New from Old: Thorectandrin Alkaloids in a Southern Australian Marine Sponge, Thorectandra choanoides (CMB-01889)

Thorectandra choanoides (CMB-01889) was prioritized as a source of promising new chemistry from a library of 960 southern Australian marine sponge extracts, using a global natural products social (GNPS) molecular networking approach. The sponge was collected at a depth of 45 m. Chemical fractionation followed by detailed spectroscopic analysis led to the discovery of a new tryptophan-derived alkaloid, thorectandrin A (1), with the GNPS cluster revealing a halo of related alkaloids 1a–1n. In considering biosynthetic origins, we propose that Thorectandra choanoides (CMB-01889) produces four well-known alkaloids, 6-bromo-1′,8-dihydroaplysinopsin (2), 6-bromoaplysinopsin (3), aplysinopsin (4), and 1′,8-dihydroaplysinopsin (10), all of which are susceptible to processing by a putative indoleamine 2,3-dioxygenase-like (IDO) enzyme to 1a–1n. Where the 1′,8-dihydroalkaloids 2 and 10 are fully transformed to stable ring-opened thorectandrins 1 and 1a–1b, and 1h–1j, respectively, the conjugated precursors 3 and 4 are transformed to highly reactive Michael acceptors that during extraction and handling undergo complete transformation to artifacts 1c–1g, and 1k–1n, respectively. Knowledge of the susceptibility of aplysinopsins as substrates for IDOs, and the relative reactivity of Michael acceptor transformation products, informs our understanding of the pharmaceutical potential of this vintage marine pharmacophore. For example, the cancer tissue specificity of IDOs could be exploited for an immunotherapeutic response, with aplysinopsins transforming in situ to Michael acceptor thorectandrins, which covalently bind and inhibit the enzyme.

Recently, a GNPS molecular networking analysis was employed on 960 Southern Australian marine sponges, to map the chemical space of natural products, which resulted in the isolation of rare indolo-imidazole alkaloids, trachycladindoles H-M [8], new sesterterpene butenolides, cacolides A-L and cacolic acids A-C [9], and new sesquiterpenes, Recently, a GNPS molecular networking analysis was employed on 960 Southern Australian marine sponges, to map the chemical space of natural products, which resulted in the isolation of rare indolo-imidazole alkaloids, trachycladindoles H-M [8], new sesterterpene butenolides, cacolides A-L and cacolic acids A-C [9], and new sesquiterpenes, dysidealactams A-F, and dysidealactones A-B [10]. In this report, we present the discovery of a new class of tryptophan-derived alkaloid, thorectandrin A (1) (Figure 1), from a Great Australian Bight specimen of Thorectandra choanoides, prioritized for chemical investigation, based on GNPS molecular networking analysis of the same library of Southern Australian sponges.

GNPS Molecular Networking to Explore New Chemistry
To search for new marine natural products, 960 n-BuOH soluble partitions from the EtOH extracts of a library of Southern Australian marine sponges and 95 authentic standards (previously isolated from marine sponges) from the Capon lab were assembled and subjected to UPLC-QTOF-MS/MS analysis. The resulting data were used to create a consolidated GNPS molecular network ( Figure S1). In this molecular network, we found a specific molecular cluster ( Figure S2) associated with Thorectandra choanoides (CMB-01889) (collected in 1995 during deep water scientific trawling in the Great Australian Bight), which did not co-correlate with any metabolites found in the other 959 sponge extracts, or any of our authentic marine natural products. Following isolation, detailed spectroscopic analysis identified a new alkaloid scaffold, thorectandrin A (1) (Figure 1), while mass spectrometry (MS) data revealed molecular formulae for a host of structurally related analogues (1a-1n) in the same GNPS cluster. Note: All Thorectandra choanoides (CMB-01889) chemistry in this report are displayed as free bases, although all were isolated, and where appropriate, characterised as the trifluoroacetic acid salts.

GNPS Molecular Networking to Explore New Chemistry
To search for new marine natural products, 960 n-BuOH soluble partitions from the EtOH extracts of a library of Southern Australian marine sponges and 95 authentic standards (previously isolated from marine sponges) from the Capon lab were assembled and subjected to UPLC-QTOF-MS/MS analysis. The resulting data were used to create a consolidated GNPS molecular network ( Figure S1). In this molecular network, we found a specific molecular cluster ( Figure S2) associated with Thorectandra choanoides (CMB-01889) (collected in 1995 during deep water scientific trawling in the Great Australian Bight), which did not co-correlate with any metabolites found in the other 959 sponge extracts, or any of our authentic marine natural products. Following isolation, detailed spectroscopic analysis identified a new alkaloid scaffold, thorectandrin A (1) (Figure 1), while mass spectrometry (MS) data revealed molecular formulae for a host of structurally related analogues (1a-1n) in the same GNPS cluster. Note: All Thorectandra choanoides (CMB-01889) chemistry in this report are displayed as free bases, although all were isolated, and where appropriate, characterised as the trifluoroacetic acid salts.
Inspired by this sequence of transformations, we hypothesised that a comparable indoleamine 2,3-dioxygenase-like enzyme in Thorectandra choanoides (CMB-01889) converts 6-bromo-1′,8-dihydroaplysinopsin (2) to its ring-opened N-formyl derivative (1a), which is then rapidly hydrolyzed to thorectandrin A (1) (Scheme 3). Although 2 is reported as a sponge natural product [7], its absolute configuration (even enantiopurity) remains unassigned. Based on our experience, a possible challenge to assigning an absolute configuration to 2 might be enantiopurity, due to a propensity for keto-enol mediated epimerisation/racemisation. Armed with knowledge of the new thorectandrin scaffold and its likely biosynthetic relationship to the vintage aplysinopsin scaffold, we turned our attention to the literature and noted a 2015 report of racemic spiroreticulatine (8) from the South China Sea marine sponge Fascaplysinopsis reticulata [13], and a subsequent 2019 report from the same source of the known sponge alkaloid (Z)-3′-deimino-3′-oxoaplysinopsin (9), as a co-metabolite with 1b [14]. Although 8 was initially ascribed a plausible biosynthesis involving condensation of indole-3-carboxaldehyde and 1,3-dimethylhydantoin, this hypothesis seems highly improbable. A far more likely pathway would see ring opening of the indole heterocycle in the cometabolite 9, delivering a reactive Michael acceptor intermediate that Inspired by this sequence of transformations, we hypothesised that a comparable indoleamine 2,3-dioxygenase-like enzyme in Thorectandra choanoides (CMB-01889) converts 6-bromo-1 ,8-dihydroaplysinopsin (2) to its ring-opened N-formyl derivative (1a), which is then rapidly hydrolyzed to thorectandrin A (1) (Scheme 3). Although 2 is reported as a sponge natural product [7], its absolute configuration (even enantiopurity) remains unassigned. Based on our experience, a possible challenge to assigning an absolute configuration to 2 might be enantiopurity, due to a propensity for keto-enol mediated epimerisation/racemisation. and one nudibranch [11], the latter most likely a dietary input from nudibranches feeding on sponges. Typical exemplars include 6-bromoaplysinopsin (3) and aplysinopsin (4). In turning our attention to the likely biosynthesis of thorectandrin A (1), we considered the metabolic relationship between L-tryptophan (5), N-formyl-L-kynurenine (6), and Lkynurenine (7), and the fact that indoleamine 2,3-dioxygenase is known to convert 5 to 6, which undergoes facile hydrolysis by a formamidase to 7 (Scheme 2) [12]. Scheme 2. Biosynthetic conversion of L-tryptophan (5) to N-formyl-L-kynurenine (6) to Lkynurenine (7).
Inspired by this sequence of transformations, we hypothesised that a comparable indoleamine 2,3-dioxygenase-like enzyme in Thorectandra choanoides (CMB-01889) converts 6-bromo-1′,8-dihydroaplysinopsin (2) to its ring-opened N-formyl derivative (1a), which is then rapidly hydrolyzed to thorectandrin A (1) (Scheme 3). Although 2 is reported as a sponge natural product [7], its absolute configuration (even enantiopurity) remains unassigned. Based on our experience, a possible challenge to assigning an absolute configuration to 2 might be enantiopurity, due to a propensity for keto-enol mediated epimerisation/racemisation. Armed with knowledge of the new thorectandrin scaffold and its likely biosynthetic relationship to the vintage aplysinopsin scaffold, we turned our attention to the literature and noted a 2015 report of racemic spiroreticulatine (8) from the South China Sea marine sponge Fascaplysinopsis reticulata [13], and a subsequent 2019 report from the same source of the known sponge alkaloid (Z)-3′-deimino-3′-oxoaplysinopsin (9), as a co-metabolite with 1b [14]. Although 8 was initially ascribed a plausible biosynthesis involving condensation of indole-3-carboxaldehyde and 1,3-dimethylhydantoin, this hypothesis seems highly improbable. A far more likely pathway would see ring opening of the indole heterocycle in the cometabolite 9, delivering a reactive Michael acceptor intermediate that Armed with knowledge of the new thorectandrin scaffold and its likely biosynthetic relationship to the vintage aplysinopsin scaffold, we turned our attention to the literature and noted a 2015 report of racemic spiroreticulatine (8) from the South China Sea marine sponge Fascaplysinopsis reticulata [13], and a subsequent 2019 report from the same source of the known sponge alkaloid (Z)-3 -deimino-3 -oxoaplysinopsin (9), as a co-metabolite with 1b [14]. Although 8 was initially ascribed a plausible biosynthesis involving condensation of indole-3-carboxaldehyde and 1,3-dimethylhydantoin, this hypothesis seems highly improbable. A far more likely pathway would see ring opening of the indole heterocycle in the cometabolite 9, delivering a reactive Michael acceptor intermediate that undergoes non-stereoselective (enzyme or non-enzyme mediated) intramolecular Michael addition to racemic 8 (see Scheme 4).

Other Thorectandrin Co-Metabolites
While the thorectandrin GNPS cluster revealed a number of related metabolites, due to low abundance and a lack of sponge biomass, it was not possible to isolate and acquire definitive spectroscopic data to secure unambiguous structure assignments. Notwith-

Other Thorectandrin Co-Metabolites
While the thorectandrin GNPS cluster revealed a number of related metabolites, due to low abundance and a lack of sponge biomass, it was not possible to isolate and acquire definitive spectroscopic data to secure unambiguous structure assignments. Notwithstanding, we did acquire molecular formulae for many minor compounds, and on the basis of these measurements and biosynthetic considerations we tentatively propose structures, as shown in Table 2 Figure 3). While these adducts 1c-1g are believed to be solvolysis artifacts induced by long-term storage of Thorectandra choanoides (CMB-01889) in aqueous ethanol, followed by n-butanol partitioning and the use of methanol to dissolve dried extract, the absence of a rational solvolysis pathway from 1a to 1c, and 1 to 1d-1g, warranted consideration. We hypothesized that in addition to 6-bromo-1 ,8-dihydroaplysinopsin (2), Thorectandra choanoides (CMB-01889) produces 6-bromoaplysinopsin (3), which was comparably transformed by an indoleamine 2,3-dioxygenase-like enzyme to reactive Michael adducts (N-formyl-∆ 1 −8 -thorectandrin A and ∆ 1 −8 -thorectandrin A), with both undergoing Michael addition solvolysis during storage, fractionation and handling, to 1c-1g (Scheme 5). A recent review highlights the prevalence of solvolysis adduct artifacts among marine natural products, including among imidazoles/imidazolones [15].

Conclusions
Our application of the GNPS molecular networking to a library of Southern Australian marine sponges reinforced the value of this approach, in both dereplicating and prioritizing extracts for detailed investigation, and in guiding the discovery of new scaffolds. It also revealed itself to be a valuable tool for interrogating the halo of co-clustering minor analogues (including solvolysis artifacts), chemistry that typically defines traditional methods of isolation and structure elucidation.

Conclusions
Our application of the GNPS molecular networking to a library of Southern Australian marine sponges reinforced the value of this approach, in both dereplicating and prioritizing extracts for detailed investigation, and in guiding the discovery of new scaffolds. It also revealed itself to be a valuable tool for interrogating the halo of co-clustering minor ana-logues (including solvolysis artifacts), chemistry that typically defines traditional methods of isolation and structure elucidation.
As members of the aplysinopsin family of marine natural product are long known for their biological properties (i.e., anticancer, antibiotic, antidepressant, antimalarial, and antimicrobial properties) [11], the realisation that they are possible substrates for indoleamine 2,3-dioxygenases is significant. With human indoleamine 2,3-dioxygenase upregulated in key human tissues (i.e., small intestine and lung), and a number of cancers (i.e., acute myeloid leukemia, ovarian, and colorectal carcinoma), knowledge that aplysinopsins are substrates, and yield potent Michael acceptors, could inform future development of this pharmacophore. For example, one might take advantage of the tissue selective abundance of human indoleamine 2,3-dioxygenases for in situ production of highly reactive Michael acceptors (i.e., as warheads within cancer cells). Alternatively, one might seek to diminish the susceptibility of aplysinopsin chemotherapeutics to indoleamine 2,3-dioxygenases, to improve in vivo pharmacokinetics. Either way, an understanding of the biotransformation of aplysinopsins to thorectandrins and spiroreticulatine, and the Michael acceptor status of key intermediates, would inform researchers seeking to exploit the therapeutic potential of these closely related and uniquely marine pharmacophores.

General Experimental Procedures
Chiroptical measurements ([α] D ) were obtained on a JASCO P-1010 polarimeter (JASCO International Co. Ltd., Tokyo, Japan) in a 100 × 2 mm cell at 23 • C. Electronic Circular Dichroism (ECD) measurement were obtained on a JASCO J-810 spectropolarimeter (JASCO International Co. Ltd., Tokyo, Japan) in a 0.1 cm path-length cell. Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker Avance 600 MHz spectrometer (Bruker Pty. Ltd., Alexandria, Australia) with a 5 mm PASEL 1 H/D-13 C Z-Gradient probe at 25 • C in methanol-d 4 by referencing to residual 1 H or 13 C signals (δ H 3.30 and δ C 49.15). Highresolution ESIMS spectra were obtained on a Bruker micrOTOF mass spectrometer (Bruker Daltonik Pty. Ltd., Preston, Australia) by direct injection in MeOH at 3 µL/min, using sodium formate clusters as an internal calibrant. Semi-preparative HPLC was performed using Agilent 1100 series HPLC instrument (Agilent Technologies Inc., Mulgrave, Australia) with corresponding detector, fraction collector and software inclusively. Analytical-grade solvents were used for extractions and partitions. Chromatography solvents were of HPLC grade and filtered/degassed through 0.45 µm polytetrafluoroethylene (PTFE) membrane prior to use. Deuterated solvents were purchased from Cambridge Isotopes (Cambridge Isotope Laboratories, Tewksbury, MA, USA). The human colorectal (SW620) and lung the manuscript, with support from S.K. and A.A.S. All authors have read and agreed to the published version of the manuscript.