Two new steroid sulfates from a cheilostome bryozoan, Calyptotheca sp.

Abstract Two new steroid sulfates 1 and 2 were obtained from a lipophilic extract of an undescribed bryozoan species in the genus Calyptotheca. The structures of compounds 1 and 2 were elucidated by spectroscopic methods and chemical modifications. Steroids 1 and 2 exhibited moderate cytotoxicity at IC50 54 and 30 µM, respectively, against NBT-T2 cells. Graphical Abstract


Introduction
From the early stage of marine natural products chemistry, coral reef organisms have provided a number of steroids such as gorgosterol (Ling et al. 1970) and aragusterol (Iguchi et al. 1993) that show unique structures or biological activity. Among the marine steroids, polyoxygenated steroids constitute a representative group. As an example, hippuristanol, isolated from the gorgonian coral Isis hippuris (Higa et al. 1981), was later rediscovered as a specific inhibitor against RNA helicase eIF4A (Bordeleau et al. 2006) and is used as a research tool in life science studies (Ishikawa et al. 2013).
Among marine invertebrates, bryozoans have also been sources of new bioactive marine natural products. Representative bryozoan molecules include the macrolide bryostatin 1. Although it failed to be an anticancer agent, it is currently on a clinical trial against Alzheimer's disease (Farlow et al. 2019).
On a continual quest for new bioactive molecules from Okinawan coral reef organisms, we have screened lipophilic extracts of marine invertebrates collected at depths of 40-70 m in upper to middle mesophotic coral ecosystems (MCEs) (Rocha et al. 2018), from where a fewer number of metabolites have been reported than from shallower coral reefs. We have discovered several unique bioactive metabolites from sponges collected in MCEs Tianero et al. 2009;Hermawan et al. 2011). During an expedition, we encountered a conspicuous red bryozoan Calyptotheca sp. (Figure S1), at Seragaki, Okinawa. Since its lipophilic extract showed moderate cytotoxicity and several unique signals in the 1 H NMR spectrum, we examined the extract to identify two new steroids whose structures are the topic of this report.

Results and discussion
The molecular formula of compound 1, C 27 H 41 NaO 5 S, was established by negative ESIMS. The presence of a sulfate group as a sodium salt was confirmed with IR absorption at 1227 cm À1 and with the presence of Na by ICP-MS. Characteristic 1 H NMR signals at d 7.12 (1H, J ¼ 10.1 Hz), 6.23 (1H, J ¼ 10.1 Hz), and 6.07 (1H, s) and 13 C NMR signals at d 187. 3, 171.1, 157.3, 127.1, and 123.4 suggested the presence of a 1,4-cyclohexadien-3-one system in ring A. This a,b,a',b'-unsaturated ketone moiety was confirmed by the characteristic carbon signal at d 187.3, a strong IR absorption band at 1662 cm À1 , and the UV absorption at 243 nm and also by comparing the spectroscopic data of 1 with the same moiety in dendronesterone C (Duh et al. 2004), chabrolosteroids A and B (Su et al. 2007), and others (Ksebati and Schmitz 1988;Seo et al. 1996;Zhang et al. 2005).
The steroidal nucleus moiety was confirmed by the interpretation of COSY and HMBC data (Table S1). Of four methyl groups, two singlets at d 0.73 and 1.24 were assigned to H-18 and H-19, respectively, by observing HMBC correlations (H-18/C-12, 13, 14, and 17 and H-19/C-1, 5, 9, and 10). Of the two remaining methyl doublets, the one at d 0.95 (H-27, J ¼ 6.7 Hz) correlated to C-24, 25 and 26. Judging from the chemical shifts of C-26 (d 73.5; d 3.79 dd, 3.91 dd), it was elucidated as an oxymethylene group likely connected to a sulfate group. The methylene protons coupled with H-25 (d 1.79 m), which in turn coupled with H-27 as shown in COSY. Another doublet methyl at d 0.90 (H-21, J ¼ 6.4 Hz) showed HMBC correlations to C-17, 20 and 22 and also a COSY correlation to a tertiary proton at H-20 (d 1.38 m). By comparing NMR data for the side chain moiety of compound 1 with those of sulfated steroids, asterasterols B and C (De Marino et al. 1997), and polyoxygenated steroids from starfishes (Finamore et al. 1991;Iorizzi et al. 1994), the moiety was confirmed.
The absolute configuration at C-25 of 1 was determined by chiral derivatization after solvolysis of 1. Compound 1 was treated in a pyridine-dioxane solution to give primary alcohol 3, which was confirmed by a negative ion [M-H]at m/z 397.3106 and higher-field chemical shifts for H-26 (d 3.30 and 3.40). MPA (methoxyphenylacetyl) esters were prepared for the determination of chirality at C-25 of 1, although MTPA (methoxytrifluorophenylacetyl) esters have more frequently been prepared for similar steroids (Iorizzi et al. 1992;Seco et al. 2004). To confirm whether or not MPA esters can be used, Sand R-MPA esters of S-2-methylbutanol were prepared. The chemical shift difference Dd 0.08 for two methylene protons at C-1 of the R-MPA ester (d 4.00, 3.92) of S-2-methylbutanol was larger than Dd 0.06 for the S-MPA ester (d 3.99, 3.93), as similarly observed for MTPA esters (Seco et al. 2004). Alcohol 3 was then treated with Sor R-MPA to give esters 5 and 6, respectively. As the chemical shift difference Dd 0.16 for H-26 (d 4.03 and 3.87) of R-MPA ester 6 was larger than that Dd 0.12 for H-26 (d 4.01 and 3.89) of S-MPA ester 5, the configuration at C-25 was determined as S (Figure 1).
The molecular formula C 27 H 39 NaO 5 S of compound 2 was determined by negative ESIMS [M-Na]at m/z 475.2548 suggesting 2 as a dehydro-analog of steroid 1. The presences of a sulfate group and Na were confirmed by IR absorption at 1216 cm À1 and ICP-MS, respectively. Most of the 1 H and 13 C NMR signals for the steroidal nucleus including the a,b,a',b'-unsaturated ketone moiety are nearly the same as for 1 (Table S1), while the main structural difference is a double bond (d 5.27 overlapped, d 139.0, 124.9). The position of the double bond was assigned at C-22 by observing HMBC correlations (H-21/C-20, 22; H-24/C-23) and COSY correlations (H-22-23/H-20, 24), and also by comparing the NMR data with those of the same moiety (De Marino et al. 1997). The absolute configuration at C-25 was solved by applying the same method after preparing MPA esters 7 and 8 from alcohol 4. As the chemical shift difference Dd 0.11 for H-26 (d 3.99 and 3.88) of R-MPA ester 8 was larger than Dd 0.06 for H-26 (d 3.97 and 3.91) of S-MPA ester 7, the configuration was assigned as S (Figure 1).

General experimental procedures
NMR spectra were recorded on a Bruker Avance III 500 spectrometer. Chemical shifts and coupling constants are reported as d and Hz, respectively. IR spectra were recorded on a Jasco FT/IR-6100 Fourier transform infrared spectrophotometer. ESI-MS spectra were measured on a Jeol JMS-T100LP instrument. ICP-MS was run on an Agilent ICP-MS 7700X instrument. Optical rotation and UV spectra were measured on a Jasco P-1010 polarimeter and a Hitachi U-2001 spectrophotometer, respectively. Purification work was done on a Hitachi HPLC instrument using Mightysil RP columns. All solvents used for experiments were reagent grade.

Animal material
The red calcareous bryozoan ( Figure S1a) was collected by hand using trimix rebreathers at 60 m depth off Seragaki, Okinawa, Japan and kept frozen until extraction. Through observation of the colony surface with a stereoscopic microscope and scanning electron microscope (Figure S1b), the animal was identified as an undescribed species in the genus Calyptotheca Harmer (Class Gymnolaemata, Order Cheilostomata, Suborder Flustrina, Superfamily Smittionoidea, Family Lanceoporidae), by one of us (MHD) (Gordon 2017). Voucher specimens are deposited (NSMT-Te 1227) in the marine invertebrate collections at The National Museum of Nature and Science, Tsukuba, Japan.

Extraction and isolation
The specimen (2.1 kg) was extracted three times with acetone (5 L). After removal of acetone under vacuum, the residue was partitioned between EtOAc and H 2 O. The EtOAc layer was concentrated to give an extract (4.69 g), which was subjected to a silica gel column to give five fractions by using solvent mixtures increasing in polarity from n-hexane to MeOH. A fraction (2.36 g) eluted with MeOH was further separated into eight fractions by using a reversed-phase (RP) silica gel column with decreasing solvent polarity (50% aq. MeOH to n-hexane). The second fraction (390 mg) was further separated by RP HPLC (H 2 O-MeOH, 1-3) to give five subfractions. From the last subfraction (128.8 mg), compound 1 (21.1 mg) was obtained after repeated RP HPLC (H 2 O-MeOH, 1-3). From the third subfractions (49.8 mg), compound 2 (5.5 mg) was obtained after RP HPLC (H 2 O-MeCN-MeOH, 1-1-2).

Solvolysis of 1 to give alcohol 3
A solution of 1 (2.1 mg) in pyridine-dioxane (1-1, 300 mL) was heated at 140 C for 4 h in a capped reaction vial (Iorizzi et al. 1994). After cooling to room temperature, two drops of water were added. The solution was then dried with N 2 flow. The crude product was purified by preparative TLC (RP18, MeOH-H 2 O, 7-3) to yield alcohol 3 (1.6 mg, 97%).

Conclusion
From an undescribed bryozoan species, Calyptotheca sp., two new steroid sulfates 1 and 2 were isolated. Their structures were found to contain an a,b,a',b'-unsaturated ketone moiety in A ring and sulfate at the end of the side chain. The chirality at C-25 was elucidated by comparing chemical shifts of diastereotopic methylene protons at C-26 after attaching either Sor R-MPA group.