Crellasterones A and B: A-Norsterol Derivatives from the New Caledonian Sponge Crella incrustans

Two new steroids, crellasterones A (1) and B (2), together with the previously reported compound chalinasterol (3) and several nucleosides (4–7), were isolated from the sponge Crella incrustans, collected in New Caledonia. The structures of the new compounds were established by extensive NMR and mass spectroscopic analysis and revealed unprecedented marine natural products with a ring-contracted A-norsterone nucleus and 2-hydroxycyclopentenone chromophore. The absolute configurations were derived from electronic circular dichroism (ECD) measurements in conjunction with high-level density functional theory (DFT) calculations.


Introduction
Sterols [1] are among the most studied groups of natural products, with interest commencing in the 19th century, running through to the 1969 Nobel Prize in Chemistry for Sir Derek Barton, and continuing to the present day. Natural products based on sterols first began to be isolated from terrestrial and marine sources several decades after the initial discovery and characterization of cholesterol [1] and include important compounds such as aplysterol [2] and 25-methylxestosterol [3]. Sponges were the first invertebrates shown to contain sterols other than cholesterol, and since then, sponges have been shown to have the most diverse array of novel sterols [4]. The presence of sterols in sponges is not only interesting from a biochemical and functional viewpoint, but also provides information relevant to the classification of these organisms (chemotaxonomy) [5]. Sponge sterols often contain unusual side-chains and modified ring systems, ranging in carbon content from C 24 to C 31 [4][5][6][7].
Sponges from the genus Crella have not been widely studied, with only a few natural products isolated, including crellastatins A-M, [8][9][10] shishicrellastatins A and B [11], norselic acids A-E [12] and benzylthiocrellidone [13]. Crellastatin A, a dimeric 4,4 -dimethylsterol from Crella sp. exhibits in vitro cytotoxic activity against a human bronchopulmonary non-small-cell lung carcinoma cell line (NSCLC-N6) at an IC 50 of 1.5 µg/mL [8]. Similarly, crellastatins B-M also exhibit in vitro antitumor activity against NSCLC cell lines with IC 50 values of 1-10 µg/mL [9,10]. The closely related shishicrellastatin A, and from Crella spinulata, B, are cathepsin B inhibitors with an IC 50 value of 8 µg/mL each [11]. Up regulation of cathepsin B is correlated with tumor cell invasiveness. Norselic acid A [12] is an anti-infective steroid isolated from the Antarctic sponge Crella sp. that possesses antibiotic activity against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive S. aureus (MSSA), vancomycin-resistant Enterococcus faecium (VRE), and Candida albicans [12]. Noreselic acids A-E were also found to be active against the Leishmania parasite at low micromolar levels. Benzylthiocrellidone, the only non-steroidal metabolite from Crella spp., was isolated from Crella spinulata, found on the Great Barrier Reef in Australia [13].

Results and Discussion
The frozen sponge was exhaustively extracted with ethanol to afford an extract that was successively partitioned with n-hexane/aqueous ethanol, ethyl acetate/aqueous ethanol and nbutanol/water. Reversed phase chromatography of the ethyl acetate extract yielded the nucleosides inosine (4), 2′-deoxyuridine (5), uridine (6) and guanosine (7). The n-hexane extract yielded chalinasterol (3) as the major metabolite, along with smaller quantities of two new compounds, crellasterones A (1) and B (2).

Results and Discussion
The frozen sponge was exhaustively extracted with ethanol to afford an extract that was successively partitioned with n-hexane/aqueous ethanol, ethyl acetate/aqueous ethanol and n-butanol/water. Reversed phase chromatography of the ethyl acetate extract yielded the nucleosides inosine (4), 2 -deoxyuridine (5), uridine (6) and guanosine (7). The n-hexane extract yielded chalinasterol (3) as the major metabolite, along with smaller quantities of two new compounds, crellasterones A (1) and B (2).
Crellasterone B (2) was also isolated as an optically active white solid. HRESIMS analysis of 2 revealed a protonated molecule [M + H] + at m/z 399.3263, indicative of a molecular formula C 27 H 42 O 2 (∆mmu 0.5). IR absorption bands at 3274, 1700 cm −1 suggested the presence of hydroxy and carbonyl functionalities. Analysis of the 1D and 2D NMR spectra of 2 (COSY, HSQC, HMBC) revealed the presence of the same steroidal ring system as in 1 (Figure 1). The 13 C NMR spectrum of 2 displayed fewer carbon resonances relative to 1, and especially notable was the absence of an oxygen-bearing methine and the ethyl group of 1, which indicated the absence of the ethoxy group. The 13 C NMR spectrum also revealed six non-protonated carbons, nine methines, seven methylenes and six methyl carbons. The other notable difference from 1 was in the side chain, with the absence of an exomethylene and the presence of two mutually coupled olefinic methines H-22 (δ H 5.13) and H-23 (δ H 5.14). Key HMBC correlations from H 3 -21 (δ H 0.97) to C-17 (δ C 55.8), C-20 (δ C 40.3) and C-22 (δ C 135.9), and from H 3 -28 (δ H 0.89) to C-23 (δ C 132.0), C-24 (δ C 43.1) and C-25 (δ C 33.2) confirmed the positioning of the double bond, between C-22 and C-23. The dd coupling of H-23 further implied a methine group at C-24 and confirmed that two methyl groups were attached to C-25. The geometry of the double bond was determined to be E ( 3 J 22,23 = 15.2 Hz). As with 1, C-20 was connected back to the steroid nucleus via HMBC correlations ( Figure 1 and Supplementary Materials, Table S2), resulting in crellasterone B being assigned structure 2.
Having established the structures of crellasterone A and B, we turned our attention to determination of their absolute stereochemistries using electronic circular dichroism (ECD). The simpler compound 2, showed a small negative specific rotation and a positive Cotton effect at 263 nm, and a negative Cotton effect at 310 nm in the ECD spectrum ( Figure 2B). Since there are no ECD spectral data reported for any related compounds, the ECD spectra could not be compared to literature values. Time-dependent density functional theory (TD-DFT) calculations (Turbomole 7.1) [16] of 2 gave very good matches for UV-Vis ( Figure 2A) and ECD ( Figure 2B) spectra for (10R)-crellasterone B (see Supplementary Materials, Figure S18). The negative Cotton effect at 300 nm and positive Cotton effect at 260 nm were confirmed to arise solely from the A-ring chromophore and the configuration at C-10 by repeating the calculation with just rings A and B (data not shown), which gave identical UV and ECD spectra.  Figure 1 for locant numbering.  (Figure 1). The 13 C NMR spectrum of 2 displayed fewer carbon resonances relative to 1, and especially notable was the absence of an oxygen-bearing methine and the ethyl group of 1, which indicated the absence of the ethoxy group. The 13 C NMR  Compound 1 is more complex, having a second stereocenter adjacent to the chromophore. While NMR spectroscopy had established the configuration at C-6, we wished to confirm this through TD-DFT calculations. Compound 1 also showed a negative specific rotation and a similar UV-Vis spectrum ( Figure 2C), but unlike 2, negative Cotton effects at 264 and 310 nm in the ECD spectrum ( Figure 2D). From the NMR spectrum, H-6 showed small couplings to H-7α and H-7β, suggesting H-6 was equatorial; thus a β-ethoxy group was placed at C-6. TD-DFT calculations of 6α-and 6β-isomers of 1 (Supplementary Materials, Figures S19-S21), supported the assignment of H-6 as α.
Only the 6β-ethoxy isomer of 1 gave negative Cotton effects at 301 and 262 nm, in agreement with the experimental ECD spectrum of 1 (Figures 2D and S21). Having established the absolute configurations at C-6 and C-10, ROESY data established the relative configurations of the remaining steroidal core of 1 (Figure 3) [17]. Correlations between H 3 -19, H-11β and H-8 demonstrated the trans-relationship of the B/C ring junction and placed the C-19 methyl group on the same face of the steroid ring as H-8. Other significant correlations observed were between H-1β and H-19, and between H-1α, H-7α and H-9, confirming that H-9 is on the α-face. The C/D ring junction was assigned based on ROESY correlations between H-8 and H-18, confirming H-18 and H-8 to be on the β-face. Due to overlapping of peaks, no correlation between H-14 and H-9 could be observed. A ROESY correlation between H-20 and H-18 suggested C-20 was R in accord with standard steroidal stereochemistry (Supplementary Materials, Figure S22). The ROESY correlations described above are consistent with the trans/trans B/C/D relationship of a regular steroid [12]. Figure S22). The ROESY correlations described above are consistent with the trans/trans B/C/D relationship of a regular steroid [12]. Other significant ROESY correlations observed were between H-1β and H3-19 (above the plane of the ring system), between H-1α, H-9 and H-11α, and between H-12α and H-21 (below the plane). The C-24 configuration of 2 was assigned as S due to the fact that the chemical shift difference between C-26 and C-27 carbon atoms was 0.5 ppm and the chemical shift of C-28 was 18.1 ppm, in accordance with the rules developed by Wright [18].

overlapping of peaks, no correlation between H-14 and H-9 could be observed. A ROESY correlation between H-20 and H-18 suggested C-20 was R in accord with standard steroidal stereochemistry (Supplementary Materials,
Compounds 1 and 2 are C27 sterols related to maltadiolone (8) and its esters, an unpublished series of semi-synthetic steroids that appear in a patent without any stereochemistry or spectral data [19]. These compounds belong to the small group of A-nor ring-contracted steroids [20,21]. The first example of a ring A-contracted steroid, bearing a one-carbon appendage at C-2 (anthosterones A and B (9)), was described by Andersen, Clardy and co-workers in 1988 as a marine natural product from Anthoracuata (Antho) graceae [22] and the same compounds, plus a range of side chain analogues, were isolated by Molinki in 2004 from Phorbas amaranthus and displayed moderate cytotoxicity against HCT-116 tumor cells [21]. Both sponges are in the same order as Crella (Poecilosclerida) and the latter is synonymous with Crella hospitalis (Schmidt, 1870) [23]. A compound with an A-nor-cholesterol core identical to that of crellasterone B, namely 3-hydroxy-4-nor-5α-cholest-3-en-2-one, but with a different side chain was reported as a side-product of bis-steroid synthesis [24]. However, the reported structure is certainly incorrect as the reported 13 C chemical shifts of C-3 and C-5 (δC 184.8 and 125.9 respectively) are not reasonable for 2-hydroxycyclopent-2-enone and would fit better for cyclopent-2-enone.
It is likely that 1 is the reaction product of an electrophilic natural product, such as 1a, with ethanol, the extraction solvent (Figure 4). A similar proposal has been shown by us in relation to the abiotic synthesis of an ethoxy substituted spiroisoxazoline alkaloid and furanones [25,26]. Similarly, the anthosterones (e.g., 9) could arise from the abiotic reaction of an intermediate peracid with methanol ( Figure 4). A possible biogenesis of 1 and 2 from chalinasterol (3β-ergosta-5,24(28)-dien-3ol) is via a Baeyer-Villiger-type oxidative ring contraction and decarboxylation (Figure 4) [27]. Using the same mechanism, but starting with spongosterol ((24S)-5α-ergost-22E-en-3β-ol) instead of chalinasterol, would lead to crellasterone B (2). Other significant ROESY correlations observed were between H-1β and H 3 -19 (above the plane of the ring system), between H-1α, H-9 and H-11α, and between H-12α and H-21 (below the plane). The C-24 configuration of 2 was assigned as S due to the fact that the chemical shift difference between C-26 and C-27 carbon atoms was 0.5 ppm and the chemical shift of C-28 was 18.1 ppm, in accordance with the rules developed by Wright [18].
Compounds 1 and 2 are C 27 sterols related to maltadiolone (8) and its esters, an unpublished series of semi-synthetic steroids that appear in a patent without any stereochemistry or spectral data [19]. These compounds belong to the small group of A-nor ring-contracted steroids [20,21]. The first example of a ring A-contracted steroid, bearing a one-carbon appendage at C-2 (anthosterones A and B (9)), was described by Andersen, Clardy and co-workers in 1988 as a marine natural product from Anthoracuata (Antho) graceae [22] and the same compounds, plus a range of side chain analogues, were isolated by Molinki in 2004 from Phorbas amaranthus and displayed moderate cytotoxicity against HCT-116 tumor cells [21]. Both sponges are in the same order as Crella (Poecilosclerida) and the latter is synonymous with Crella hospitalis (Schmidt, 1870) [23]. A compound with an A-nor-cholesterol core identical to that of crellasterone B, namely 3-hydroxy-4-nor-5α-cholest-3-en-2-one, but with a different side chain was reported as a side-product of bis-steroid synthesis [24]. However, the reported structure is certainly incorrect as the reported 13 C chemical shifts of C-3 and C-5 (δ C 184.8 and 125.9 respectively) are not reasonable for 2-hydroxycyclopent-2-enone and would fit better for cyclopent-2-enone.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1010 Polarimeter (Jasco, Tokyo, Japan) and electronic circular dichroism spectra were acquired on a Jasco J-810 spectropolarimeter (Jasco). Infrared spectra were recorded on a Nicolet iS10 ATR FTIR Spectrometer (Thermo Scientific, Waltham, MA, USA). NMR spectra were acquired at 25 °C on a Bruker Avance AVII 600 MHz NMR spectrometer in CDCl3, processed using Bruker Topspin 3.5 software (Bruker, Fällanden, Switzerland) and referenced to residual solvent (CHCl3 δΗ 7.24; δC 77.01). LC-MS was performed on an Agilent 1260 (Agilent, Santa Clara, CA, USA) Infinity UHPLC coupled to an Agilent 6130 single quadrupole mass spectrometer by electrospray ionization in positive and negative polarity modes. UV-Vis spectra were acquired on an Agilent 1260 Infinity diode array detector. High-resolution mass spectra were acquired on a Thermo Scientific LTQ Orbitrap XL or a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) by direct infusion in a positive polarity mode. HPLC separations were achieved on a Gilson 506C HPLC system (Gilson, Middleton, WI, USA) with UNIPOINT v5.11 software. Analytical and preparative HPLC were performed on Synergi Max-RP HPLC columns (Phenomenex): Synergi 10 μ MAX-RP, 250 × 4.6 mm and Synergi 10 μ MAX-RP, 250 × 21.2 mm respectively. LC-MS was performed on a C18 analytical column (Phenomenex Gemini 3 μ C18, 150 × 2.0 mm) with a gradient from 5 to 100% CH3CN in H2O with 0.025% formic acid; flow rate 0.2 mL/min and UV detection at 254 nm. In this sponge, the known compound chalinasterol (3) was present as the major component, making up 5% of the n-hexane extract. The structure was identified by comparison with published spectroscopic data [28]. The polar fractions contained nucleosides, including inosine (4), 2 -deoxyuridine (5), uridine (6) and guanosine (7), (Scheme 1) identified on the basis of 1 H NMR spectra and mass spectrometry.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1010 Polarimeter (Jasco, Tokyo, Japan) and electronic circular dichroism spectra were acquired on a Jasco J-810 spectropolarimeter (Jasco). Infrared spectra were recorded on a Nicolet iS10 ATR FTIR Spectrometer (Thermo Scientific, Waltham, MA, USA). NMR spectra were acquired at 25 • C on a Bruker Avance AVII 600 MHz NMR spectrometer in CDCl 3 , processed using Bruker Topspin 3.5 software (Bruker, Fällanden, Switzerland) and referenced to residual solvent (CHCl 3 δ H 7.24; δ C 77.01). LC-MS was performed on an Agilent 1260 (Agilent, Santa Clara, CA, USA) Infinity UHPLC coupled to an Agilent 6130 single quadrupole mass spectrometer by electrospray ionization in positive and negative polarity modes. UV-Vis spectra were acquired on an Agilent 1260 Infinity diode array detector. High-resolution mass spectra were acquired on a Thermo Scientific LTQ Orbitrap XL or a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) by direct infusion in a positive polarity mode. HPLC separations were achieved on a Gilson 506C HPLC system (Gilson, Middleton, WI, USA) with UNIPOINT v5.11 software. Analytical and preparative HPLC were performed on Synergi Max-RP HPLC columns (Phenomenex): Synergi 10 µ MAX-RP, 250 × 4.6 mm and Synergi 10 µ MAX-RP, 250 × 21.2 mm respectively. LC-MS was performed on a C 18 analytical column (Phenomenex Gemini 3 µ C18, 150 × 2.0 mm) with a gradient from 5 to 100% CH 3 CN in H 2 O with 0.025% formic acid; flow rate 0.2 mL/min and UV detection at 254 nm.

Animal Material
The sponge was collected by self-contained underwater breathing apparatus (SCUBA) in New Caledonia. The samples were frozen upon collection, and stored at −20 • C. The sponge was identified as Crella incrustans (class Demospongiae, order Poecilosclerida, family Crellidae). A voucher specimen under the registration number (NC-10) is kept in the collections at the University of Auckland, New Zealand. Another voucher specimen has been deposited at the Department of Chemistry & Biomolecular Sciences, Macquarie University, Sydney, Australia, also under NC-10.

Conclusions
Marine sponges of the genus Crella have been the subject of relatively few natural products studies, with only six papers published in the past 50 years. Herein, we have reported two new unusual sterols, crellasterones A and B, from Crella incrustans, collected in New Caledonia. These sterols represent a new type of A-norsteroid not previously reported as natural products. The absolute configurations of the new compounds were determined on the basis of ECD in conjunction with DFT calculations.