New Hygrocins K–U and Streptophenylpropanamide A and Bioactive Compounds from the Marine-Associated Streptomyces sp. ZZ1956

Marine-derived Streptomyces actinomycetes are one of the most important sources for the discovery of novel bioactive natural products. This study characterized the isolation, structural elucidation and biological activity evaluation of thirty compounds, including twelve previously undescribed compounds, namely hygrocins K–U (5–13, 17 and 18) and streptophenylpropanamide A (23), from the marine-associated actinomycete Streptomyces sp. ZZ1956. Structures of the isolated compounds were determined by a combination of extensive NMR spectroscopic analyses, HRESIMS data, the Mosher’s method, ECD calculations, single crystal X-ray diffraction and comparison with reported data. Hygrocins C (1), D (2), F (4), N (8), Q (11) and R (12), 2-acetamide-6-hydroxy-7-methyl-1,4-naphthoquinone (22), echoside C (27), echoside A (28) and 11,11′-O-dimethylelaiophylin (30) had antiproliferative activity (IC50: 0.16–19.39 μM) against both human glioma U87MG and U251 cells with hygrocin C as the strongest active compound (IC50: 0.16 and 0.35 μM, respectively). The analysis of the structure–activity relationship indicated that a small change in the structures of the naphthalenic ansamycins had significant influence on their antiglioma activities. Hygrocins N (8), O (9), R (12), T (17) and U (18), 2-amino-6-hydroxy-7-methyl-1,4-naphthoquinone (21), 2-acetamide-6-hydroxy-7-methyl-1,4-naphthoquinone (22), 3′-methoxy(1,1′,4′,1″-terphenyl)-2′,6′-diol (26), echoside C (27) and echoside A (28) showed antibacterial activity against methicillin-resistant Staphylococcus aureus and Escherichia coli with MIC values of 3–48 μg/mL.


Introduction
Compound 3-amino-5-hydroxy benzoic acid (3, is the precursor of a big group of natural products, including the ansamycins, the mitomycins and the unique saliniketals (degraded ansamycins) [1]. The ansamycins have two characteristic structural features: an aromatic core and a so-called ansa bridge containing a lactam moiety, whose two ends link to two nonadjacent positions of the aromatic core [2]. The ansamycins can be divided into naphthalenic or benzenic depending on the nature of their aromatic ring. The naphthalenic ansamycins include rifamycins, ansalactams, chaxamycins, divergolides, hygrocins, naphthomycins, rubradirins and streptovaricins. Their structural characteristic is a 1,4-naphtoquinone or a 1,4-hydroxynaphtalene chromophore. Benzenic ansamycins have a 1,4-benzoquinone or a 1,4-hydrobenzoquinone chromophore and include ansatrienins, cebulactams, cytotrienins, geldanamycins, herbimycins, macbecins, maytansines (ansamitocins) and tetrapetalones. Precursor feeding experiments and genetic and biochemical methods have been applied to investigate the biosynthesis of the ansamycins, demonstrating that AHBA is the source of the chromophore and the aliphatic ansa chains are derived from acetate, propionate, isobutyrate or glycolate units [1,2].
Compound 5 had the same molecular formula C 28 H 31 NO 8 and very similar UV characteristic absorptions as hygrocins C-F (1-4), indicating that they are isomers. Careful analyses of the 1 H, 13 C, HMQC, COSY, HMBC and NOESY NMR spectra of 5 demonstrated that its structure was different from those of 1-4 in the configurations at C-2 and C 3 -C 4 double bond as well as the position of the lactone ring formation. The configuration at C-2 was established as R based on a strong NOE correlation observed between H-2 and H 3 -6a ( Figure 2). A strong NOE correlation of H-2 with H 3 -6a was an indication of 2Rconfiguration in 1 and 4, compared to the 2S-configuration in 2 and 3 without the NOE correlation of H-2 and H 3 -6a [21]. The chemical shift value (δ C 13.7) of C-4a in 5 indicated a 3E-configuration, compared to the downfield chemical shift values (δ C 21.1-22.2) (Tables S3 and S4) of C-4a for a 3Z-configuration in 1, 2 and 4 [21]. Observed strong NOE (for 7, 8,13), no NOE (for 10-12) or weak NOE (5,6,9) correlation between H-3 and H 3 -4a also supported the assignment of the 3E-or 3Z-configuration. In addition, the trans-coupling constant value of 16.0 Hz ( 3 J H8/H9 ) indicated an 8E-configuration and the small vicinal coupling constant value of 2.8 Hz ( 3 J H6/H7 ) suggested a syn orientation between H-6 and H-7 [21]. HMBC correlations ( Figure 2) of H-6 (δ H 4.65) with C-5 (δ C 167.7) established the linkage of C 5 and C 6 through an oxygen to form the lactone ring. It is known that hygrocins C-E (1-3) have the same 6S, 7S, 10S, 19R-configuration. Therefore, we proposed compound 5 to have the same 6S, 7S, 10S, 19R-configuration as 1-3 based on a common biosynthetic origin. Based on the above analyses, it can be concluded that the structure of 5 is similar to that of 1 with the only difference being the configuration of the C 3 -C 4 double bond. Therefore, the structure of 5 was elucidated as a previously undescribed member of the naphthoquinone ansamycins, named hygrocin K. Its 13 C and 1 H NMR data are reported in Tables 1 and 2. Antibiotics 2022, 11, x FOR PEER REVIEW 4 of 17
Compound 5 had the same molecular formula C28H31NO8 and very similar UV characteristic absorptions as hygrocins C-F (1-4), indicating that they are isomers. Careful analyses of the 1 H, 13 C, HMQC, COSY, HMBC and NOESY NMR spectra of 5 demonstrated that its structure was different from those of 1-4 in the configurations at C-2 and C3-C4 double bond as well as the position of the lactone ring formation. The configuration at C-2 was established as R based on a strong NOE correlation observed between H-2 and H3-6a ( Figure 2). A strong NOE correlation of H-2 with H3-6a was an indication of 2Rconfiguration in 1 and 4, compared to the 2S-configuration in 2 and 3 without the NOE correlation of H-2 and H3-6a [21]. The chemical shift value (δC 13.7) of C-4a in 5 indicated a 3E-configuration, compared to the downfield chemical shift values (δC 21.1-22.2) (Tables  S3 and S4) of C-4a for a 3Z-configuration in 1, 2 and 4 [21]. Observed strong NOE (for 7, 8,13), no NOE (for 10-12) or weak NOE (5,6,9) correlation between H-3 and H3-4a also supported the assignment of the 3E-or 3Z-configuration. In addition, the trans-coupling constant value of 16.0 Hz ( 3 JH8/H9) indicated an 8E-configuration and the small vicinal coupling constant value of 2.8 Hz ( 3 JH6/H7) suggested a syn orientation between H-6 and H-7 [21]. HMBC correlations ( Figure 2) of H-6 (δH 4.65) with C-5 (δC 167.7) established the linkage of C5 and C6 through an oxygen to form the lactone ring. It is known that hygrocins C-E (1-3) have the same 6S, 7S, 10S, 19R-configuration. Therefore, we proposed compound 5 to have the same 6S, 7S, 10S, 19R-configuration as 1-3 based on a common biosynthetic origin. Based on the above analyses, it can be concluded that the structure of 5 is similar to that of 1 with the only difference being the configuration of the C3-C4 double bond. Therefore, the structure of 5 was elucidated as a previously undescribed member of the naphthoquinone ansamycins, named hygrocin K. Its 13 C and 1 H NMR data are reported in Tables 1 and 2.    13 C NMR data of compounds 5-13 (150 MHz,δ C C 172.5,C 173.1,C 171.6,C 171.6,C 171.5,C 171.7,C 171.7,C 2 56.9,CH 56.9,CH 121.9,C 122.6,C 126.8,C 126.8,C 126.8,C 126.8,C 126.6,C 3 133.1,CH 133.6,CH 128.9,CH 127.5,CH 129.7,CH 129.7,CH 129.7,CH 129.7,CH 129.7,C 4 133.9,C 133.5,C 137.5,C 137.8 3 a,b The data with the same labels in each column may be interchanged. Compound 6 had the same molecular formula C 28 H 31 NO 8 as 1-5 deduced from its HRESIMS ion peak at m/z 508.1979 [M-H] − and 13 C NMR data. Careful analyses of its 1Dand 2D-NMR spectra determined that 6 and hygrocins F (4) had the same planar structure.
Antibiotics 2022, 11, 1455 6 of 17 As described above for 5, no NOE correlation between H-2 with H 3 -6a and weak NOE correlation between H-3 with H 3 -4a as well as the relative upfield shift value at δ C 13.6 for C-4a indicated that 6 had 2S-and 3E-configurations [21]. The structure of 6 was thus identified as a previously undescribed naphthoquinone ansamycin, named hygrocin L. Its 13 C and 1 H NMR data (Tables 1 and 2) were assigned based on the HMQC, COSY and HMBC correlations ( Figure 2). Compounds 7 and 8 had very similar UV absorptions and the same molecular formula C 28 H 29 NO 7 deduced from their HRESIMS ion peaks at m/z 490.1875 and 490.1871 [M-H] − , respectively, 18 mass units lower than that of 1-6, corresponding to the loss of a H 2 O molecule. Detailed analyses of the 1D-and 2D-NMR spectra of 7 and 8 as well as comparison of their NMR data with those of 1-6 demonstrated that the methine at C-2 and the non-protonated oxygenated carbon at C-19 in 1-6 were replaced by two non-protonated olefinic carbons at C-2 (δ C 121.9 in 7 or δ C 122.6 in 8) and C-19 (δ C 134.8 in 7 or δ C 136.4 in 8). Therefore, both 7 and 8 had a C 2 -C 19 Table S15) indicated a 6S-configuration for 8. Therefore, compound 7 should have the same 6S, 7S-configuration as compounds 1-3 and 5 and compound 8 should have the same 6S, 7R-configuration as compounds 4, 6 and 16 [21] based on their shared biogenesis, the structures of the reported compounds and the Mosher's method results of 8. The structures of 7 and 8 were thus elucidated as two previously unreported naphthoquinone ansamycins, named hygrocin M (7) and hygrocin N (8). The 13 C and 1 H NMR data (Tables 1 and 2)   Compound 6 had the same molecular formula C28H31NO8 as 1-5 deduced from its HRESIMS ion peak at m/z 508.1979 [M-H] − and 13 C NMR data. Careful analyses of its 1Dand 2D-NMR spectra determined that 6 and hygrocins F (4) had the same planar structure. As described above for 5, no NOE correlation between H-2 with H3-6a and weak NOE correlation between H-3 with H3-4a as well as the relative upfield shift value at δC 13.6 for C-4a indicated that 6 had 2S-and 3E-configurations [21]. The structure of 6 was thus identified as a previously undescribed naphthoquinone ansamycin, named hygrocin L. Its 13 C and 1 H NMR data (Tables 1 and 2) were assigned based on the HMQC, COSY and HMBC correlations ( Figure 2). Compounds 7 and 8 had very similar UV absorptions and the same molecular formula C28H29NO7 deduced from their HRESIMS ion peaks at m/z 490.1875 and 490.1871 [M-H] − , respectively, 18 mass units lower than that of 1-6, corresponding to the loss of a H2O molecule. Detailed analyses of the 1D-and 2D-NMR spectra of 7 and 8 as well as comparison of their NMR data with those of 1-6 demonstrated that the methine at C-2 and the non-protonated oxygenated carbon at C-19 in 1-6 were replaced by two non-protonated olefinic carbons at C-2 (δC 121.9 in 7 or δC 122.  Table S15) indicated a 6S-configuration for 8. Therefore, compound 7 should have the same 6S, 7S-configuration as compounds 1-3 and 5 and compound 8 should have the same 6S, 7R-configuration as compounds 4, 6 and 16 [21] based on their shared biogenesis, the structures of the reported compounds and the Mosher's method results of 8. The structures of 7 and 8 were thus elucidated as two previously unreported naphthoquinone ansamycins, named hygrocin M (7) and hygrocin N (8). The 13 C and 1 H NMR data (Tables 1 and 2) of 7 and 8 were assigned based on the HMQC, COSY and HMBC correlations (Figures 2 and 3). Compounds 9 and 10 were obtained as a red amorphous powder and had very similar UV characteristic absorptions (around 201 and 335 nm) to those of 7 and 8, suggesting that they were analogues. Both 9 and 10 had the same molecular formula C28H31NO8 deduced from their 13   Compounds 9 and 10 were obtained as a red amorphous powder and had very similar UV characteristic absorptions (around 201 and 335 nm) to those of 7 and 8, suggesting that they were analogues. Both 9 and 10 had the same molecular formula C 28 H 31 NO 8 deduced from their 13 C NMR data and HRESIMS ion peaks at m/z 508.1975 [M-H] − in 9 and 508.1974 [M-H] − in 10, 18 mass units higher than those of 7 and 8. Compared to 7 and 8, one additional aromatic hydrogen signal at δ H 7.43 (s) in 9 or δ H 7.42 (s) in 10 was observed in their 1 H NMR spectra. However, the 13 C NMR signal at δ C 212.9 in 7 or δ H 212.0 in 8 for the ketone group at C-13 was replaced in both 9 and 10 by upfield shifted signals at δ C 177.7 in 9 or δ C 177.8 in 10. Further analyses of their HMQC, COSY and HMBC correlations ( Figure 4) as well as consideration of their molecular formula and 14 degrees of unsaturation required by the molecular formula demonstrated that 9 and 10 were derivatives of 7 and 8, respectively, with ring opening between C-13 and C-14. The chemical shift at δ C 16.4 for C-4a and no NOE or weak NOE correlation between H-3 and H 3 -4a in 9 or 10 indicated a 3E-configuration, compared to the downfield shift values at δ C 21.1 and 22.1 (Table 1) for the C-4a and the strong NOE correlation between H-3 and H 3 -4a in 7 and 8 with a 3Z-configuration. Therefore, the structures of 9 and 10 were elucidated as two previously reported naphthoquinone ansamycins, named hygrocin O (9) and hygrocin P (10). Their 13 C and 1 H NMR data are reported in Tables 1 and 3. one additional aromatic hydrogen signal at δH 7.43 (s) in 9 or δH 7.42 (s) in 10 was observed in their 1 H NMR spectra. However, the 13 C NMR signal at δC 212.9 in 7 or δH 212.0 in 8 for the ketone group at C-13 was replaced in both 9 and 10 by upfield shifted signals at δC 177.7 in 9 or δC 177.8 in 10. Further analyses of their HMQC, COSY and HMBC correlations ( Figure 4) as well as consideration of their molecular formula and 14 degrees of unsaturation required by the molecular formula demonstrated that 9 and 10 were derivatives of 7 and 8, respectively, with ring opening between C-13 and C-14. The chemical shift at δC 16.4 for C-4a and no NOE or weak NOE correlation between H-3 and H3-4a in 9 or 10 indicated a 3E-configuration, compared to the downfield shift values at δC 21.1 and 22.1 (Table 1) for the C-4a and the strong NOE correlation between H-3 and H3-4a in 7 and 8 with a 3Z-configuration. Therefore, the structures of 9 and 10 were elucidated as two previously reported naphthoquinone ansamycins, named hygrocin O (9) and hygrocin P (10). Their 13 C and 1 H NMR data are reported in Tables 1 and 3.      Figure 4) demonstrated that 11 and 12 were the methyl esters of 9 and 10, respectively. The structures of 11 and 12 were thus identified as two previously undescribed naphthoquinone ansamycins, named hygrocin Q (11) and R (12). The 13 C and 1 H NMR data of 11 and 12 are reported in Tables 1 and 3. It should be noted that 11 and 12 may be the artificial products of methyl esterification of 9 and 10, respectively, originated in the extraction and separation process.
The HRESIMS spectrum of compound 13 gave an ion peak at m/z 508.1975 [M-H] − , corresponding to a molecular formula C 28 H 31 NO 8 , which was the same as those of 9 and 10. Detailed analyses of the 1D-and 2D-NMR spectra of 13 determined that 9 and 13 had the same planar structure and their structural difference was only the different configuration of the C 3 -C 4 double bond. The downfield shift value at δ C 21.1 for C-4a and a strong NOE correlation between H-3 and H 3 -4a ( Figure 5) suggested a 3Z-configuration in 13. The structure of 13 was thus assigned as a previously undescribed naphthoquinone ansamycin, named hygrocin S (13). Its 13 C and 1 H NMR data (Tables 1 and 3) were assigned based on the HMQC, COSY and HMBC correlations ( Figure 5).
Compounds 11 and 12 were also obtained as a red amorphous powder and had same molecular formula C29H33NO8 deduced from their 13 C NMR data and HRESIMS ion peaks at m/z 522.2125 [M-H] − in 11 and 522.2132 [M-H] − in 12, 14 mass units higher than those of 9 and 10. Compared to the 13 C and 1 H NMR data of 9 and 10, both 11 and 12 had additional NMR signals for a methoxy group at δC 52.1 and δH 3.57 (3H, s) in 11 and δC 52.2 and δH 3.65 (3H, s) in 12. A HMBC correlation of H3-24 (δH 3.57) with C-13 (δC 176.0) in 11 and H3-24 (δH 3.65) with C-13 (δC 176.0) in 12 established the position of the methoxy group. Further analyses of their HMQC, COSY, HMBC and NOE correlations (Figure 4) demonstrated that 11 and 12 were the methyl esters of 9 and 10, respectively. The structures of 11 and 12 were thus identified as two previously undescribed naphthoquinone ansamycins, named hygrocin Q (11) and R (12). The 13 C and 1 H NMR data of 11 and 12 are reported in Tables 1 and 3. It should be noted that 11 and 12 may be the artificial products of methyl esterification of 9 and 10, respectively, originated in the extraction and separation process.
The HRESIMS spectrum of compound 13 gave an ion peak at m/z 508.1975 [M-H] − , corresponding to a molecular formula C28H31NO8, which was the same as those of 9 and 10. Detailed analyses of the 1D-and 2D-NMR spectra of 13 determined that 9 and 13 had the same planar structure and their structural difference was only the different configuration of the C3-C4 double bond. The downfield shift value at δC 21.1 for C-4a and a strong NOE correlation between H-3 and H3-4a ( Figure 5) suggested a 3Z-configuration in 13. The structure of 13 was thus assigned as a previously undescribed naphthoquinone ansamycin, named hygrocin S (13). Its 13 C and 1 H NMR data (Tables 1 and 3) were assigned based on the HMQC, COSY and HMBC correlations ( Figure 5). Compound 17 was obtained as a yellow amorphous powder and its molecular formula C27H31NO7 was determined based on the HRESIMS ion peak at m/z 480.2024 [M-H] − and 13 C NMR data. Interpretation of the 1 H, 13 C and HMQC NMR spectra of 17 indicated that its twenty-seven carbons (Table 4) were assigned to four carbonyls, six pairs of double bonds, two oxymethines, one methine, four methylenes and four methyls. These carbon types of 17 were very similar to those of hygrocin B (15). Compared to 15, the NMR spectra of 17 showed additional signals for one non-protonated olefinic carbon and one methylene group at δC 37.7 and δH 2.90 (2H, d, J = 7.2 Hz) ( Table 4) and lacked the signals for one carbonyl carbon, one protonated olefinic carbon and the non-protonated carbon at δC 52.6 (C-4) (Table S7), which were observed in the NMR spectra of 15. Further analyses of the HMQC, COSY and HMBC correlations ( Figure 5) of 17 indicated that 17 had a C3-C4 double bond, but did not have the lactone structure existed in 15. Therefore, compound 17 was a seco-derivative of 15. A shared biogenesis suggested that 17 and 15 should have the same 14S, 17S, 18S-configuration. Analyses described above resulted in the Compound 17 was obtained as a yellow amorphous powder and its molecular formula C 27 H 31 NO 7 was determined based on the HRESIMS ion peak at m/z 480.2024 [M-H] − and 13 C NMR data. Interpretation of the 1 H, 13 C and HMQC NMR spectra of 17 indicated that its twenty-seven carbons (Table 4) were assigned to four carbonyls, six pairs of double bonds, two oxymethines, one methine, four methylenes and four methyls. These carbon types of 17 were very similar to those of hygrocin B (15). Compared to 15, the NMR spectra of 17 showed additional signals for one non-protonated olefinic carbon and one methylene group at δ C 37.7 and δ H 2.90 (2H, d, J = 7.2 Hz) ( Table 4) and lacked the signals for one carbonyl carbon, one protonated olefinic carbon and the non-protonated carbon at δ C 52.6 (C-4) (Table S7), which were observed in the NMR spectra of 15. Further analyses of the HMQC, COSY and HMBC correlations ( Figure 5) of 17 indicated that 17 had a C 3 -C 4 double bond, but did not have the lactone structure existed in 15. Therefore, compound 17 was a seco-derivative of 15. A shared biogenesis suggested that 17 and 15 should have the same 14S, 17S, 18S-configuration. Analyses described above resulted in the identification of 17 as a previously undescribed naphthoquinone ansamycin, named hygrocin T. Its 13 C and 1 H NMR data (Table 4) were assigned based on the HMQC, COSY and HMBC correlations ( Figure 5). Compound 18 was obtained as a yellow amorphous powder and its HRESIMS gave an ion peak at m/z [M-H] − 282.0770, corresponding to a molecular formula C 16 H 13 NO 4 with eleven degrees of unsaturation. Based on the analyses of its 1 H, 13 C, DEPT and HMQC NMR spectra, the sixteen carbons were assigned to three carbonyls (δ C 184. 2, 177.4, 169.3), five pairs of double bonds, one methylene (δ C 36.4) and two methyl groups (δ C 20.8, 16.3) ( Table 5). The three carbonyl and five pairs of double bonds accounted for eight out of the eleven degrees of unsaturation required by the molecular formula, suggesting that 18 had a structure with three rings. Above evidence, together with further analyses of its COSY and HMBC correlations (Figure 5), demonstrated that the core structure of 18 was similar to that of 17 with the only difference being the absence in 18 of the side chain attached. Therefore, the structure of 18 was elucidated as a previously unreported naphthoquinone ansamycin, named hygrocin U. The 13 C and 1 H NMR data (Table 5) assignment of 18 was made based on the HMQC, COSY and HMBC correlations ( Figure 5).  13 C NMR data. The twelve carbons in 23 were assigned to one carbonyl (δ C 176.0), eight olefinic carbons, one oxymethine (δ C 71.9), one methylene (δ C 37.5) and one methyl group (δ C 18.7). COSY correlations ( Figure 6) of H-11 (δ H 6.09, 1H, m) with H- 10 (δ H 6.74,1H,dd,15.8,1.8 Hz) and H [3][4][5][6][7][8][9][10][11][12]3H,dd,6.5,1.8 Hz) as well as HMBC correlations (Figure 6) of H-12 with C-10 (δ C 128.6) and C-11 (δ C 126.7) indicated the existence of a 1-propen-1-yl group. Similarly, a "-CH 2 -CH(OH)-CO-" structural fragment was established based on the COSY correlations of 1H,ddd,9.3,6.2,1H,dd,14.1,3.6 Hz;2.64,1H,dd,14.1,9.3 Hz) and 1H,d,6.2 Hz) as well as HMBC correlations of H-7 with C-9 (δ C 176.0) and OH-8 with C-7 (δ C 37.5), C-8 (δ C 71.9) and C-9. In the downfield area (δ H 6.09-7.60) of the 1 H NMR spectrum of 23, there were signals for six olefinic protons. The 1-propen-1-yl group accounted for two olefinic protons and two olefinic carbons and the remaining four olefinic protons and six olefinic carbons were assigned to an aromatic ring. HMBC correlations of H-10 with C-1 (δ C 135.6), C-2 (δ C 136.6) and C-3 (δ C 125.3) and H- 3 (δ H 7.41, 1H, d, 7.5 Hz) with C-10 established the linkage of the 1-propen-1-yl at C-2. In the same way, the positioning of the "-CH 2 -CH(OH)-CO-" group at C-1 was indicated by HMBC correlations of H-6 (δ H 7.15, 1H, m) with C-7, H-7 with C-2 and C-6 (δ C 130.7) and H-8 with C-1. In addition, the 1 H NMR spectrum of 23 showed two noncarbonated proton signals at δ H 7.25 (1H, s) and 7.16 (1H, s), which were assigned to NH 2 -9. The HRESIMS data also supported a -NH 2 group at C-9, rather than a -OH group. A trans-coupling constant value of 15.8 Hz ( 3 J H10/H11 ) indicated a 10E-configuration, while the absolute configuration at C-8 was determined based on the results ( Figure 6 and Tables S16-S19) from ECD calculations. The ECD spectrum of 23 displayed positive and negative Cotton effects at 215 and 244 nm, respectively, which closely matched those of the ECD curve calculated for 8R-23. Based on the foregoing evidence, the structure of 23 was identified as a previously undescribed phenylpropanamide analogue, named streptophenylpropanamide A. Its 13 C and 1 H NMR data are reported in Table 5.
H-8 with C-1. In addition, the 1 H NMR spectrum of 23 showed two noncarbonated proton signals at δH 7.25 (1H, s) and 7.16 (1H, s), which were assigned to NH2-9. The HRESIMS data also supported a -NH2 group at C-9, rather than a -OH group. A trans-coupling constant value of 15.8 Hz ( 3 JH10/H11) indicated a 10E-configuration, while the absolute configuration at C-8 was determined based on the results (Figure 6 and Tables S16-S19) from ECD calculations. The ECD spectrum of 23 displayed positive and negative Cotton effects at 215 and 244 nm, respectively, which closely matched those of the ECD curve calculated for 8R-23. Based on the foregoing evidence, the structure of 23 was identified as a previously undescribed phenylpropanamide analogue, named streptophenylpropanamide A. Its 13 C and 1 H NMR data are reported in Table 5.

Biological Activity Evaluation
Sulforhodamine B (SRB) assay was applied to determine the activity of all thirty isolated compounds (1-30) against the proliferation of glioma cells. Doxorubicin was used as a positive control. The results ( Table 6)

Biological Activity Evaluation
Sulforhodamine B (SRB) assay was applied to determine the activity of all thirty isolated compounds (1-30) against the proliferation of glioma cells. Doxorubicin was used as a positive control. The results ( Table 6) indicated that compounds 1, 2, 4, 8, 11, 12, 22 and 30 showed potent antiproliferative activity against both glioma U87MG and U251 cells with IC 50 values ranging from 0.16 to 10.46 µM. Compounds 27 and 28 also had activity in inhibiting the proliferation of glioma U87MG and U251 cells with IC 50 values of 11.18 and 19.39 µM, respectively. Among all the active compounds, hygrocin C (1) showed the strongest activity (IC 50 : 0.16 and 0.35 µM), followed by hygrocin D (2) (IC 50 : 0.39 and 2.63 µM). Compounds 1-18 were eighteen naphthoquinone ansamycins. It was noted that the active ring closed compounds hygrocins C (1), D (2) and F (4) had a 3Z-configuration, compared to the inactive ring closed compounds hygrocins E (3), K (5) and L (6) with a 3Econfiguration. However, although both hygrocins M (7) and N (8) had the 3Z-configuration, they exhibited significantly different activities due to the different positioning of the ring closure at the C-6 or C-7 position. In addition, most of the ring open compounds hygrocins O (9), P (10), S (13) and T (17) and degrahygrocin A (14) were inactive. However, the ring open compounds hygrocins Q (11) and R (12), the methyl esters of hygrocins O (9) and P (10), respectively, were active. These analyses of the structure-activity relationship indicated that a small change in the structure of this class of compounds had significant influence on their antiglioma activities. The activity of compounds 1-30 in inhibiting the growth of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli was also evaluated. The results ( Table 6) showed that compounds 8, 9,12,17,18,21,22 and 26-28 exhibited antibacterial activity against both MRSA and E. coli with MIC values of 3-48 µg/mL.

Isolation and Taxonomic Identity of Streptomyces sp. ZZ1956
The Streptomyces sp. ZZ1956 strain was isolated from a marine mud sample collected from the mangrove area (4.15 • S, 119.61 • E) of Pangkep District South Sulawesi Province, Indonesia in September 2018. Briefly, the sample (1.0 g) was suspended in sterile water to make dilutions of 10 −2 , 10 −3 and 10 −4 g/mL. Each dilution of 200 µL was spread over the surface of solid Gauze's medium in a Petri dish and then incubated for 10 days at 28 • C. The single ZZ1956 colony from a Petri dish with the 10 −2 g/mL dilution was transferred to a Gauze's agar plate. After growth for another 7 days at 28 • C, the pure strain ZZ1956 colony ( Figure S2) was transferred onto Gauze s agar slants and stored at 4 • C for later use. The 16S rDNA sequence analysis of the strain ZZ1956 was conducted by Legenomics (Hangzhou, China). The 16S rDNA sequence of the strain ZZ1956 was deposited in GenBank with an accession number of MT672495. The voucher strain of Streptomyces sp. ZZ1956 was preserved at the Laboratory of the Institute of Marine Biology and Pharmacology, Ocean College, Zhoushan campus, Zhejiang University, Zhoushan, China.

Mass Culture of the Strain ZZ1956
Colonies of the strain ZZ1956 from the Gauze's agar plate were inoculated into 500 mL Erlenmeyer flasks, each containing 250 mL of sterile GYM liquid medium and then incubated at 28 • C for 3 days on a rotary shaker (180 rpm) to prepare the seed broth. The seed broth (10 mL) was then transferred into a 500 mL Erlenmeyer flask containing 250 mL sterilized GYM liquid medium. A total of 60 L (240 bottles) of culture was prepared for this study and incubated at 28 • C for 15 days under shaking (180 rpm) condition.

Extraction and Isolation of Compounds 1-30
The 60-L culture of strain ZZ1956 was centrifuged to yield supernatant and mycelia. The mycelia were extracted with MeOH three times (3 L, each time) to give a MeOH extract solution. The supernatant was applied to a Dianion HP-20 column eluted with water and then MeOH to obtain a MeOH elution. The MeOH extract solution and MeOH elution were combined and dried in vacuo to give a crude extract, which was further partitioned with EtOAc three times to give an EtOAc extract (24 g

MTPA Esterification Hygrocin N (8)
Hygrocin N (8, 3 mg) was dissolved in 2 mL anhydrous pyridine. Half of the sample solution was added either (R)-or (S)-α-methoxy-α-(trifluoromethyl)-phenylacetyl chloride (MTPA-Cl, 45 µL). The mixture was stirred at room temperature for 24 h and then added MeOH (0.5 mL) to stop the reaction. The reaction products were separated by HPLC using column B with a flow rate of 1 mL/min and UV detection of 210 nm to furnish (S)-MTPA ester 8s (1.0 mg, t R

ECD Calculations
The details of ECD calculations for compound 23 were described as our previous publications [19,20].

Sulforhodamine B (SRB) Assay
The culture of human glioma cells and the SRB assay were according to our previous reports [19,33].

Antibacterail Activity Determination
The antibacterial activity of the tested compounds against MRSA and E. coli was evaluated by the micro broth dilution method [34] using vancomycin and gentamicin as positive controls and DMSO as a negative control.