Isomalabaricane Triterpenes from the Marine Sponge Rhabdastrella sp.

The marine sponge of the genus Geodia, Jaspis, Rhabdastrella, and Stelletta are characterized chemically by a variety of isomalabaricane triterpenes. This class of compounds drew spotlights in marine lead discovery due to their profound anti-proliferative properties. Further research on exploring its chemical diversity led to the identifications of two new isomalabaricane-type triterpenes rhabdastin H (1) and rhabdastin I (2). Their structures were unraveled using a series of spectroscopic approaches. These isolates were found to exhibit unique structural features with the only reported tetrahydrofuran functionality among all marine-derived isomalabaricanes. Both compounds 1 and 2 showed activities against K562 (IC50 11.7 and 9.8 μM) and Molt4 (IC50 16.5 and 11.0 μM) leukemic cells in MTT cell proliferative assay.


Introduction
Marine sponges continue to act as a fruitful source of bioactive and unusual metabolites. Among these metabolites are the sponge-derived isomalabaricanes. They were reported to exhibit potent anti-proliferative properties against a series of cancer cells, including colorectal carcinoma [1], ovarian carcinoma [2], acute promyelocytic leukemia [3], acute lymphoblastic leukemia [4], prostate carcinoma [3], gastric adenocarcinoma [3], ductal carcinoma [3], hepatocellular carcinoma [3], cervix carcinoma [3], papillomavirus-related endocervical adenocarcinoma [5], and malignant melanoma [4]. The isomalabaricane-type triterpenoids are characterized by an α-methyl group at C-8, owning to their trans-syn-trans tricyclic ring junction instead of trans-anti-trans of their isomers, malabaricanes [1]. Isomalabaricanes are only found in sponges and are considered as chemotaxonomic markers of Rhabdastrella sp. [1] Despite the significant difficulties in the isolation and characterization of isomalabaricanes due to light induced 13-E/Z isomerization [6], their significant cytotoxic activity prompted extensive research on this class of compounds aiming to find new drug leads.
The current study aimed to explore novel isomalabaricanes from marine sources. We isolated two unusual compounds with tetrahydrofuran functional group, the moiety that has been reported previously in malabaricane-type triterpenoids but without any cytotoxic assessments [7,8]. Then, the isolated compounds were evaluated against several cancer cell lines using MTT assay to further interpret their anti-proliferative properties.

Results
The freeze-dried specimen of the wild-type sponge Rhabdastrella sp. (Figure 1) was extracted with a 1:1 mixture of methanol (MeOH) and dichloromethane (CH 2 Cl 2 ) (1:1) to provide the crude extract. The obtained crude extract was further fractionated and purified using normal and reversed-phase column chromatography yielding two isomalabaricanes, rhabdastin H (1) and rhabdastin I (2). These isolates demonstrated unique structural features with the presence of the first identified tetrahydrofuran moiety in this class of compounds. The current study aimed to explore novel isomalabaricanes from marine sources. We isolated two unusual compounds with tetrahydrofuran functional group, the moiety that has been reported previously in malabaricane-type triterpenoids but without any cytotoxic assessments [7,8]. Then, the isolated compounds were evaluated against several cancer cell lines using MTT assay to further interpret their anti-proliferative properties.

Results
The freeze-dried specimen of the wild-type sponge Rhabdastrella sp. (Figure 1) was extracted with a 1:1 mixture of methanol (MeOH) and dichloromethane (CH2Cl2) (1:1) to provide the crude extract. The obtained crude extract was further fractionated and purified using normal and reversed-phase column chromatography yielding two isomalabaricanes, rhabdastin H (1) and rhabdastin I (2). These isolates demonstrated unique structural features with the presence of the first identified tetrahydrofuran moiety in this class of compounds. The molecular formula of 1 was suggested as C32H50O5 based on 13 C NMR and HRESIMS data that showed a molecular ion peak at m/z 537.3539 [M + Na] + implying eight degrees of unsaturation. The IR spectrum of 1 revealed the presence of carbonyl groups from absorptions at 1742, 1726, and 1703 cm −1 . Its 13 C NMR spectrum of 1 (Table 1), measured in CDCl3, showed the presence of thirty-two carbon signals, which were assigned by the assistance of DEPT data to nine methyl groups, eight sp 3 methylenes, six sp 3 methines (including two oxymathines), four sp 3 quaternary carbons, one sp 2 methine and four sp 2 quaternary carbons (including two ketone carbonyl). The NMR signals at δC 169.8 (C) and 20.9 (CH3) and δH 2.12 (3H, s) and the IR absorption at 1742 cm −1 (Table 1).
Based on the above results and with the assistance of 1 H-1 H COSY and HMBC experiments ( Figure 2), the planar structure of 1 was determined. To establish the proton sequences in 1, the 1 H-1 H COSY spectrum analysis established five proton sequences from H2-1 to H2-2, H-5 to H2-7, and H2-11 to H2-12, H2-15 to H-21, and H-23 to H-24. These data, together with the HMBC correlations ( Figure 2) from H2-1 and H2-2 to C-3, H2-11 and H2- The molecular formula of 1 was suggested as C 32 H 50 O 5 based on 13 C NMR and HRESIMS data that showed a molecular ion peak at m/z 537.3539 [M + Na] + implying eight degrees of unsaturation. The IR spectrum of 1 revealed the presence of carbonyl groups from absorptions at 1742, 1726, and 1703 cm −1 . Its 13 C NMR spectrum of 1 (Table 1), measured in CDCl 3 , showed the presence of thirty-two carbon signals, which were assigned by the assistance of DEPT data to nine methyl groups, eight sp 3 methylenes, six sp 3 methines (including two oxymathines), four sp 3 quaternary carbons, one sp 2 methine and four sp 2 quaternary carbons (including two ketone carbonyl). The NMR signals at δ C 169.8 (C) and 20.9 (CH 3 ) and δ H 2.12 (3H, s) and the IR absorption at 1742 cm −1 suggested the presence of an acetoxy group. Carbon signals of the eight methyl groups (δ C 31.  (Table 1).
Based on the above results and with the assistance of 1 H-1 H COSY and HMBC experiments (Figure 2), the planar structure of 1 was determined. To establish the proton sequences in 1, the 1 H-1 H COSY spectrum analysis established five proton sequences from H 2 -1 to H 2 -2, H-5 to H 2 -7, and H 2 -11 to H 2 -12, H 2 -15 to H-21, and H-23 to H-24. These data, together with the HMBC correlations ( Figure 2) from H 2 -1 and H 2 -2 to C-3, H 2 -11 and H 2 -12 to C-9, H 2 -12 to C-8 and C-13, H 3 -19 to C-1, C-5, C-9 and C-10, H 3 -28 and H 3 -29 to C-3, C-4 and C-5 and H 3 -30 to C-7, C-8, C-9 and C-13 established the connectivity within the 6-membered (A), 6-membered (B), and 5-membered (C) rings.  Ring A of 1 was found to possess one ketone at C-3 and two methyl groups (C-28 and C-29), one methyl group (C-19), and one methyl groups (C-30) attached at C-4, C-8, and C-10, respectively. The key HMBC correlations suggested the connection of H 3 -18 to C-13, C-14, and C-15, H-20 to C-16 and C-17; H 3 -21 to C-17, C-20, and C-22; H-23 to C-22; H 3 -26 and H 3 -27 to C-24 and C-25. Thus, the side chain C-20 to C-27 was found to possess one double bond at C-24/C-25, one ketone group at C-22, two methyl groups at C-25, and one methyl group at C-20. One acetoxy group at C-23 was confirmed by the HMBC correlation between an oxymethine proton resonating at δ H 6.09 (H-23) and the protons of an acetate methyl (δ H 2.12) to the ester carbonyl carbon at δ C 169.8. An ether linkage was proposed between C-14 and C-17 forming a tetrahydrofuran ring based on the degrees of unsaturation and molecular formula. Based on the above analysis, the gross structure of 1 was established unambiguously and named rhabdastin H following up the previous investigation of cytotoxic isomalabaricanes with oxygenated and olefinic side chains from the sponge Rhabdastrella globostellata [9]. Mar. Drugs 2021, 19, x FOR PEER REVIEW 4 of 8 The relative configurations of the eight chiral centers at C-5, C-8, C-9, C-10, C-13, C-14, C-17, and C-20 in 1 were elucidated by the following NOE analysis (Figure 3). It was found that H3-19 (δH 0.79, s) showed NOE interaction with H-9 (δH 1.44, m) and H-9 with H3-18.  The relative configurations of the eight chiral centers at C-5, C-8, C-9, C-10, C-13, C-14, C-17, and C-20 in 1 were elucidated by the following NOE analysis (Figure 3). It was found that H 3 -19 (δ H 0.79, s) showed NOE interaction with H-9 (δ H 1.44, m) and H-9 with H 3 -18. The relative configurations of the eight chiral centers at C-5, C-8, C-9, C-10, C-13, C-14, C-17, and C-20 in 1 were elucidated by the following NOE analysis (Figure 3). It was found that H3-19 (δH 0.79, s) showed NOE interaction with H-9 (δH 1.44, m) and H-9 with H3-18.  Since all naturally occurring isomalabaricanes displayed that H-5 is trans to Me-19, we assumed the β-orientation of H 3 -19. Thus, H 3 -18 and H-9 were suggested to be positioned on the β-face. One of the methyl groups (H 3 -29) at C-4, which showed a NOE correlation with H 3 -19, was β-oriented and the other one (H 3 -28) was α-oriented. The
The HR-ESI-MS of rhabdastin I (2) showed a molecular ion peak at (m/z 539.3696 [M + Na] + ) and the molecular formula C 32 H 52 O 5 was suggested based on the HRESIMS and 13 C NMR data. The IR spectrum of 2 showed the absorption of carbonyl groups (1745 and 1725 cm −1 ) and a hydroxy group (3436 cm −1 ). The 1 H and 13 C NMR spectroscopic data of 2 ( Table 2) and 1 ( Table 1) indicated similarity in structure except that the ketocarbonyl carbon (C-3) in 1 was replaced by a hydroxy group-bearing methine carbon in 2. In the 13 C NMR spectrum, the signal at δ c 220.3 was replaced by a signal at δ c 79.5, and in the 1 H NMR spectrum, a signal at δ H 3.24 (dd, J = 11.5, 6.5 Hz) was attributed to a hydroxybearing methine at C-3. H-3 showed an NOE correlation with H-5 (δ H 1.54, m) suggesting a β-orientation of the hydroxy group at C-3. Therefore, 2 was identified as the 2S-hydroxy derivative of 1.  The plausible biosynthesis route of the obtained isomalabaricanes presented in Figure 4. The main isomalabaricane 6/6/5 carbocyclic skeleton might be derived from 2,3S-oxidosqualene through hydroxylation and electrocyclizations. The attached tetrahydrofuran moiety and the subsequent side chain were suggested to go through a hydroxylation, an electrocyclization, dehydration, oxidation, and acetylation, forming the first identified isomalabaricane with tetrahydrofuran functionality.
Mar. Drugs 2021, 19, x FOR PEER REVIEW 6 of 8 The plausible biosynthesis route of the obtained isomalabaricanes presented in Figure 4. The main isomalabaricane 6/6/5 carbocyclic skeleton might be derived from 2,3Soxidosqualene through hydroxylation and electrocyclizations. The attached tetrahydrofuran moiety and the subsequent side chain were suggested to go through a hydroxylation, an electrocyclization, dehydration, oxidation, and acetylation, forming the first identified isomalabaricane with tetrahydrofuran functionality. To further clarify the anti-proliferative potential of the isolated compounds, four cancer cell lines (DLD-1: colorectal adenocarcinoma; T-47D: ductal carcinoma; Molt4: acute lymphoblastic leukemia; K562: chronic myelogenous leukemia) were used for MTT screening (Table 3). Compounds 1 and 2 were found to exhibit anti-proliferative activities against Molt4 and K562 leukemic cells with the IC50 value ranging from 9.81 to 16.54 μM.  To further clarify the anti-proliferative potential of the isolated compounds, four cancer cell lines (DLD-1: colorectal adenocarcinoma; T-47D: ductal carcinoma; Molt4: acute lymphoblastic leukemia; K562: chronic myelogenous leukemia) were used for MTT screening (Table 3). Compounds 1 and 2 were found to exhibit anti-proliferative activities against Molt4 and K562 leukemic cells with the IC 50 value ranging from 9.81 to 16.54 µM. Table 3. Anti-proliferative activities of compounds 1 and 2.

Conclusions
The current study highlighted the discovery of the first isomalabaricane derivatives with tetrahydrofuran moiety. Although the identified functionalities did not lead to a dramatic increase in the anti-proliferative properties, the chemical diversity of this class of triterpenes was enriched by these compounds with such unique structures.