Streptoglycerides E–H, Unsaturated Polyketides from the Marine-Derived Bacterium Streptomyces specialis and Their Anti-Inflammatory Activity

Four new streptoglycerides E–H (1–4), with a rare 6/5/5/-membered ring system, were isolated from a marine-derived actinomycete Streptomyces specialis. The structures of 1–4 were elucidated by detailed analysis of HRESIMS, 1D and 2D NMR data and ECD spectra as well as comparison of their spectroscopic data with those reported in literature. Compounds 1–4 showed significant anti-inflammatory activity by inhibiting lipopolysaccharide (LPS)-induced nitric oxide (NO) production in Raw 264.7 cells with IC50 values ranging from 3.5 to 10.9 µM. Especially, 2 suppressed mRNA expression levels of iNOS and IL-6 without cytotoxicity.


Introduction
Inflammation is a normal defense mechanism that occurs to cope with tissue damage and microbial infection [1]. When tissues are exposed to LPS, NO and cytokines such as interleukins (IL-6 and IL-1β), and tumor necrosis factor-α (TNF-α) are produced naturally [2,3]. However, the uncontrolled cytokine overproduction promotes a variety of diseases [4]. NO and interleukins play a central role in the pathogenesis of inflammation owing to overproduction under abnormal physiological conditions [5,6]. Therefore, NO and interleukins are considered as a critical indicator for developing drugs for inflammatory diseases [7,8].
Up to now, there have been many reports on marine natural products with antiinflammatory activity [9][10][11]. Marine organisms inhabiting extreme environments have adopted unique survival strategies for growing and reproducing under hostile conditions, biosynthesizing molecules valuable for pharmaceutical applications [12,13]. Marine Streptomyces are an especially attractive source for exploring marine natural products as they produce various kinds of secondary metabolites with potent biological activities [14,15].
Recently, we reported streptoglycerides A-D with a quite rare 6/5/5 heterocyclic scaffold and anti-inflammatory activity isolated from a strain of marine-derived Streptomyces sp. [16]. During our ongoing investigation of secondary metabolites from marine microorganisms, we encountered another strain, designated as Streptomyces specialis 208DD-067, isolated from a sediment sample collected off Dokdo, South Korea, which produced new derivatives of streptoglycerides (1)(2)(3)(4). Herein, we report the isolation, structure elucidation and anti-inflammatory activity of streptoglycerides E-H (1-4) (Figure 1).
The planar structure of 1 was elucidated by detailed analysis of 1 H-1 H COSY and HMBC data. The structure of ring A was determined as a 2,3,4-substituted-5-methyl-tetrahydropyran by the continuous COSY correlations from H-1a,b to H-4 through H-2, H-

Structural Elucidation
Compound 1 was isolated as a white powder. The molecular formula of 1 was determined to be C 16 Table 1). The 13 C NMR and HSQC spectra of 1 exhibited the presence of two methyl carbons at δ C 13.0 and 18.4; three oxygenated methylene carbons at δ C 62.3, 78.4 and 79.7; an oxygenated methine carbon at δ C 80.0; eight methine carbons at δ C 32.1, 54.3, 130.3, 131.5, 131.8, 132.9, 136.0 and 133.6; and two nonprotonated carbons at δ C 87.4 and 106.1 ( Table 1).
The planar structure of 1 was elucidated by detailed analysis of 1 H-1 H COSY and HMBC data. The structure of ring A was determined as a 2,3,4-substituted-5-methyltetrahydropyran by the continuous COSY correlations from H-1a,b to H-4 through H-2, H-3, and H 3 -16 and the HMBC correlations from H-1a,b to C-5 ( Figure 2). The HMBC correlations from H-13a,b to C-14 and C-15; and H-15a,b to C-13 and C-14 and their chemical shifts of δ C 79.7 (C-13), 87.4 (C-14), and 78.4 (C-15) identified a partial structure corresponding to a 2-C-substituted glycerol moiety. The HMBC correlations from H-13a,b to C-3 and C-4 and from H-15a,b to C-4 and C-5 confirmed the connection of the glycerol moiety to the ring A via ether linkages of C-3/C-13 and C-5/C15 and the linkage between C-4 and C-14 was also determined by the HMBC correlations from H-13a,b to C-4 and C-14 and H-15a,b to C-4 and C-14. The presence of a triene side chain was determined by the continuous COSY correlations from H-6 to H 3 -12 and the connection of the side chain to the ring A at C-5 was confirmed by the HMBC correlations from H-6 to C-4 and C-5 and from H-7 to C-5. Thus, the planar structure of 1 was determined as 2-methylstreptoglyceride B which is closely related to streptoglyceride B, recently isolated from Streptomyces sp. [16]. , and 78.4 (C-15) identified a partial structure corresponding to a 2-C-substituted glycerol moiety. The HMBC correlations from H-13a,b to C-3 and C-4 and from H-15a,b to C-4 and C-5 confirmed the connection of the glycerol moiety to the ring A via ether linkages of C-3/C-13 and C-5/C15 and the linkage between C-4 and C-14 was also determined by the HMBC correlations from H-13a,b to C-4 and C-14 and H-15a,b to C-4 and C-14. The presence of a triene side chain was determined by the continuous COSY correlations from H-6 to H3-12 and the connection of the side chain to the ring A at C-5 was confirmed by the HMBC correlations from H-6 to C-4 and C-5 and from H-7 to C-5. Thus, the planar structure of 1 was determined as 2-methylstreptoglyceride B which is closely related to streptoglyceride B, recently isolated from Streptomyces sp. [16]. The relative configuration of 1 was determined by NOESY correlations and coupling constant analysis ( Figure 3). The geometries of double bonds in the triene chain were deduced as E-configuration on the basis of their large coupling constants (J ≥14 Hz, Table 1). The NOESY correlations from H-4 to H-2, H-3, H-6, and H-7 suggested that these protons had a cofacial relationship. The coupling constant (7.0 Hz) for H-3 and H-4 also indicated these two protons are located as a syn-arrangement.  The relative configuration of 1 was determined by NOESY correlations and coupling constant analysis ( Figure 3). The geometries of double bonds in the triene chain were deduced as E-configuration on the basis of their large coupling constants (J ≥14 Hz, Table 1). The NOESY correlations from H-4 to H-2, H-3, H-6, and H-7 suggested that these protons had a cofacial relationship. The coupling constant (7.0 Hz) for H-3 and H-4 also indicated these two protons are located as a syn-arrangement. The relative configuration of 1 was determined by NOESY correlations and coupling constant analysis ( Figure 3). The geometries of double bonds in the triene chain were deduced as E-configuration on the basis of their large coupling constants (J ≥14 Hz, Table 1). The NOESY correlations from H-4 to H-2, H-3, H-6, and H-7 suggested that these protons had a cofacial relationship. The coupling constant (7.0 Hz) for H-3 and H-4 also indicated these two protons are located as a syn-arrangement. Furthermore, the fact that the furofuran ring system (rings B and C) is a rigid structure and always has a cis-fused relationship, on the basis of general stereochemical considerations, and the lack of NOE correlations from H-6 to H-13b and H-15b allowed us to determine the relative configuration of C-14 (OH-14 and H-4 had a cis-relationship) Furthermore, the fact that the furofuran ring system (rings B and C) is a rigid structure and always has a cis-fused relationship, on the basis of general stereochemical considerations, and the lack of NOE correlations from H-6 to H-13b and H-15b allowed us to determine the relative configuration of C-14 (OH-14 and H-4 had a cis-relationship) [17,18]. Therefore, the relative configuration of C-2, C-3, C-4, C-5 was determined as 2S*, 3S*, 4R*, 5S* as depicted in Figure 3. To further confirm this fact, the 3D models and conformational analysis of two possible relative configurations of 1 (2S*, 3S*, 4R*, 5S*, 14S*) and 1' (2S*, 3S*, 4R*, 5S*, 14R*) were built by Conflex program and their 1 H and 13 C chemical shifts were calculated by Gaussian software. Using both 1 H and 13 C NMR chemical shifts in the DP4+ probability output resulted in a 100% preference for the 1 diastereomer over the 1' diastereomer ( Figure 4, Figure S33 in the supplementary). This result also supported the fact that H-4 and OH-14 are cofacial. Thus, there were only two possible absolute configurations of the 6/5/5 tricyclic ring system for 1 as 1A (2S, 3S, 4R, 5S, 14S) or 1B (2R, 3R, 4S, 5R, 14R).
Mar. Drugs 2021, 19, x FOR PEER REVIEW 4 of 12 [17,18]. Therefore, the relative configuration of C-2, C-3, C-4, C-5 was determined as 2S*, 3S*, 4R*, 5S* as depicted in Figure 3. To further confirm this fact, the 3D models and conformational analysis of two possible relative configurations of 1 (2S*, 3S*, 4R*, 5S*, 14S*) and 1' (2S*, 3S*, 4R*, 5S*, 14R*) were built by Conflex program and their 1 H and 13 C chemical shifts were calculated by Gaussian software. Using both 1 H and 13 C NMR chemical shifts in the DP4+ probability output resulted in a 100% preference for the 1 diastereomer over the 1' diastereomer (  The absolute configuration of 1 was determined by ECD spectrum calculations. The theoretical ECD spectra of 1A (2S, 3S, 4R, 5S, 14S) and its enantiomer 1B (2R, 3R, 4S, 5R, 14R) were generated by the Gaussian 16 program. The experimental ECD spectrum of 1 showed a good agreement with the calculated ECD spectrum of 1A ( Figure 5). Therefore, the absolute configuration of 1 was determined as 2S, 3S, 4R, 5S, 14S, which was same to other natural products with the 6/5/5 tricyclic ring skeleton (diocollettine A, streptoglycerides A-D, and bafilomycins P-Q) [16,17,19]. Thus, 1 was determined as a new derivative of streptoglyceride B and named streptoglyceride E. The absolute configuration of 1 was determined by ECD spectrum calculations. The theoretical ECD spectra of 1A (2S, 3S, 4R, 5S, 14S) and its enantiomer 1B (2R, 3R, 4S, 5R, 14R) were generated by the Gaussian 16 program. The experimental ECD spectrum of 1 showed a good agreement with the calculated ECD spectrum of 1A ( Figure 5). Therefore, the absolute configuration of 1 was determined as 2S, 3S, 4R, 5S, 14S, which was same to other natural products with the 6/5/5 tricyclic ring skeleton (diocollettine A, streptoglycerides A-D, and bafilomycins P-Q) [16,17,19]. Thus, 1 was determined as a new derivative of streptoglyceride B and named streptoglyceride E. The absolute configuration of 1 was determined by ECD spectrum calculations. The theoretical ECD spectra of 1A (2S, 3S, 4R, 5S, 14S) and its enantiomer 1B (2R, 3R, 4S, 5R, 14R) were generated by the Gaussian 16 program. The experimental ECD spectrum of 1 showed a good agreement with the calculated ECD spectrum of 1A ( Figure 5). Therefore, the absolute configuration of 1 was determined as 2S, 3S, 4R, 5S, 14S, which was same to other natural products with the 6/5/5 tricyclic ring skeleton (diocollettine A, streptoglycerides A-D, and bafilomycins P-Q) [16,17,19]. Thus, 1 was determined as a new derivative of streptoglyceride B and named streptoglyceride E. Compound 2 was also isolated as a white powder. The molecular formula of 2 was determined to be the same as that of 1, C16H22O4 by HRESIMS data (m/z 301.1417 [M+Na] + , calcd. for C16H22O4Na, 301.1416). The 1D and 2D NMR data of 2 were similar but not identical to those of 1, and by detailed analysis of HMBC and COSY data, the planar structure of 2 was determined to be the same as 1, suggesting that 2 is a diastereomer of 1. The relative configuration of 2 was also determined by analysis of NOESY data and coupling constants. The strong NOESY correlations between H-4 and H-3, H-6 and H3-16 indicated that these protons were located on the same face of the molecule. The significant difference between 2 and 1 was the methyl group (H3-16) located on the same face with Compound 2 was also isolated as a white powder. The molecular formula of 2 was determined to be the same as that of 1, C 16 , 301.1416). The 1D and 2D NMR data of 2 were similar but not identical to those of 1, and by detailed analysis of HMBC and COSY data, the planar structure of 2 was determined to be the same as 1, suggesting that 2 is a diastereomer of 1. The relative configuration of 2 was also determined by analysis of NOESY data and coupling constants. The strong NOESY correlations between H-4 and H-3, H-6 and H 3 -16 indicated that these protons were located on the same face of the molecule. The significant difference between 2 and 1 was the methyl group (H 3 -16) located on the same face with H-4 in 2, while H-4 and H 3 -16 had a trans-relationship in 1. Furthermore, the fact that H-4 and OH-14 had a co-facial relationship was determined by a similar procedure to that of 1. Therefore, the relative configuration of 2 was determined as 2S*, 3R*, 4S*, 5R*, 14R* (2A) (Figure 3).
The absolute configuration of 2 was determined by comparison of its experimental ECD spectrum with calculated ECD spectra of 2A (2S, 3R, 4S, 5R, 14R) and its enantiomer 2B (2R, 3S, 4R, 5S, 14S). The experimental ECD spectrum of 2 matched well with the calculated ECD spectrum of 2A ( Figure 6). Therefore, the absolute configuration of 2 was determined as 2S, 3R, 4S, 5R, 14R. Thus, 2 was a new diastereomer of 1 and named streptoglyceride F. Compound 3 was isolated as a white powder. The molecular formula of 3 was determined to be C 17 H 24 O 4 by HR-ESIMS data (m/z 315.1570 [M+Na] + , calcd. for C 17 H 24 O 4 Na 315.1572), one methylene group (-CH 2 -) more than that of 1. The 1 H and 13 C NMR data of 3 were almost identical to that of 1, except for the presence of one more methylene group at δ C 21.5 (C-16) and δ H 1.31 (H-16a) and 1.40 (H-16b). Furthermore, the continuous COSY correlations from H 3 -17 (δ H 0.97) to H-2 (δ H 1.69) via H-16a,b suggested that an ethyl group was substituted at C-2 in 3 instead of a methyl substitution in 1. Thus, the planar structure of 3 was determined as 2-ethylstreptoglyceride B (Figure 2).
The absolute configuration of 2 was determined by comparison of its experimental ECD spectrum with calculated ECD spectra of 2A (2S, 3R, 4S, 5R, 14R) and its enantiomer 2B (2R, 3S, 4R, 5S, 14S). The experimental ECD spectrum of 2 matched well with the calculated ECD spectrum of 2A ( Figure 6). Therefore, the absolute configuration of 2 was determined as 2S, 3R, 4S, 5R, 14R. Thus, 2 was a new diastereomer of 1 and named streptoglyceride F. Compound 3 was isolated as a white powder. The molecular formula of 3 was determined to be C17H24O4 by HR-ESIMS data (m/z 315.1570 [M+Na] + , calcd. for C17H24O4Na 315.1572), one methylene group (-CH2-) more than that of 1. The 1 H and 13 C NMR data of 3 were almost identical to that of 1, except for the presence of one more methylene group at δC 21.5 (C-16) and δH 1.31 (H-16a) and 1.40 (H-16b). Furthermore, the continuous COSY correlations from H3-17 (δH 0.97) to H-2 (δH 1.69) via H-16a,b suggested that an ethyl group was substituted at C-2 in 3 instead of a methyl substitution in 1. Thus, the planar structure of 3 was determined as 2-ethylstreptoglyceride B (Figure 2).
The relative configuration of 3 was determined to be the same as 1 by the same aforementioned procedure for 1, (2S*, 3S*, 4R*, 5S*, 14S*, (3A)) (Figure 3), and the experimental ECD spectrum of 3 was compared to the calculated ECD spectra of 3A and its enantiomer 3B. The experimental ECD spectrum of 3 was well-matched to the calculated ECD spectrum of 3A (Figure 7). Therefore, the absolute configuration of 3 was determined as 2S, 3S, 4R, 5S, 14S. Thus, the structure of 3 was determined as a new derivative of streptoglyceride B and named streptoglyceride G. The relative configuration of 3 was determined to be the same as 1 by the same aforementioned procedure for 1, (2S*, 3S*, 4R*, 5S*, 14S*, (3A)) ( Figure 3), and the experimental ECD spectrum of 3 was compared to the calculated ECD spectra of 3A and its enantiomer 3B. The experimental ECD spectrum of 3 was well-matched to the calculated ECD spectrum of 3A (Figure 7). Therefore, the absolute configuration of 3 was determined as 2S, 3S, 4R, 5S, 14S. Thus, the structure of 3 was determined as a new derivative of streptoglyceride B and named streptoglyceride G. The relative configuration of 4 was determined to be the same as 2 by the same abovementioned procedure for 2 (2S*, 3R*, 4S*, 5R*, 14R*, (4A)) and the experimental ECD spectrum of 4 was compared to the calculated ECD spectra of 4A (2S, 3R, 4S, 5R, 14R) and its enantiomer 4B (2R, 3S, 4R, 5S, 14S). The experimental ECD spectrum of 4 was well matched to the calculated ECD spectrum of 4A (Figure 8). Therefore, the absolute configuration of 4 was determined as 2S, 3R, 4S, 5R, 14R. Thus, 4 was determined as a new diastereomer of 3 and named streptoglyceride H. The relative configuration of 4 was determined to be the same as 2 by the same abovementioned procedure for 2 (2S*, 3R*, 4S*, 5R*, 14R*, (4A)) and the experimental ECD spectrum of 4 was compared to the calculated ECD spectra of 4A (2S, 3R, 4S, 5R, 14R) and its enantiomer 4B (2R, 3S, 4R, 5S, 14S). The experimental ECD spectrum of 4 was well matched to the calculated ECD spectrum of 4A (Figure 8). Therefore, the absolute configuration of 4 was determined as 2S, 3R, 4S, 5R, 14R. Thus, 4 was determined as a new diastereomer of 3 and named streptoglyceride H.

Bioactivities
Compounds 1-4 were screened for their effects on the production of NO in LPS-stimulated RAW 264.7 mouse macrophage cell line. All four compounds showed moderate inhibitory effects with IC50 values ranging from 3.5 to 10.9 M ( Table 2). The test was performed four times to confirm the reproducibility and statistical analyses were conducted using a t-test.

Bioactivities
Compounds 1-4 were screened for their effects on the production of NO in LPSstimulated RAW 264.7 mouse macrophage cell line. All four compounds showed moderate inhibitory effects with IC 50 values ranging from 3.5 to 10.9 µM ( Table 2). The test was performed four times to confirm the reproducibility and statistical analyses were conducted using a t-test. To further investigate the anti-inflammatory effects of 2, we examined the effect of 2 on LPS-induced production of inflammatory mediators, including NO and Interleukin-6 (IL-6), in RAW 264.7 cells. As shown in Figure 9A,B, the treatment of RAW 264.7 cells with LPS increased the accumulation of nitrite and IL-6, and 2 dose-dependently inhibited LPS-induced production of nitrite and IL-6 in LPS-stimulated RAW 264.7 cells. To further investigate whether the effects of 2 were due to its effects on the mRNA expression of cognate genes, we examined the effect of 2 on the mRNA expression of inducible nitric oxide synthase (iNOS) and IL-6 by qPCR. The mRNA levels of iNOS and IL-6 were induced by LPS treatment, and this induction was suppressed by 2 in a dose-dependent manner ( Figure 9D,E). The concentrations of 2 used in this study had no cytotoxic effect on the viability of RAW 264.7 cells. Additionally, the mitogen-activated protein kinase (MAPK) activation study showed that phosphorylation of extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK) and p38 proteins was inhibited by 2 ( Figure 9). The ERK, JNK and p38 proteins belonging to the MAPK superfamily are phosphorylated in the cytoplasm of stimulated cells by LPS. The activated p-ERK, p-JNK and p38 proteins activate transcription factors related with inflammation in their nucleus. Thus, 2 is considered to have antiinflammatory activity by inhibiting the activation of the MAPK pathway. The concentrations of 2 used in this study had no cytotoxic effect on the viability of RAW 264.7 cells. Additionally, the mitogen-activated protein kinase (MAPK) activation study showed that phosphorylation of extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK) and p38 proteins was inhibited by 2 ( Figure 9). The ERK, JNK and p38 proteins belonging to the MAPK superfamily are phosphorylated in the cytoplasm of stimulated cells by LPS. The activated p-ERK, p-JNK and p38 proteins activate transcription factors related with inflammation in their nucleus. Thus, 2 is considered to have anti-inflammatory activity by inhibiting the activation of the MAPK pathway.

Isolation and Cultivation of the Strain 208DD-067
The strain 208DD-067 was isolated from a sediment sample collected in Dokdo, South Korea in August 2020. The strain was identified as Streptomyces specialis on the basis of 16S rRNA gene sequence analysis (GenBank accession number OL691077). The seed and mass cultures of the strain 208DD-067 were performed in Bennett's medium (BN broth, 1% glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, 3.2% sea salt, pH 7.0 before sterilization). A single colony of the strain from the agar plate was inoculated aseptically into a 2 L flask filled with 1 L of BN broth. After that, the strain was incubated at 24 • C for 7 days on a rotary shaker at 120 rpm and then the culture broth was transferred to a 100 L fermenter filled with 70 L of BN broth. The mass culture was incubated for 21 days at 28 • C and then harvested.