverse natural products are generated nonenzymatically from metabolites with inherent nucleophilic reactivity include the discoipyrroles4 and the ammosamides.5 Chemical complexity can also be created by electrophilic processes such as formaldehyde-mediated dimerization of the bohemamine alkaloids.6 These types of nonenzymatic pathways provide a potential route for both natural or directed efforts to enhance the chemical diversity of gene-encoded small molecules. In the current study, an extract of the marine sponge Dactylia sp., which appeared active in an assay for inhibitors of © XXXX American Chemical Society

Received: May 8, 2019

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DOI: 10.1021/acs.orglett.9b01636 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

48.0, C-8), and two methylene carbons [δC 36.2 (C-6), 34.3 (C-7)]. HMBC correlations (Figure 2) from H-9/H-13 to C-11 and from H-10/H-12 to C-8 established a 4,4-disubstituted

Figure 1. Structure of spirodactylone (1).

degenerate olefinic protons at δH 7.21 (2H, d, J = 10.1 Hz, H9/H-13) and 6.18 (2H, d, J = 10.1 Hz, H-10/H-12), and three para-substituted benzene groups at δH 7.05 (2H, d, J = 8.8 Hz, H-28/H-32), 6.57 (2H, d, J = 8.6 Hz, H-16/H-20), 6.56 (2H, d, J = 8.8 Hz, H-29/H-31), 6.50 (2H, d, J = 8.6 Hz, H-22/H26), 6.31 (2H, d, J = 8.6 Hz, H-23/H-25), and 6.23 (2H, d, J = 8.6 Hz, H-17/H-19) (Table 1). The 13C NMR spectrum Table 1. NMR Spectroscopic Data for Spirodactylone (1) in CD3OD no. 1 2 3 4 5 6 7 8 9/13 10/12 11 14 15 16/20 17/19 18 21 22/26 23/25 24 27 28/32 29/31 30

δC

δNa

δH, mult (J in Hz)

130.4 169.6, C 133.3, C 143.2, C 136.8, C 36.2, CH2 34.3, CH2 48.0, C 154.9, CH 129.8, CH 187.7, C 126.1, C 126.9, C 132.4, CHb 115.0, CH 158.6, C 125.2, C 131.9, CH 115.9, CH 158.0, C 122.7, C 132.4, CHb 116.1, CH 159.8, C

Figure 2. Key 2D NMR correlations for spirodactylone (1).

cyclohexadienone moiety. Additional HMBC correlations from H2-7 to C-8, C-9/13, and C-14, and from H-9/13 to C-7, C-8, and C-14 revealed connections between C-7 and C-8 and between C-8 and C-14. This indicated a spiro junction between rings B and C. The deshielded chemical shift of H2-6 (δH 4.00) suggested an adjacent N atom, and a 1H−15N HMBC correlation from H2-7 to N-1 (δN 130.4) confirmed the location of the lone nitrogen in 1. HMBC correlations from H2-6 to C-2 and C-5 led to the establishment of three C−N bonds between C-2, C-5, C-6, and N-1. Three disubstituted aryl groups with para-hydroxy substituents were readily established from their characteristic 1 H−1H COSY couplings and 1H−13C HMBC data. HMBC correlations from H-16/20 to C-14, H-22/26 to C-4, and H28/32 to C-3 revealed the position of the phenol substituents. These data and the fact that the 13C chemical shift values of C2, C-3, C-4, and C-5 in 1 were similar to those reported for denigrin B (2),10 a marine alkaloid that contains a diaryl pyrrole-2-one moiety, were sufficient to allow the structure of spirodactylone (1) to be proposed. However, due to the presence of 9 contiguous nonprotonated carbons in 1, unambiguous definition of the tricyclic core comprised of rings A, B, and C was lacking. Therefore, we utilized the recently reported LR-HSQMBC NMR experiment to further characterize 1.11 The LR-HSQMBC pulse sequence is a highsensitivity experiment designed to facilitate detection of 4bond and 5-bond heteronuclear couplings. These long-range correlations can complement the 2,3JC,H couplings normally observed in an HMBC spectrum, to help define the structure of proton-deficient molecular scaffolds. Successful application of this NMR technique, and the importance of the additional heteronuclear correlations it provides is illustrated by several recent studies of challenging new natural product structures.12 Numerous 4- and 5-bond C−H couplings were detected in the

4.00, t (6.1) 2.28, t (6.1) 7.21, d (10.1) 6.18, d (10.1)

6.57, d (8.6) 6.23, d (8.6)

6.50, d (8.6) 6.31, d (8.6)

7.05, d (8.8) 6.56, d (8.8)

N assignments were based on 1H−15N HMBC correlations. The δN values were not calibrated to an external standard but were referenced to neat NH3 (δ 0.00) using the standard Bruker parameters. bSignals overlapped. a15

revealed signals for one cross-conjugated ketone (δC 187.7, C11), one amide (δC 169.6, C-2), ten nonprotonated sp2 carbons [δC 159.8 (C-30), 158.6 (C-18), 158.0 (C-24), 143.2 (C-4), 136.8 (C-5), 133.3 (C-3), 126.9 (C-15), 126.1 (C-14), 125.2 (C-21), 122.7 (C-27)], eight degenerate sp2 methine signals that reflected 16 aromatic carbons [δC 154.9 (C-9/C-13), 132.4 (C-16/C-20 and C-28/C-32), 131.9 (C22/C-26), 129.8 (C-10/12), 116.1 (C-29/C-31), 115.9 (C23/C-25), 115.0 (C-17/C-19)], one quaternary sp3 carbon (δC B

DOI: 10.1021/acs.orglett.9b01636 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters LR-HSQMBC spectrum of 1 (optimized for nJC,H = 2 Hz), including key correlations from the aryl substituents to C-2, C3, C-4, C-5, and C-14 in the tricyclic core of the molecule (Figure 2). This provided powerful supporting spectroscopic evidence for the assigned structure of spirodactylone (1). The tetrasubstituted pyrrole derivative denigrin B (2) was also isolated from the Dactylia extract, and it was identified by comparison of the spectroscopic data we measured with published data.10 Compound 2 was previously obtained from an extract of the marine sponge Dendrilla nigra, and the first total synthesis was recently reported.13 The core of denigrin B (2) is a 2-oxo-3,4-diaryl pyrrole that is further elaborated by Nethyl phenol and 5-vinylphenol substituents. Comparison of their structures suggested that spirodactylone (1) might be derived from denigrin B (2) via an oxidative cyclization mechanism. Oxidative cyclizations of electron-rich arenes have been studied extensively as preparative reactions and invoked in the biosynthesis of natural products, including the cephalotaxus alkaloids.14−17 Although examples involving enamines are rare,18 we reasoned that this might be a promising approach to explore. To address the chemical feasibility of this transformation, we invesitgated the conversion of 2 to 1 using LC-MS to monitor the progress of small scale (0.1 μmol, 50 μg) reactions (Table 2). To our delight, we found that mild oxidative conditions,

conversion, while 1 equiv moderately improved product formation (entries 8 and 9). With these optimized reaction conditions in hand (1 equiv of NBS, CH2Cl2, rt, 14 h), the reaction was conducted on 3.4 μmol of denigrin B (2) and a 12% yield of spirodactylone (1) was obtained following HPLC purification. As depicted in Scheme 1, we propose the following mechanism for this Scheme 1. Generation of Spirodactylone (1) from Denigrin B (2) via Electophilic Halogenation-Initiated Dearomative Spirocyclization

Table 2. Screening of Halogenation-Initiated Dearomative Spirocyclization Conditions of Denigrin B (2)

entrya

electrophile (equiv)

temp (°C)

ratio of 2/1b

1 2 3 4 5 6 7c 8 9

NBS (2) PhI(OAc)2 NIS (2) I2 (2) NCS (2) NBS (2) NBS (2) NBS (4) NBS (1)

rt rt rt rt rt 50 rt rt rt

1/1 other products 9/1 9/1 >10:1 9/1 9/1 3/2 2/3

conversion. First, the selective formation of the bromonium species at the electron-rich enamine position provides intermediate A. Subsequent intramolecular ipso-cyclization provides B, and final elimination of HBr gives spirodactylone (1). The tricyclic core of spirodactylone (1) is comprised of a bicyclic indolizidone ring system spiro-fused to a cyclohexadieneone ring. This constellation of rings and functional groups is unprecedented in natural products; however, the tricyclic carbon−nitrogen framework has been reported in several synthetic studies.19,20 The fact that denigrin B (2) could be converted to 1 under mild oxidative conditions suggests that 2 may be a biogenic precursor produced by the sponge or associated symbiotic microbes. Under appropriate cellular or environmental conditions, enzyme-directed assembly of 2 could be followed by nonenzymatic oxidation and intramolecular cyclization to generate spirodactylone (1). This may be another example where the inherent reactivity of enzymatically assembled secondary metabolites provides further chemical diversity, including in this case the generation of an unprecedented alkaloid skeleton.

a

All reactions were run under the following conditions, unless otherwise indicated: 0.1 mmol of 2 in CH2Cl2 (1.0 mM) at rt for 14 h. b Ratios were determined by comparing the total ion counts of 2 and 1. cReactions were performed at 0.1 M in CH2Cl2.

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ASSOCIATED CONTENT

S Supporting Information *

specifically 2 equiv of NBS in CH2Cl2 (1.0 mM) at room temperature for 14 h provided the oxidative ion signal consistent with natural spirodactylone (entry 1, Table 2). HPLC and NMR analysis was then used to confirm that this reaction indeed provided authentic 1 (Supporting Information). Other oxidants were also tested, including PhI(OAc)2, NIS, I2, and NCS, but these resulted in worse conversion or alternative, and as yet unidentified, products (entries 2−5). Elevating the reaction temperature to 50 °C or decreasing the reaction concentration reduced conversion to spirodactylone (entries 6−7). Finally, the loading of NBS was investigated, and we found that using 4 equiv of NBS modestly decreased

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01636. Experimental procedures and full spectroscopic data for spirodactylone (1) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

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

DOI: 10.1021/acs.orglett.9b01636 Org. Lett. XXXX, XXX, XXX−XXX

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Martin J. Schnermann: 0000-0002-0503-0116 Kirk R. Gustafson: 0000-0001-6821-4943 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and with federal funds from the National Cancer Institute, National Institutes of Health, under Contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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DOI: 10.1021/acs.orglett.9b01636 Org. Lett. XXXX, XXX, XXX−XXX