Elucidation of Spirodactylone, a Polycyclic Alkaloid from the Sponge Dactylia sp., and Nonenzymatic Generation from the Cometabolite Denigrin B

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.

Graphical Abstract

The 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 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.

Figure 1.

Structure of spirodactylone (1).

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 δ_H 4.00 (2H, t, J = 6.1 Hz, H₂-6), 2.28 (2H, t, J = 6.1 Hz, H₂-7), two pairs of degenerate olefinic protons at δ_H 7.21 (2H, d, J = 10.1 Hz, H-9/H-13) and 6.18 (2H, d, J = 10.1 Hz, H-10/H-12), and three para-substituted benzene groups at δ_H 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/H-26), 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 revealed signals for one cross-conjugated ketone (δ_C 187.7, C-11), one amide (δ_C 169.6, C-2), ten nonprotonated sp² carbons [δ_C 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 [δ_C 154.9 (C-9/C-13), 132.4 (C-16/C-20 and C-28/C-32), 131.9 (C-22/C-26), 129.8 (C-10/12), 116.1 (C-29/C-31) 115.9 (C-23/C-25), 115.0 (C-17/C-19)], one quaternary sp³ carbon (δ_C 48.0, C-8), and two methylene carbons [δ_C 36.2 (C-6), 34.3 (C-7)].

Table 1.

NMR Spectroscopic Data for Spirodactylone (1) in CD₃OD

no.	δC	δNa	δH, mult (J in Hz)
1		130.4
2	169.6, C
3	133.3, C
4	143.2, C
5	136.8, C
6	36.2, CH2		4.00, t (6.1)
7	34.3, CH2		2.28, t (6.1)
8	48.0, C
9/13	154.9, CH		7.21, d (10.1)
10/12	129.8, CH		6.18, d (10.1)
11	187.7, C
14	126.1, C
15	126.9, C
16/20	132.4, CHb		6.57, d (8.6)
17/19	115.0, CH		6.23, d (8.6)
18	158.6, C
21	125.2, C
22/26	131.9, CH		6.50, d (8.6)
23/25	115.9, CH		6.31, d (8.6)
24	158.0, C
27	122.7, C
28/32	132.4, CHb		7.05, d (8.8)
29/31	116.1, CH		6.56, d (8.8)
30	159.8, C

^a15N assignments were based on ¹H–¹⁵N HMBC correlations. The δ_N values were not calibrated to an external standard but were referenced to neat NH₃ (δ 0.00) using the standard Bruker parameters.

^bSignals overlapped.

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 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 (δ_H 4.00) suggested an adjacent N atom, and a ¹H–¹⁵N HMBC correlation from H₂-7 to N-1 (δ_N 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.

Figure 2.

Key 2D NMR correlations for spirodactylone (1).

Three disubstituted aryl groups with para-hydroxy substituents were readily established from their characteristic ¹H–¹H COSY couplings and ¹H–¹³C HMBC data. HMBC correlations from H-16/20 to C-14, H-22/26 to C-4, and H-28/32 to C-3 revealed the position of the phenol substituents. These data and the fact that the ¹³C chemical shift values of C-2, 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 4-bond and 5-bond heteronuclear couplings. These long-range correlations can complement the ^2,3J_C,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 LR-HSQMBC spectrum of 1 (optimized for ⁿJ_C,H = 2 Hz), including key correlations from the aryl substituents to C-2, C-3, 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 N-ethyl 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, 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 conversion, while 1 equiv moderately improved product formation (entries 8 and 9).

Table 2.

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

entrya	electrophile (equiv)	temp (°C)	ratio of 2/1b
1	NBS (2)	rt	1/1
2	PhI(OAc)2	rt	other products
3	NIS (2)	rt	9/1
4	I2 (2)	rt	9/1
5	NCS (2)	rt	>10:1
6	NBS (2)	50	9/1
7c	NBS (2)	rt	9/1
8	NBS (4)	rt	3/2
9	NBS (1)	rt	2/3

^aAll 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.

^bRatios were determined by comparing the total ion counts of 2 and 1.

^cReactions were performed at 0.1 M in CH₂Cl₂.

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 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).

Scheme 1.

Generation of Spirodactylone (1) from Denigrin B (2) via Electophilic Halogenation-Initiated Dearomative Spirocyclization

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.