New Ceramides and Cerebrosides from the Deep-Sea Far Eastern Starfish Ceramaster patagonicus

Three new ceramides (1–3) and three new cerebrosides (4, 8, and 9), along with three previously known cerebrosides (ophidiocerebrosides C (5), D (6), and CE-3-2 (7)), were isolated from a deep-sea starfish species, the orange cookie starfish Ceramaster patagonicus. The structures of 1−4, 8, and 9 were determined by the NMR and ESIMS techniques and also through chemical transformations. Ceramides 1–3 contain iso-C21 or C23 Δ9-phytosphingosine as a long-chain base and have C16 or C17 (2R)-2-hydroxy-fatty acids of the normal type. Cerebroside 4 contains C22 Δ9-sphingosine anteiso-type as a long-chain base and (2R)-2-hydroxyheptadecanoic acid of the normal type, while compounds 8 and 9 contain saturated C-17 phytosphingosine anteiso-type as a long-chain base and differ from each other in the length of the polymethylene chain of (2R)-2-hydroxy-fatty acids of the normal type: C23 in 8 and C24 in 9. All the new cerebrosides (4, 8, and 9) have β-D-glucopyranose as a monosaccharide residue. The composition of neutral sphingolipids from C. patagonicus was described for the first time. The investigated compounds 1–3, 5–7, and 9 exhibit slight to moderate cytotoxic activity against human cancer cells (HT-29, SK-MEL-28, and MDA-MB-231) and normal embryonic kidney cells HEK293. Compounds 2, 5, and 6 at a concentration of 20 µM inhibit colony formation of MDA-MB-231 cells by 68%, 54%, and 68%, respectively. The colony-inhibiting activity of compounds 2, 5, and 6 is comparable to the effect of doxorubicin, which reduces the number of colonies by 70% at the same concentration.

The polymethylene chain length of LCB and FA and the absolute configuration of the ceramide 1 were determined as follows. When 1 was methanolyzed with methanolic hydrochloric acid, fatty acid methyl ester (FAME) was obtained together with LCB. A gas chromatography-mass spectrometry (GC-MS) analysis of FAME showed the existence of one component that was characterized as saturated methyl 2-hydroxyhexadecanoate of normal type (FAME-1). The normal type of FAME-1 was also confirmed by 1 H-NMR spectra, which consisted only of one triplet terminal methyl group at δ H 0.89. The optical rotation of FAME-1 ([α] D 25 -3.5 • (c = 1.0, CHCl 3 )) is consistent with the data [α] D 25 -3.21 • reported in the literature [23]; therefore, the absolute stereochemistry at C-2 is suggested to be R. Based on this suggestion, as well as on NMR and mass spectrometric data, we assumed LCB of ceramide 1 to have 21 carbon atoms and iso-type of unsaturated polymethylene chain. The geometry of the double bond in LCB can be determined on the basis of the 13 C-NMR chemical shift of the methylene carbon adjacent to the olefinic carbon (δ C ≈ 27 for (Z) isomers and δ C ≈ 32 for (E) isomers [24]). The 13 C-NMR spectrum of compound 1 indicated the presence of two characteristic allyl carbons, C-8 (δ C 27.5) and C-11 (δ C 27.3). Thus, the olefinic group in 1 was determined to have a cis (Z) geometry. The location of the double bond in the LCB moiety at position C-9 was determined through 1 H-1 H COSY, HMBC, and 2D TOCSY NMR experiments (  Based on all above-mentioned data, we determined the structure of 1 to be (2S,3S,4R,9Z)-2-[(2R)-2-hydroxyhexadecanoylamino]-19-methyl-9-icosen-1,3,4-triol. As far as we know, a ceramide with such a chemical structure was isolated for the first time.
The IR spectrum of compound 2 showed the presence of hydroxyl (3404 cm −1 ) and amide (1657, 1522 cm −1 ) groups. The molecular formula of compound 2 was determined as C 39 H 77 NO 5 from the [M + Na] + sodium adduct ion peak at m/z 662.5324 in the (+)HRESIMS and the [M -H] − deprotonated molecular ion peak at m/z 638.5364 in the (-)HRESIMS (Figures S9 and S10). A comparison of the 1 H-, 13 C-NMR spectra and an extensive 2D NMR analysis of compounds 1, 2, and 3 revealed that the unsaturated phytosphingosine-type ceramide with a 2-hydroxy fatty acid of 2 and 3 is identical to that of compound 1, while the polymethylene chain lengths of LCB and/or FA of 1-3 differ from each other (Figures 1 and S11-S15, Table 1). A comparison of the molecular weights (MWs) of 1 and 2 showed that they differed by 28 amu.
The FA unit in 2 was identified by GC analysis and the mass spectra of the FAME-2 derivative were measured by GC-MS similarly to compound 1. The GC-MS analysis showed that FAME-2 was identical to FAME-1. Moreover, the normal type of FAME-2 was also confirmed by the 1 H-NMR spectrum, which consisted of only one triplet terminal methyl group at δ H 0.89. Thus, the FA of ceramide 2 was determined to be (2R)-2-hydroxyhexadecanoic acid. Based on this finding, as well as on the NMR and mass spectrometry data, we suggested that LCB of ceramide 2 has 23 carbon atoms and an isotype of unsaturated polymethylene chain. Accordingly, the structure of 2 was determined to be (2S,3S,4R,9Z)-2-[(2R)-2-hydroxyhexadecanoylamino]-21-methyl-9-docosen-1,3,4-triol.
Compound 3 was characterized from a mixture with compound 2 at a ratio of 2:1 on the basis of the evaluation of the ion peak intensities in ESI mass-spectra. The IR spectrum of compound 3 showed the presence of hydroxyl (3407 cm −1 ) and amide (1655, 1522 cm −1 ) groups. The positive HRESI mass spectrum of this mixture showed two [M + Na] + ion peaks at m/z 662.5324 corresponding to compound 2 and at m/z 676.5463 corresponding to compound 3. Therefore, the molecular formula of compound 3 was determined as C 40 H 79 NO 5 from the [M + Na] + sodium adduct ion peak at m/z 676.5463 in the (+)HRESIMS and the [M -H] − deprotonated molecular ion peak at m/z 652.5520 in the (-)HRESIMS (Figures S16 and S17). The NMR spectra of compounds 3 and 2 were almost identical ( Figures S18-S22), but the MWs of 3 and 2 differed by 14 amu. A GC-MS analysis and mass spectra of fatty acid methyl esters obtained from the mixture of 3 and 2 showed the presence of FAME-2 containing saturated methyl 2-hydroxyhexadecanoate of the normal type and FAME-3 containing methyl 2-hydroxyheptadecanoate of the normal type. Thus, compounds 2 and 3 differed from each other by FA residues, C 16 in 2 and C 17 in 3, and had an identical C 23 unsaturated phytosphingosine-type LCB. Thus, the structure of 3 was determined to be (2S, The IR spectrum of compound 4 showed the presence of hydroxyl (3383 cm −1 ) and amide (1649, 1538 cm −1 ) groups. The molecular formula of compound 4 was determined as C 45 H 85 NO 9 Table 2, Figures S25 and S26). Thus, the presence of characteristic signals of an unsaturated sphingosine-type ceramide including the 2-hydroxy FA residue in the 1 H-and 13 C-NMR spectra of the ceramide part of 4 was shown ( Figure 1). Moreover, the ceramide moiety of 4 had normal and anteiso-types of side chains because the carbon atom signals of the terminal methyl groups were observed at δ C 14.0 (normal form) and 11.3 and 19.1 (anteiso-form) in the 13 C-NMR spectrum ( Table 2). The 1 H-1 H COSY and HSQC correlations in the NMR spectra of 4 indicated the corresponding sequences of protons at C-1 to C-11; C-21 to C-22 through C-19 and C-20; C-2 to NH; C-2 to C-4 , and C-16 to C-14 (Table 2, Figures 2B, S27 and S28). The key HMBC cross-peaks such as Hb-1/C-2, C-3; H-2/C-1 ; H-3/C-2, C-4; H-4/C-5, C-6; H-5/C-6, C-7; H-8/C-6, C-7, C-10; H-9/C-8, C-10, C-11; H-11/C-9; H 3 -21/C-19, C-20; H 3 -22/C-19; NH/C-2, C-1 ; H-2 /C-1 ; Ha-3 /C-1 , C-2 , C-4 ; H 2 -16 /C-15 ; and H 3 -17 /C-15 , C-16 confirmed the overall structure of the ceramide part of 4 ( Figures 2B and S29).
A GC-MS analysis of FAME-4 showed the existence of one component belonging to saturated methyl 2-hydroxyheptadecanoate of the normal type. Based on this finding, as well as on the NMR and mass spectrometry data, we assumed that the LCB of the ceramide part of 4 has 22 carbon atoms and an anteiso-type of unsaturated polymethylene chain. The E-configuration of the 4(5)-double bond in LCB was determined on the basis of the coupling constant between H-4 and H-5 (15.8 Hz) in the 1 H-NMR spectrum of 4 ( Table 2). The geometry of the 9(10)-double bond in LCB was characterized as Z on the basis of the 13 C-NMR chemical shifts of methylene carbons at δ C 27.2 (C-8) and δ C 27.0 (C-11) [19]. The location of the double bond in the LCB moiety was determined through 1 H-1 H COSY and HMBC NMR experiments (Table 2, Figures 2B, S27 and S29).
The absolute configuration of C-2 and C-3 in LCB of the ceramide part of 4 is suggested to be (2S,3R) according to the similarities of the 1 H-NMR data with the previously known asteriacerebroside G with a (2S,3R)-configuration of asymmetric centers [18]. In addition to the above-mentioned signals, the 1 H-NMR spectrum of 4 exhibited one resonance in the de-shielded region due to the anomeric proton of the monosaccharide unit at δ H 4.92 that correlated in the HSQC experiment with a carbon signal at δ C 105.4 ( Table 2). The 1 H-1 H COSY, HSQC, HMBC, and ROESY experiments led to the assignment of all the proton and carbon signals to the carbohydrate residue of 4 ( Table 2, Figures 2B and S25-S29). The coupling constant (7.8 Hz) of the anomeric proton was indicative of a β-configuration of the glycosidic bond. The NMR spectroscopic data of the monosaccharide moiety strictly coincided with those of a β-glucopyranosyl residue of the known asteriacerebroside G from A. amurensis [18]. The attachment of the monosaccharide to the ceramide part of 4 was deduced from the long-range correlations in the HMBC spectrum. There were cross-peaks between H-1" of Glcp and C-1 of aglycon, as well as between H-1 of the ceramide part and C-1" of Glcp ( Figure 2B). Acid hydrolysis of cerebroside 4 with 2M TFA was carried out to confirm the identification of its monosaccharide unit as glucose. An alcoholysis of sugar by ©-(−)-2-octanol followed by acetylation, a GC analysis, and a comparison with the corresponding derivatives of standard monosaccharides allowed us to identify the D-configuration for the β-glucopyranosyl residue of 4.
tive of a β-configuration of the glycosidic bond. The NMR spectroscopic data of the monosaccharide moiety strictly coincided with those of a β-glucopyranosyl residue of the known asteriacerebroside G from A. amurensis [18]. The attachment of the monosaccharide to the ceramide part of 4 was deduced from the long-range correlations in the HMBC spectrum. There were cross-peaks between H-1″ of Glcp and C-1 of aglycon, as well as between H-1 of the ceramide part and C-1″ of Glcp ( Figure 2B). Acid hydrolysis of cerebroside 4 with 2M TFA was carried out to confirm the identification of its monosaccharide unit as glucose. An alcoholysis of sugar by ©-(−)-2-octanol followed by acetylation, a GC analysis, and a comparison with the corresponding derivatives of standard monosaccharides allowed us to identify the D-configuration for the β-glucopyranosyl residue of 4. type ceramide residue containing a 2-hydroxy FA (Figure 1). Moreover, the ceramide part of 8 had the normal and anteiso-types of side chains because the carbon atom signals of the terminal methyl groups were observed at δ C 14.0 (normal form) and 11.3 and 19.2 (anteiso-form) in the 13 C-NMR spectrum ( Table 2). The 1 H-1 H COSY and HSQC correlations in the NMR spectra of 8 indicated the corresponding sequences of protons at C-1 to C-5; C-16 to C-17 through C-15 and C-14; C-2 to NH; C-2 to C-4 , and C-22 to C-20 (Table 2, Figures 2C, S34 and S35). The key HMBC cross-peaks such as Hb-1/C-2, C-3; H-2/C-1 ; H-3/C-4; H-4/C-5; H 3 -16/C-14, C-15; H 3 -17/C-14; NH/C-2, C-1 ; H-2 /C-1 ; Ha-3 /C-1 , C-2 , C-4 ; H 2 -21 /C-20 ; H 3 -22 /C-21 confirmed the common structure of the ceramide part of 8 ( Figures 2C and S36).   A GC-MS analysis of FAME-5 showed the existence of one component that belonged to a saturated methyl 2-hydroxytricosanoate of the normal type. Based on this finding, as well as on the NMR and mass spectrometric data, we assumed that the LCB of the ceramide part of 8 has 17 carbon atoms and the anteiso-type of saturated polymethylene chain. The absolute configuration of LCB of the ceramide moiety of 8 is suggested to be D-ribo-(2S,3S,4R) on the basis of the similarity of its 1 H-NMR spectroscopic data with those of the LCB of ceramide 1. The attachment of the monosaccharide to the ceramide part of 8 was deduced from long-range correlations in the HMBC spectrum. There were cross-peaks Mar. Drugs 2022, 20, 641 9 of 16 between H-1" of Glcp and C-1 of aglycon, as well as between H-1 of the ceramide part and C-1" of Glcp ( Figures 2C and S36).

HN
The presence of a monosaccharide unit and the structure of the ceramide part of  Figures S37 and S38). Based on a thorough 2D NMR analysis of cerebrosides 9 and 8, we suggested that the ceramide part of 9 is almost identical to those of compound 8 ( Figure S39-S43). However, a comparison of the molecular weights of 8 and 9 showed that they differ in MW by 14 amu. A GC-MS analysis of FAME-6 showed the presence of one component that was characterized as saturated methyl 2-hydroxytetracosanoate normal type.
The presence of a monosaccharide unit and the structure of the ceramide part of
Since compounds 1-3, 5-7, and 9 inhibited the viability of breast cancer cells MDA-MB-231, we then tested their ability to inhibit colony formation of MDA-MB-231 cells using the soft agar assay. The colony formation assay, also referred to as soft agar assay, allows for screening the therapeutic efficacy of compounds for anchorage-independent cell growth, which is one of the hallmark characteristics of cellular transformation and uncontrolled growth of cancer cells [26].
Mar. Drugs 2022, 20, x FOR PEER REVIEW 11 of 17 age-independent cell growth, which is one of the hallmark characteristics of cellular transformation and uncontrolled growth of cancer cells [26]. As a result, we found that compounds 1, 3, 7, and 9 at a concentration of 20 µM had a comparable effect on MDA-MB-231 colony formation and decreased the number of colonies by 46%, 48%, 44%, and 50%, respectively. Compounds 2, 5, and 6 (20 µM) inhibited colony formation of MDA-MB-231 cells by 68%, 54%, and 68%, respectively. The colony-inhibiting activity of compounds 2, 5, and 6 (20 µM) was comparable with the effect of doxorubicin that reduced the number of colonies by 70% at a concentration of 20 µM (Figure 4).  1-3, 5-7, and 9 on colony formation of human breast cancer cells MDA-MB-231 (2.4 × 10 4 ) that were exposed to PBS (control), Doxo (5, 10, and 20 µM), or the compounds under study (5,10, and 20 µM) and placed on dishes with 0.3% Basal Medium Eagle (BME) agar containing 10% fetal bovine serum FBS, 2 mM L-glutamine, and 25 µg/mL gentamicin. After 14 days of incubation, the number of colonies was counted under a microscope using the ImageJ software program.

General Procedures
Optical rotations were determined on a PerkinElmer 343 polarimeter (Waltham, MA, USA). UV spectra were recorded on a Shimadzu UV-1601 PC spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were recorded using a Bruker Equinox 55 spectrophotometer in CDCl 3 (Bruker, Göttingen, Germany). The 1 H-and 13 C-NMR spectra were obtained on Bruker Avance III 700 spectrometer (Bruker BioSpin, Bremen, Germany) at 700.13 and 176.04 MHz, respectively; chemical shifts were referenced to the corresponding residual solvent signals (δ H 7.21/δ C 123.5 for C 5 D 5 N). The HRESIMS spectra were recorded on a Bruker Impact II Q-TOF mass spectrometer (Bruker, Bremen, Germany); the samples were dissolved in MeOH (c 0.001 mg/mL). HPLC separations were carried out on an Agilent 1100 Series chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a differential refractometer and with the following columns used: Diasfer-110-C18

Extraction and Isolation
The fresh C. patagonicus specimens (3 kg wet weight) were cut into small pieces and extracted with CHCl 3 :MeOH (2:1) followed by further extraction with CHCl 3 :MeOH (1:1) and EtOH. The combined extracts were concentrated in vacuo to a residue of 159.5 g. This residue was separated between H 2 O (1.5 L) and AcOEt:BuOH (2:1) (4.5 L), and the organic layer was concentrated in vacuo to obtain a less polar fraction (51.5 g), which was washed with cold acetone (1 L). The acetone-soluble fraction (28.5 g) was chromatographed on a Si gel column (19 × 4.5 cm) using CHCl 3 Table 2.  Table 2.  Table 2.

Methanolysis of Compounds 1-9 and Analysis of FAMEs
Compounds 1-9 (1 mg) were heated with 1 N HCl in 80% aqus. MeOH (1.0 mL) at 80 • C for 4 h. The reaction mixtures were then extracted with n-hexane and the extracts were concentrated in vacuo to yield FAME-1-FAME-9. The FAMEs were analyzed on Supelcowax 10 columns at 200 • C. Helium was used as the carrier gas at a linear velocity of 30 cm/s. Mass spectra were recorded at 70 eV. The obtained mass spectra were compared with the NIST library and a FA mass spectra archive accessible online.

Acid Hydrolysis and Determination of Absolute Configurations of Monosaccharides
The acid hydrolysis of 4 (0.5 mg) was carried out in a solution of 2 M trifluoroacetic acid (TFA) (1 mL) in a sealed vial on an H 2 O bath at 100 • C for 2 h. The H 2 O layer was washed with CHCl 3 (3 × 1.0 mL) and concentrated in vacuo. One drop of concentrated TFA and 0.5 mL of R-(-)-2-octanol (Sigma Aldrich) were added to the sugar fraction, and the sealed vial was heated in a glycerol bath at 130 • C for 6 h. The solution was evaporated in vacuo and exposed to a mixture of pyridine/acetic anhydride (1:1, 0.5 mL) for 24 h at room temperature. The acetylated 2-octylglycosides were analyzed by GC using the corresponding authentic samples prepared by the same procedure. The following peaks were detected in the hydrolysate of 4: D-glucose (t R 24.24, 24.84, 25.08, and 25.38 min). The retention times of the authentic samples were as follows: D-glucose (t R 24.23, 24.83, 25.06, and 25.37 min), L-glucose (t R 24.39, 24.63, 24.83, and 25.06 min).

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.