Isolation of Scalimides A–L: β-Alanine-Bearing Scalarane Analogs from the Marine Sponge Spongia sp.

A chemical investigation of a methanol extract of Spongia sp., a marine sponge collected from the Philippines, identified 12 unreported scalarane-type alkaloids—scalimides A–L (1–12)—together with two previously described scalarin derivatives. The elucidation of the structure of the scalaranes based on the interpretation of their NMR and HRMS data revealed that 1–12 featured a β-alanine-substituted E-ring but differed from each other through variations in their oxidation states and substitutions occurring at C16, C24, and C25. Evaluation of the antimicrobial activity of 1–12 against several Gram-positive and Gram-negative bacteria showed that 10 and 11 were active against Micrococcus luteus and Bacillus subtilis, respectively, with MIC values ranging from 4 to 16 μg/mL.


Introduction
Scalaranes are one of the most prevalent groups of sesterterpenes, and they are primarily found in marine invertebrates [1][2][3]. Since scalarin, the first reported natural scalaranetype marine product, was discovered in the marine sponge Cacospongia scalaris in 1972 [4], several hundreds of scalarane sesterterpenes have been identified from sponges belonging to the order Dicytoceratida [5]. This class of scalaranes is characterized as a trans-fused 6/6/6/6 carbocyclic ring system and, in many cases, contains an additional pentacyclic E ring [6]. The general structure of the E ring contains an oxygen atom in the form of a lactone or furan, but certain reports have shown that a nitrogen-rich environment can produce a pyrrole or lactam [7]. The first example of nitrogen-bearing scalarane was found in the case of molliorins that were reported from 1977 to 1979. A series of investigations on the marine sponge Cacospongia mollior resulted in consecutive isolations of five pyrrole-bearing scalaranes-molliorins A-E [8][9][10][11]. Plausible biosynthesis of pyrrole in molliorins that involved condensation reactions of scalaridial-which is a common intermediate-with the corresponding amines was proposed, and this could be proved by the synthesis of molliorins after the proposed condensation under acidic conditions.
Since the mid-2000s, scalaranes containing lactams or cyclic imides have been identified. Hyatelactam, which was isolated from the marine sponge Hyatella intestinalis in 2006, is the first example of a lactam-bearing scalarane [12]. Following this report, 17 additional scalaranes belonging to this class were isolated from the marine sponges Hyatella sp., Hyrtios sp., Petrosaspongia sp., and Spongia sp. [13][14][15][16][17]. In these cases, a maleimide or α,β-unsaturated γ-butyrolactam moiety appeared as a structural feature of the E ring, but only hyrtioscalarin A contained a linear amide terminus ( Figure 1). Although the pharmacological activity of the scalarane alkaloids has not been thoroughly investigated, some interesting biological properties have been reported, such as the anticancer activities of hyatelactam against HT-29 (human colorectal adenocarcinoma cell line, GI 50 = 8.1 µM) [7], hyrtioscalarin D against A549 (adenocarcinomic human alveolar basal epithelial cell, IC 50 = 5.4 µM), and hyrtioscalarin G against A549 (IC 50 = 6.9 µM) and PC3 (human prostate The marine sponge of the genus Spongia, which belongs to the order Dicytoceratida, is known as a rich source of scalarane sesterterpenes. To date, various scalarane sesterterpenes exhibiting cytotoxicity or antagonistic activity against FXR (farnesoid X receptor), such as scalarolide [18], isoscalarafurans A-B [19], scalalactams A-D [16], and 12-O-deacetyl-12-epi-19-deoxy-21-hydroxyscalarin [20], have been isolated from marine sponges [21,22]. Our preliminary biological evaluation of the methanol extract of Spongia sp. collected from the Bohol province in the Philippines revealed a mild antimicrobial activity, prompting an investigation of bioactive secondary metabolites of the sponge. In this study, we report the identification of 12 undescribed scalarane alkaloids-scalimides A-L (1-12)-that contain a β-alanine moiety and their antimicrobial activity against several strains of Gram-positive and Gram-negative bacteria ( Figure 2).  The marine sponge of the genus Spongia, which belongs to the order Dicytoceratida, is known as a rich source of scalarane sesterterpenes. To date, various scalarane sesterterpenes exhibiting cytotoxicity or antagonistic activity against FXR (farnesoid X receptor), such as scalarolide [18], isoscalarafurans A-B [19], scalalactams A-D [16], and 12-O-deacetyl-12-epi-19-deoxy-21-hydroxyscalarin [20], have been isolated from marine sponges [21,22]. Our preliminary biological evaluation of the methanol extract of Spongia sp. collected from the Bohol province in the Philippines revealed a mild antimicrobial activity, prompting an investigation of bioactive secondary metabolites of the sponge. In this study, we report the identification of 12 undescribed scalarane alkaloids-scalimides A-L (1-12)-that contain a β-alanine moiety and their antimicrobial activity against several strains of Gram-positive and Gram-negative bacteria ( Figure 2).

Elucidation of the Structure
The complete structure could be proven by observing the HMBC correlations from H-16 to C-17 (δC 140.5)/C-18 (δC 151.1) and from H-14 (δH 2.03) to C-16 (δC 60.2). Additionally, the configuration of C-16 was further supported by the absence of the NOESY correlation between H-14 and H-16 and the higher chemical shift value of H-14 (δH 2.03 vs. δH 1.66 in 1), which resulted from an increasing 1,3-diaxial interaction on the D ring due to an axial substitution of OH-16.
Scalimide C (3) was isolated as an amorphous solid. Its molecular formula was determined to be C31H45NO7 by HRESIMS (m/z 566.3087 [M + Na] + , as calculated for C31H45NO7Na, 566.3088), corresponding to 10 degrees of unsaturation. The 1 H and 13 C NMR spectra of 3 resembled those of 1, except for the presence of a methoxy group at δH 3.52/δC 58.0, suggesting that 3 was a methyl ether of 1 at C-16, which was consistent with its being 14 mass units higher than that of 1 in the HRESIMS spectrum ( Table 1). The location of the methoxy group was confirmed by the HMBC correlation from the methyl singlet at δH 3.52 to C-16 (δH 4.29/δC 74.9) (Figure 3). The configuration of C-16 was assigned as β based on the NOESY correlation between H-14 and H-16, as well as the large coupling constant for H-16 (δH 4.29, JH-16-H-15 = 9.1, 7.0 Hz).
Scalimide D (4) was isolated as an amorphous solid. The molecular formula of 4 was not only identical to that of 3, but its 1 H NMR spectrum also showed a high degree of similarity with that of 2, except for the presence of a methoxy group at δH 3.46/δC 57.9, suggesting that 4 was the OMe-16 analog of 2 (Table 1, Figure S25). The HMBC correlation from the methyl singlet at δH 3.46 to C-16 (δC 70.1) confirmed the substitution of OMe at C-16, and its α orientation of the methoxy group was determined by the coupling constants of H-16 (δH 4.18, dd, JH-16-H-15 = 4.0, 1.6 Hz).
Scalimide E (5) was isolated as an amorphous solid, and its molecular mass was 14 mass units higher than those of 3 and 4, as determined by HRESIMS (m/z 580.3240 [M + Na] + , as calculated for C32H47NO7Na, 580.3245). Compared with that of 4, the 1 H NMR spectrum of 5 showed an additional methyl singlet at δH 3.63, suggesting that 5 is a methyl ester of 4 (Table 1, Figure S32). The HMBC correlation from the methyl group at δH 3.63 to C-3′ (δC 173.0) confirmed the presence of a methyl ester moiety in the side chain of 5 (Figure 3). Scalimide E (5) could be an artifact generated from 4 during the course of purification using MeOH and TFA, but this possibility was not investigated in this study.
Scalimide F (6) was isolated as an amorphous solid. Its molecular formula was determined to be C30H41NO6 by HRESIMS (m/z 534.2817 [M + Na] + , as calculated for C30H41NO6Na, 534.2826), corresponding to 11 degrees of unsaturation. The analysis of the   1), which resulted from an increasing 1,3-diaxial interaction on the D ring due to an axial substitution of OH-16.
Scalimide C (3) was isolated as an amorphous solid. Its molecular formula was determined to be C 31 H 45 NO 7 by HRESIMS (m/z 566.3087 [M + Na] + , as calculated for C 31 H 45 NO 7 Na, 566.3088), corresponding to 10 degrees of unsaturation. The 1 H and 13 C NMR spectra of 3 resembled those of 1, except for the presence of a methoxy group at δ H 3.52/δ C 58.0, suggesting that 3 was a methyl ether of 1 at C-16, which was consistent with its being 14 mass units higher than that of 1 in the HRESIMS spectrum ( Table 1). The location of the methoxy group was confirmed by the HMBC correlation from the methyl singlet at δ H 3.52 to C-16 (δ H 4.29/δ C 74.9) (Figure 3). The configuration of C-16 was assigned as β based on the NOESY correlation between H-14 and H-16, as well as the large coupling constant for H-16 (δ H 4.29, J H-16-H-15 = 9.1, 7.0 Hz).
Scalimide D (4) was isolated as an amorphous solid. The molecular formula of 4 was not only identical to that of 3, but its 1 H NMR spectrum also showed a high degree of similarity with that of 2, except for the presence of a methoxy group at δ H 3.46/δ C 57.9, suggesting that 4 was the OMe-16 analog of 2 (Table 1, Figure S25). The HMBC correlation from the methyl singlet at δ H 3.46 to C-16 (δ C 70.1) confirmed the substitution of OMe at C-16, and its α orientation of the methoxy group was determined by the coupling constants of H-16 (δ H 4.18, dd, J H-16-H-15 = 4.0, 1.6 Hz).
Scalimide E (5) was isolated as an amorphous solid, and its molecular mass was 14 mass units higher than those of 3 and 4, as determined by HRESIMS (m/z 580.3240 [M + Na] + , as calculated for C 32 H 47 NO 7 Na, 580.3245). Compared with that of 4, the 1 H NMR spectrum of 5 showed an additional methyl singlet at δ H 3.63, suggesting that 5 is a methyl ester of 4 (Table 1, Figure S32). The HMBC correlation from the methyl group at δ H 3.63 to C-3 (δ C 173.0) confirmed the presence of a methyl ester moiety in the side chain of 5 ( Figure 3). Scalimide E (5) could be an artifact generated from 4 during the course of purification using MeOH and TFA, but this possibility was not investigated in this study.
Scalimide G (7) was isolated as an amorphous solid. Its molecular formula was determined to be C 31 H 47 NO 7 by HRESIMS (m/z 568.3238 [M + Na] + , as calculated for C 31 H 47 NO 7 Na, 568.3245), corresponding to nine degrees of unsaturation. Analysis of the 1D and 2D NMR data revealed a similar pentacyclic framework to that of 4 ( Table 2, Figures  S46-S51). However, one additional sp 3 oxymethine at δ H 5.39/δ C 81.7 was observed as a substitution for one of the carbonyl groups of a pyrrole-2,5-dione in 4. This information indicates the reduction of a carbonyl group in the pyrrole-2,5-dione of 4 to a hydroxyl group whose position was determined to be at C-24, as observed from the HMBC correlations from H-16 (δ H 4.02)/H 2 -1 (δ H 3.68/3.53) to C-24 (δ C 81.7) (Figures S2 and S50). Similarly, the small coupling constant of H-16 (dd, J H-16-H-15 = 4.4, 1.3 Hz) could be a sign of the α-orientation of OMe-16. Furthermore, the 24-OH configuration was assigned as a βorientation based on the NOESY correlation between H-24 and 16α-OMe.  Scalimide H (8) was isolated as an amorphous solid. The 1 H and 13 C NMR data of 8 differed from those of 7 only in the presence of an additional methyl singlet at δ H 3.04/δ C 50.5, which was found to be in agreement with the HRESIMS result, indicating a molecular mass that was 14 units higher than that of 7 (Table 2, Figures S53, S54 and S60). The HMBC correlations from δ H 3.04 to C-24 (δ C 87.0) and from H-24 (δ H 5.44) to δ C 50.5 could be evidence of the OMe-24 substituent ( Figure 5). Since OMe-24 exhibited a cross-peak with H 3 -23 in the NOESY spectrum, the OMe-24 was determined to be β-oriented ( Figure S3).   Scalimide I (9) was isolated as an amorphous solid. Its molecular formula was determined to be C 30 H 45 NO 6 13 C NMR data revealed that 9 had the same scaffold as 1, but with one methylene at δ H 4.07/3.97 instead of a carbonyl group (Table 2, Figures S61 and S62). The HMBC correlations from δ H 4.07/3.97 to C-17 (δ C 153.9)/C-18 (δ C 140.1)/C-25 (δ C 171.7) indicated a γ-lactam moiety, and the NOESY correlation between one of the methylene protons at δ H 3.97 and H-16 suggested the location of the methylene at C-24 to determine a 24-2H-pyrrol-25-one ( Figure 5 and Figure S3). In addition, the β-configuration of OH-16 was confirmed by the large coupling constant of H-16 (dd, J H-16-H-15 = 9.8, 6.6 Hz).
Scalimide L (12) was isolated as an amorphous solid. The 1 H NMR spectrum of 12 was almost identical to that of 11, except for an additional methyl singlet at δH 3.65, suggesting that 12 was a methyl ester of 11 (Table 2, Figure S82). The presence of an additional methyl group was consistent with the molecular formula of C31H45NO5, as determined in an HRESIMS analysis (m/z 512.3366 [M + H] + , as calculated for C31H46NO5, 512.3371). Therefore, the HMBC correlation from δH 3.65 to C-3′ (δC 174.4) confirmed the presence of the methyl propionate side chain, which may have been an artifact generated during the separation steps when using MeOH as an eluent.

Biological Activities
As the MeOH extract of Spongia sp. exhibited mild antimicrobial activity in our preliminary screening, the minimal inhibitory concentrations (MICs) of scalimides A-L (1-12) were evaluated by using several strains of Gram-positive and Gram-negative bacteria (Table 3). Interestingly, 1-12 were more potent against Gram-positive bacteria but were mostly inactive against Gram-negative bacteria. Among these compounds, 10 displayed Scalimide K (11) was isolated as an amorphous solid. Its molecular formula was determined to be C 30 H 43 NO 5 by HRESIMS (m/z 520.3030 [M + Na] + , as calculated for C 30 H 43 NO 5 Na, 520.3033), corresponding to 10 degrees of unsaturation. The 1 H NMR spectra of 11 in combination with the HSQC data showed olefinic protons at δ H 6.25/6.03 and a methylene proton at δ H 4.06 ( Scalimide L (12) was isolated as an amorphous solid. The 1 H NMR spectrum of 12 was almost identical to that of 11, except for an additional methyl singlet at δ H 3.65, suggesting that 12 was a methyl ester of 11 (Table 2, Figure S82). The presence of an additional methyl group was consistent with the molecular formula of C 31 H 45 NO 5 , as determined in an HRESIMS analysis (m/z 512.3366 [M + H] + , as calculated for C 31 H 46 NO 5 , 512.3371). Therefore, the HMBC correlation from δ H 3.65 to C-3 (δ C 174.4) confirmed the presence of the methyl propionate side chain, which may have been an artifact generated during the separation steps when using MeOH as an eluent.

Biological Activities
As the MeOH extract of Spongia sp. exhibited mild antimicrobial activity in our preliminary screening, the minimal inhibitory concentrations (MICs) of scalimides A-L (1-12) were evaluated by using several strains of Gram-positive and Gram-negative bacteria (Table 3). Interestingly, 1-12 were more potent against Gram-positive bacteria but were mostly inactive against Gram-negative bacteria. Among these compounds, 10 displayed the broadest spectrum of inhibitory effects, especially against Micrococcus luteus and Bacillus subtilis, with MIC values of 8 and 4 µg/mL, respectively. Although most of the isolated compounds displayed weak to moderate antibacterial activity, the structural diversity of the scalimides-arising from different substitutions on C-16, C-24, and C-25-provided useful information regarding the structure-activity relationship (SAR): (1) Regarding the MIC values of 1-4, the methyl ether at C-16 appeared to be more potent than the hydroxyl group for the activity toward B. subtilis; (2) when comparing 4 with 5 and 11 with 12, the carboxylic acid moiety at C-3 was identified as a crucial factor for the antimicrobial activity; (3) reduction of the carbonyl group at C-24 to a hydroxyl group (4 to 7) and a methylene group (1 to 9) decreased antibacterial activity; (4) reduction of the carboxyl group of the imide at C-25 to a lactam (1 vs. 10 and 6 vs. 11) increased antibacterial activity at least four-fold. Additionally, the cytotoxicity of 1-12 against MCF7 (a breast cancer cell line) was evaluated to identify it as a potent anticancer agent, but only moderate anticancer effects were observed for all of the tested compounds.

Biological Material
Biological material was collected in March 2015 in the Philippines (9 • 45 32.31" N 124 • 35 53.60" E) at a depth of 15 m by scuba diving. The sponges were frozen at −20 • C until they were identified as Spongia sp. and chemically analyzed. A voucher sample (153PIL-209) was stored at the Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology (KIOST).
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20110726/s1, Tables S1-S12: 13   Data Availability Statement: All data presented in this report are available with permission from the corresponding author upon request.