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

Abstract

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.

1. Introduction

Compound 3-amino-5-hydroxy benzoic acid (3,5-AHBA) 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].

While most of the ansamycins were isolated from actinomycetes, the class of maytansines was also found in higher plants and mosses [3,4]. However, the first maytansines found in plants [4] are now known to be produced by the interplay amongst bacteria in the root system [5]. It was reported that nearly 300 ansamycins have been identified from natural sources [6] and more and more ansamycins continue to be reported, such as the recently described olimycins from the ovmO-inactivated mutant strain Streptomyces olivaceus SCSIO T05 [7] and ansaseomycins from a heterologous mutant strain of Streptomyces seoulensis [8]. The ansamycins exhibit diverse biological activities, such as antibacterial (naphthomycin A, rifamycin, rubradirin and streptovaricin A), anticancer (ansamitocins P-3, ansatrienins A, geldanamycin, mitomycin C, saliniketals A and B), lipoxygenase inhibitory (tetrapetalones A and B) and antiviral (divergolide O) activities [1,9]. The well-known drugs of the ansamycin family were the first-line anti-tuberculous drug rifamycin and the antibody–drug conjugate Kadcyla (emtansine).

As part of an ongoing project to discover novel antiglioma natural products from marine microorganisms [10,11,12,13,14,15,16,17,18,19,20], we isolated an actinomycete from a sediment sample collected from an intertidal mangrove area at the Pacific Ocean close to South Sulawesi, Indonesia. This actinomycete was assigned as Streptomyces sp. ZZ1956 based on its 16S rDNA sequence analysis (Figure S1 and Table S1). An extract prepared from the culture of the strain ZZ1956 in GYM liquid medium exhibited inhibitory activity against the proliferation of glioma U251 and U87MG cells with inhibition rates of over 90%. Chemical investigation of this active extract resulted in the isolation and identification of thirty compounds 1–30, including eleven new naphthalenic ansamycin analogues hygrocins K–U (5–13, 17, 18) and one new phenylpropanamide derivative streptophenylpropanamide A (23) (Figure 1). Herein, we described the isolation and culture of the strain ZZ1956 as well as the isolation, structure elucidation and bioactive evaluation of these isolated compounds.

2. Results and Discussion

2.1. Structure Elucidation of the Isolated Compounds

After analyses of the NMR spectroscopic data and comparison with related data of references, eighteen known compounds were elucidated to be hygrocins C–F (1–4) [21], degrahygrocin A (14) [22], hygrocin B (15) [22], hygrocin G (16) [21], benzoxazolone (19) [23], coixol (20) [24], 2-amino-6-hydroxy-7-methyl-1,4-naphthoquinone (21) [25], 2-acetamide-6-hydroxy-7-methyl-1,4-naphthoquinone (22) [26], 1H-isoindole-1,3(2H)-dione (24) [27], 3-(3′-amino-3′-oxoprop-1′-en-2′-yl)oxy benzamide (25) [28], 3′-methoxy(1,1′,4′,1″-terphenyl)-2′,6′-diol (26) [29], echoside C (27) [30], echoside A (28) [30], pteridic acid hydrate (29) [31] and 11,11′-O-dimethylelaiophylin (30) [32]. The structure of hygrocin C (1) was confirmed by single crystal X-ray diffraction (Table S2). Degrahygrocin A (14) was previously reported as an alkaline hydrolytic product of hygrocin A [22]; while 2-acetamide-6-hydroxy-7-methyl-1,4-naphthoquinone (22) was an intermediate compound of chemical synthesis of ansalactam A [26]. Therefore, both compounds 14 and 22 are reported as natural products for the first time. The NMR data of these known compounds are presented in Tables S3–S14.

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 ¹H, ¹³C, 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 2R-configuration 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 (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 H₃-4a also supported the assignment of the 3E- or 3Z-configuration. In addition, the trans-coupling constant value of 16.0 Hz (³J_H8/H9) indicated an 8E-configuration and the small vicinal coupling constant value of 2.8 Hz (³J_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 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 ¹³C and ¹H NMR data are reported in

Table 1. ¹³C NMR data of compounds 5–13 (150 MHz, in MeOH-d₄, δ_C).

No.	5	6	7	8	9	10	11	12	13
1	177.3, C	177.1, 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, C	137.6 a, C	137.7 a, C	137.6 a, C	137.7 a, C	138.3, C
4a	13.7, CH3	13.6, CH3	21.1, CH3	22.1, CH3	16.4, CH3	16.4, CH3	16.4, CH3	16.4, CH3	21.1, CH3
5	167.7, C	167.3, C	167.6, C	168.3, C	168.1, C	167.9, C	168.1, C	167.9, C	168.5, C
6	75.3, CH	69.5, CH	74.7, CH	68.5, CH	75.6, CH	69.9, CH	75.5, CH	69.9, CH	75.2, CH
6a	15.2, CH3	19.1, CH3	13.5, CH3	19.1, CH3	16.4, CH3	19.6, CH3	16.2, CH3	19.6, CH3	15.0, CH3
7	74.2, CH	77.9, CH	71.3, CH	80.6, CH	75.4, CH	81.4, CH	75.3, CH	81.2, CH	74.4, CH
8	128.2, CH	125.3, CH	128.7, CH	124.4, CH	131.5, CH	127.9, CH	131.5, CH	127.9, CH	130.7, CH
9	138.3, CH	136.5, CH	137.3, CH	139.9, CH	138.3, CH	141.3, CH	138.1, CH	140.9, CH	137.9, CH
10	41.9, CH	41.9, CH	43.8, CH	43.4, CH	45.6, CH	45.8, CH	45.6, CH	45.8, CH	45.5, CH
10a	26.3, CH2	26.6, CH2	26.6, CH2	29.2, CH2	29.2, CH2	29.1, CH2	29.2, CH2	29.1, CH2	29.1, CH2
10b	10.6, CH3	12.4, CH3	12.7, CH3	11.5, CH3	12.3, CH3	12.3, CH3	12.3, CH3	12.3, CH3	12.2, CH3
11	29.2, CH2	33.0, CH2	32.1, CH2	28.9, CH2	31.1, CH2	31.2, CH2	31.0, CH2	31.0, CH2	30.9, CH2
12	41.8, CH2	42.2, CH2	39.4, CH2	42.5, CH2	33.0, CH2	33.2, CH2	33.0, CH2	33.0, CH2	33.0, CH2
13	212.0, C	211.2, C	212.9, C	212.0, C	177.7, C	177.8, C	176.0, C	176.0, C	177.7, C
14	130.1, C	127.2, C	129.9, C	129.2, C	113.9, CH	113.9, CH	113.9, CH	113.9, CH	113.7, CH
15	153.0, C	153.7, C	158.7, C	158.0, C	159.8, C	159.8, C	159.9, C	159.9, C	159.5, C
16	133.1 a, C	133.4, C	133.3, C	133.7, C	131.9 b, C	131.9, C	131.9, C	131.9, C	132.2, C
16a	17.0, CH3	16.9, CH3	17.3, CH3	17.2, CH3	16.8, CH3	16.8, CH3	16.8, CH3	16.8, CH3	16.7, CH3
17	131.5, CH	132.1, CH	131.3, CH	130.7, CH	131.7 b, CH	131.6, CH	131.6, CH	131.6, CH	131.7, CH
18	133.0 a, C	134.0, C	131.2, C	130.1, C	122.8, C	122.8, C	122.7, C	122.8, C	123.2, C
19	75.4, C	75.8, C	134.8, C	136.4, C	137.4 a, C	137.4 a, C	137.4 a, C	137.4 a, C	139.6, C
20	164.6, C	164.5, C	154.8, C	155.1, C	154.0, C	154.0, C	154.0, C	154.0, C	154.5, C
21	105.1, CH	105.3 CH	106.0, CH	105.8, CH	106.3, CH	106.4, CH	106.3, CH	106.4, CH	106.0, CH
22	185.6, C	185.9, C	187.4, C	187.0, C	186.5, C	186.4, C	186.4, C	186.4, C	186.7, C
23	129.7, C	131.1, C	129.3, C	128.4, C	131.9 b, C	131.9, C	131.9, C	131.9, C	131.7, C
24	-	-	-	-	-	-	52.1, CH3	52.2, CH3	-

^a,b The data with the same labels in each column may be interchanged.

and

Table 2. ¹H NMR data of compounds 5–8 (600 MHz, in MeOH-d₄, δ_H, multi., J in Hz).

No.	5	6	7	8
2	4.06, d (11.1)	4.04, d (11.1)	-	-
3	6.09, dd (11.1, 1.8)	6.10, dd (11.1, 1.4)	6.90, s	6,81, s
4a	2.06, d (1.8)	2.03, s	2.21, s	2.19, s
6	4.65, m	3.67, m	4.87 a, m	3.69, m
6a	1.15, d (6.4)	1.02, d (6.7)	0.91, d (6.0)	0.95, d (6.4)
7	3.90, dd (6.8, 2.8)	5.11, t (5.0)	3.86, m	4.97, t (5.8)
8	4.92, dd (16.0, 6.8)	5.26, dd (16.1, 5.0)	4.59, d (15.1)	4.41, dd (15.6, 6.6)
9	5.50, dd (16.0, 5.5)	5.06, dd (16.1, 6.4)	5.30, dd (15.1, 9.2)	5.36, dd (15.6, 5.8)
10	1.84, m	1.69, m	1.55, m	1.56, m
10a	1.49, m; 1.23, m	1.32, m	1.48, m; 0.92, m	1.30, m; 1.14, m
10b	0.71, t (7.3)	0.82, t (7.4)	0.69, t (7.2)	0.76, t (7.2)
11	1.45, m; 1.19, m	1.55, m; 1.38, m	1.28, m; 1.18, m	1.53, m; 1.35, m
12	2.71, m; 2.59, m	2.67, m; 2.49, m	2.75, d (11.6)	2.81, dd (16.8, 8.5); 2.43, dd (16.8, 10.0)
16a	2.29, s	2.27, s	2.21, s	2.15, s
17	7.22, s	7.28, s	7.57, s	7.65, s
21	5.87, s	5.93, s	5.85, s	5.81, s

^a The data was overlapped with that of H₂O.

.

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 ¹³C NMR data. Careful analyses of its 1D- and 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 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 ¹³C and ¹H NMR data (Table 1 and Table 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 (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 C₂-C₁₉ double bond. The downfield shift values of C-4a at δ_C 21.1 in 7 or δ_C 22.1 in 8 and the strong NOE correlation between H-3 and H₃-4a (Figure 2 and Figure 3) indicated that they had a 3Z-configuration; while the trans-coupling constant values of 15.1 Hz in 7 and 15.6 Hz in 8 between H-8 and H-9 suggested that both 7 and 8 had an 8E-configuration. HMBC correlations of H-6 (δ_H 4.87) with C-5 (δ_C 167.6) in 7 and H-7 (δ_H 4.97) with C-5 (δ_C 168.3) in 8 established the position of the lactone ring formation. The Mosher’s method was used to determine the configuration at C-6 in 8. The results (Figure 3 and 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 ¹³C and ¹H NMR data (Table 1 and Table 2) of 7 and 8 were assigned based on the HMQC, COSY and HMBC correlations (Figure 2 and Figure 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 ¹³C 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 ¹H NMR spectra. However, the ¹³C 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 H₃-4a 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 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 ¹³C and ¹H NMR data are reported in Table 1 and

Table 3. ¹H NMR data of compounds 9–13 (600 MHz, in MeOH-d₄, δ_H, multi., J in Hz).

No.	9	10	11	12	13
3	7.53, s	7.54, s	7.51, s	7.54, d (1.4)	7.54, s
4a	1.92, s	1.92, s	1.92, s	1.93, d (1.4)	2.24, s
6	5.05, m	3.93, m	5.06, m	3.92, m	4.84, m
6a	1.34, d (6.4)	1.24, d (6.6)	1.33, d (6.4)	1.24, d (6.4)	1.14, d (6.4)
7	4.19, t (6.3)	5.23, t (6.6)	4.19, t (6.1)	5.24, t (6.3)	4.03, t (6.0)
8	5.54, dd (15.5, 6.3)	5.57, m	5.52, dd (15.5, 6.1)	5.56, m	5.30, dd (15.5, 6.0)
9	5.48, dd (15.5, 8.6)	5.57, m	5.45, dd (15.5, 8.6)	5.57, m	5.24, dd (15.5, 8.8)
10	1.95, m	1.95, m	1.90, m	1.96, m	1.71, m
10a	1.43, m; 1.28, m	1.49, m; 1.31, m	1.45, m; 1.26, m	1.48, m; 1.29, m	1.26, m; 1.18, m
10b	0.87, t (7.1)	0.86, t (7.3)	0.86, t (7.2)	0.86, t (7.4)	0.76, t (7.4)
11	1.74, m; 1.46, m	1.76, m; 1.47, m	1.72, m; 1.41, m	1.77, m; 1.52, m	1.57, m; 1.09, m
12	2.30, m; 2.21, m	2.30, m; 2.23, m	2.28, m; 2.24, m	2.30, m; 2.26, m	2.10, m; 2.02, m
14	7.43, s	7.42, s	7.43, s	7.43, s	7.46, s
16a	2.24, s	2.21, s	2.23, s	2.21, s	2.28, s
17	7.42, s	7.39, s	7.40, s	7.40, s	6.83, s
21	5.88, s	5.88, s	5.87, s	5.88, s	5.88, s
24	-	-	3.57, s	3.65, s	-

.

Compounds 11 and 12 were also obtained as a red amorphous powder and had same molecular formula C₂₉H₃₃NO₈ deduced from their ¹³C 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 ¹³C and ¹H NMR data of 9 and 10, both 11 and 12 had additional NMR signals for a methoxy group at δ_C 52.1 and δ_H 3.57 (3H, s) in 11 and δ_C 52.2 and δ_H 3.65 (3H, s) in 12. A HMBC correlation of H₃-24 (δ_H 3.57) with C-13 (δ_C 176.0) in 11 and H₃-24 (δ_H 3.65) with C-13 (δ_C 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 ¹³C and ¹H NMR data of 11 and 12 are reported in Table 1 and Table 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 δ_C 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 ¹³C and ¹H NMR data (Table 1 and Table 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 ¹³C NMR data. Interpretation of the ¹H, ¹³C and HMQC NMR spectra of 17 indicated that its twenty-seven carbons (

Table 4. ¹³C NMR (150 MHz) and ¹H NMR (600 MHz) data of compound 17 (in DMSO-d₆).

No.	δC, Type	δH, Multi. (J in Hz)	No.	δC, Type	δH, Multi. (J in Hz)
1	171.8, C	-	10	180.1, C	-
2	37.7, CH2	2.90, d (7.2)	10a	138.6, C	-
3	125.5, CH	5.83, td (7.2, 1.2)	11	208.5, C	-
4	136.4, C	-	12	42.5, CH2	2.76, m; 2.67, m
4a	130.1, C	-	13	29.7, CH2	1.95, m; 1.67, m
4b	21.4, CH3	2.13, s	14	45.2, CH	2.00, m
5	185.4, C	-	14a	29.2, CH2	1.49, m; 1.28, m
5a	131.8, C	-	14b	12.3, CH3	0.90, t (7.5)
6	124.3, C	-	15	138.6, CH	5.47, dd (15.8, 6.8)
7	158.8, C	-	16	131.3 a, CH	5.43, dd (15.8, 6.3)
8	133.0, C	-	17	78.6, CH	3.76, t (6.3)
8a	17.0, CH3	2.35, s	18	72.0, CH	3.53, m
9	131.3 a, CH	7.92, s	18a	19.4, CH3	1.05, d (6.3)
9a	131.2 a, C	-

^a Interchangeable chemical shifts.

) 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 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 identification of 17 as a previously undescribed naphthoquinone ansamycin, named hygrocin T. Its ¹³C and ¹H 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 C₁₆H₁₃NO₄ with eleven degrees of unsaturation. Based on the analyses of its ¹H, ¹³C, 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. ¹³C NMR (150 MHz) and ¹H NMR (600 MHz) data of compounds 18 and 23 (in DMSO-d₆).

No.	18		No.	23
	δC, Type	δH, Multi. (J in Hz)		δC, Type	δH, Multi. (J in Hz)
1	169.3, C	-	1	135.6, C	-
2	36.4, CH2	2.83, d (7.1)	2	136.6, C	-
3	123.7, CH	5.79, t (7.1)	3	125.3, CH	7.41, d (7.5)
4	134.2, C	-	4	126.4 b, CH	7.13, m
4a	127.8, C	-	5	126.3 b, CH	7.12, m
4b	20.8, CH3	2.09, s	6	130.7, CH	7.15, m
5	184.2, C	-	7	37.5, CH2	3.08, dd (14.1, 3.6); 2.64, dd (14.1, 9.3)
5a	132.9, C	-	8	71.9, CH	3.92, ddd (9.3, 6.2, 3.6)
6	112.1, CH	7.19, s	9	176.0, C	-
7	165.6 a, C	-	10	128.6, CH	6.74, dd (15.8, 1.8)
8	131.1, C	-	11	126.7, CH	6.09, dq (15.8, 6.5)
8a	16.3, CH3	2.19, s	12	18.7, CH3	1.86, dd (6.5, 1.8)
9	129.1, CH	7.75, s	OH-8	-	5.42, d (6.2)
9a	119.6 a, C	-	NH2-9	-	7.25, s; 7.16, s
10	177.4, C	-
10a	137.6, C	-
OH-7	-	9.34, 1H, s

^a The data were observed from HMBC correlations; ^b The data may be interchanged.

). 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 ¹³C and ¹H NMR data (Table 5) assignment of 18 was made based on the HMQC, COSY and HMBC correlations (Figure 5).

The molecular formula C₁₂H₁₅NO₂ of 23 was determined by its HRESIMS ion peaks at m/z 206.1175 [M+H]⁺ and 228.0995 [M+Na]⁺ as well as ¹³C 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 H₃-12 (δ_H 1.86, 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 “-CH₂-CH(OH)-CO-” structural fragment was established based on the COSY correlations of H-8 (δ_H 3.92, 1H, ddd, 9.3, 6.2, 3.6 Hz) with H-7 (δ_H 3.08, 1H, dd, 14.1, 3.6 Hz; 2.64, 1H, dd, 14.1, 9.3 Hz) and OH-8 (δ_H 5.42, 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 ¹H 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 “-CH₂-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 ¹H 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 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 (³J_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 ¹³C and ¹H NMR data are reported in Table 5.

2.2. 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. Antiglioma and antibacterial activities of compounds.

Compounds	Glioma Cells (IC50: μM)		Microorganisms (MIC: μg/mL)
	U87MG	U251	MRSA	Escherichia coli
1	0.16 ± 0.01	0.35 ± 0.01	NA	NA
2	0.39 ± 0.04	2.63 ± 0.47	NA	NA
4	0.57 ± 0.09	7.33 ± 0.20	NA	NA
8	8.17 ± 0.17	7.04 ± 0.28	15	8
9	NA	NA	24	20
11	8.81 ± 0.80	10.46 ± 0.27	NA	NA
12	8.32 ± 0.38	7.86 ± 0.26	9	16
17	NA	NA	44	25
18	34.68 ± 0.58	>50	3	6
21	NA	NA	10	16
22	6.18 ± 0.18	8.13 ± 0.56	3	8
26	NA	NA	5	12
27	11.18 ± 0.92	14.64 ± 1.73	6	28
28	19.39 ± 0.67	13.42 ± 1.71	8	48
30	1.64 ± 0.06	1.35 ± 0.05	NA	NA
Doxorubicin	0.43 ± 0.01	4.18 ± 0.39	NT	NT
Vancomycin	NT	NT	0.25	NT
Gentamicin	NT	NT	0.50	0.25

NA: No activity at a concentration of 50 μM or 50 μg/mL; NT: No testing.

) indicated that compounds 1, 2, 4, 8, 11, 12, 22 and 30 showed potent antiproliferative activity against both glioma U87MG and U251 cells with IC₅₀ 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 IC₅₀ values of 11.18 and 19.39 μM, respectively. Among all the active compounds, hygrocin C (1) showed the strongest activity (IC₅₀: 0.16 and 0.35 μM), followed by hygrocin D (2) (IC₅₀: 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 3E-configuration. 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.

3. Experimental Section

3.1. General Procedures

Optical rotation (OR), ultraviolet–visible (UV), electronic circular dichroism (ECD) and infrared (IR) spectra were recorded on an Autopol I polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA), a METASH UV-8000 spectrometer (Shanghai METASH Instruments Co. Ltd., Shanghai, China), a JASCO J-815 spectropolarimeter (JASCO Co., Tokyo, Japan) and a Nicolet^TM IS^TM 10 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. HRESIMS data were acquired on an Agilent 6230 TOF LC/MS spectrometer (Agilent Technologies Co. Ltd., Santa Clara, CA, USA). NMR spectra were obtained on a JEOL 600 spectrometer (JEOL Co. Ltd., Tokyo, Japan) using standard programs and acquisition parameters and chemical shift values were expressed in δ (ppm) relative to DMSO-d₆ (δ_C 39.5, δ_H 2.50), MeOH-d₄ (δ_C 49.15, δ_H 3.31) or acetone-d₆ (δ_C 29.8, δ_H 2.05). Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), silica gel (100–200 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China), octadecyl-functionalized silica gel (ODS, Cosmosil 75C₁₈-Prep, Nacalai Tesque Inc., Kyoto, Japan) and sephadex LH-20 (GE Healthcare, Waukesha, WI, USA) were used for column chromatography. HPLC separation was performed on a CXTH LC-3000 preparative HPLC system (Beijing Chuangxin Tongheng Science & Technology Co. Ltd., Beijing, China) with column A (CT-30, 280 × 30 mm, 10 µm, Fuji-C₁₈) and an Agilent 1260 infinity HPLC system (Agilent Technologies Co. Ltd., Santa Clara, CA, USA) using Zorbax SB-C₁₈ columns (column B: 250 × 9.4 mm, 5 µm or column C: 250 × 4.6 mm, 5 µm) with a DAD detector. All solvents used for this study were purchased from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Glioma U87MG (JDS-2568) and U251 (XB-0439) cells used in the experiment were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 and Escherichia coli ATCC 25922 were gifts from Dr. Zhongjun Ma and Dr. Pinmei Wang, respectively. Doxorubicin (DOX) was purchased from Solarbio Science & Technology Co. Ltd. (Beijing, China). Vancomycin and gentamicin were ordered from Meilune Biotechnology Co., Ltd. (Dalian, China). Gauze’s agar medium was ordered from the Guangdong Huankai Microbial Science and Technology Co., Ltd. (Guangzhou, China). GYM liquid medium (glucose 4 g, yeast extract 10 g, malt extract 10 g, tap water 1.0 L) was made in the authors’ laboratory.

3.2. 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.

3.3. 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.

3.4. 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). The EtOAc extract was subjected to a column of silica gel eluted with mixtures of cyclohexane/EtOAc (10/1, 8/1, 5/1, 2/1, 1/1, v/v), EtOAc, and MeOH to give ten fractions (Frs. A–J) based on the results of TLC and HPLC analyses.

Fr. A was purified by HPLC using column C (mobile phase: MeCN/H₂O, 65/35; flow rate: 0.8 mL/min; UV detection: 210 nm) to give 26 (4.8 mg, t_R 15.4 min). Fractions B and D were separated on HPLC column B (flow rate: 1 mL/min; UV detection: 210 nm) to give 24 (3.0 mg, t_R 25.0 min, MeOH/H₂O, 40/60) and 22 (4.4 mg, t_R 24.4 min, MeOH/H₂O, 67/37), respectively.

Fr. C was subjected to a sephadex LH-20 column eluted with 70% MeOH to yield three subfractions (Frs. C₁–C₃). Fr. C₁ was further separated on column C (mobile phase: MeOH/H₂O, 34/66; flow rate: 0.8 mL/min; UV detection: 210 nm) to give 19 (1.2 mg, t_R 15.7 min) and 20 (3.8 mg, t_R 18.6 min). Compound 18 (1.7 mg, t_R 25.5 min) was obtained from Fr. C₂ through HPLC purification using column B (mobile phase: MeOH/H₂O, 67/33; flow rate: 1 mL/min; UV detection: 280 nm).

Each of Fr. E, Fr. F and Fr. H was separated by preparative HPLC using column A (flow rate: 10 mL/min; UV detection: 210 nm). Compounds 21 (4.9 mg, t_R 19.5 min), 15 (21.0 mg, t_R 24.3 min) and 16 (7.4 mg, t_R 44.1 min) were obtained from Fr. E using a mobile phase (MeOH/0.1% HOAc in H₂O, 70/30), 14 (8.0 mg, t_R 26.2 min, MeOH/0.1% HOAc in H₂O, 65/35) was purified from Fr. H and five subfractions (Frs. F₁–F₅) were obtained from Fr. F using a gradient mobile phase (MeOH/0.1% HOAc in H₂O, 30/70–100/0) in 40 min. Each of Frs. F₁–F₅ was purified by HPLC column B (flow rate: 1 mL/min; UV detection: 210 nm) to give 1 (28.0 mg, t_R 23.1 min, MeCN/H₂O, 33/67), 7 (3.3 mg, t_R 23.7 min, MeOH/H₂O, 55/45), 3 (6.0 mg, t_R 57.5 min, MeCN/H₂O, 30/70), 2 (19.0 mg, t_R 26.3 min, MeCN/H₂O, 45/55) and 8 (3.3 mg, t_R 33.3 min, MeOH/H₂O, 66/34).

Fr. G was fractionated by an ODS column eluted with 65%, 75% and 100% MeOH to give three subfractions (Frs. G₁–G₃) based on the results of TLC and HPLC analyses. Compound 29 (4.4 mg, t_R 28.2 min) was obtained from Fr. G₂ through HPLC purification using column B (mobile phase: MeCN/0.1% HOAc in H₂O, 30/70; flow rate: 1 mL/min; UV detection: 256 nm). Fr. G₃ was further separated by HPLC column A (mobile phase: MeOH/0.1% HOAc in H₂O, 70/30; flow rate: 10 mL/min; UV detection: 210 nm) to give six subfractions (Frs. G_3a–G_3f). Fr. G_3a continued to be separated on the same column A with the same flow rate and UV detection to give Fr. G_3aa and Fr. G_3ab (MeOH/0.1% HOAc in H₂O, 55/45). Further purification by HPLC column B (flow rate: 1 mL/min; UV detection: 256 nm) yielded compounds 5 (5.9 mg, t_R 48.8 min, MeCN/H₂O, 27/73) from Fr. G_3aa, 6 (2.3 mg, t_R 43.7 min, MeCN/H₂O, 33/67) from Fr. G_3ab, 23 (2.0 mg, t_R 21.9 min, MeOH/0.1% HOAc in H₂O, 65/35) from Fr. G_3b, 4 (16.0 mg, t_R 24.1 min, MeCN/H₂O, 45/55) from Fr. G_3c, 13 (3.0 mg, t_R 32.3 min) and 17 (8.0 mg, t_R 37.1 min, MeCN/0.1% HOAc in H₂O, 39/61) from Fr. G_3d, 9 (2.8 mg, t_R 30.0 min), 10 (2.8 mg, t_R 38.5 min), 12 (2.5 mg, t_R 64.2 min) and 11 (2.2 mg, t_R 77.1 min, MeOH/0.1% HOAc in H₂O, 70/30) from Fr. G_3f.

Similarly, Fr. I was also applied to an ODS column eluted with 50%, 70% and 100% MeOH to yield three subfractions (Frs. I₁–I₃). Fr. I₁ was further separated on HPLC column A (flow rate: 10 mL/min; UV detection: 210 nm) with a gradient mobile phase from 40% to 100% MeOH in 40 min to give 25 (2.0 mg, t_R 15.8 min); while compound 30 (20.0 mg, t_R 32.4 min) was obtained from Fr. I₃ by separating on column B (mobile phase: MeOH/H₂O, 95/5; flow rate: 1 mL/min; UV detection: 256 nm).

Finally, Fr. J was fractionated on an ODS column eluted with 30%, 40%, 60%, 70% and 100% MeOH to give Fr. J₁ and Fr. J₂ based on the results of HPLC analyses. Fr. J₁ was further separated on column A (flow rate: 10 mL/min; UV detection: 210 nm) with a gradient mobile phase from 40% to 100% MeOH in 40 min to give Fr. J_1a and Fr. J_1b. Compounds 27 (9.0 mg, t_R 24.3 min) and 28 (2.0 mg, t_R 42.3 min) were obtained from Fr. J_1a through HPLC purification using column B (mobile phase: ACN/H₂O, 33/67; flow rate: 1 mL/min; UV detection: 210 nm).

3.5. Compound Characterization Data

Hygrocin K (5): Light yellow oil; molecular formula C₂₈H₃₁NO₈; [α]_D²⁰ –65.5 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 203 (4.39), 271 (4.04), 304 (4.00) nm; IR (ATR) ν_max 3320, 2962, 2929, 2870, 1662, 1627, 1569, 1322, 1237, 1190, 1131, 1051, 976, 857, 733 cm⁻¹; ¹³C NMR data (150 MHz), Table 1, ¹H NMR data (600 MHz), Table 2; HRESIMS m/z 508.1969 [M–H]⁻ (calcd for C₂₈H₃₀NO₈⁻, 508.1971).

Hygrocin L (6): Light yellow oil; molecular formula C₂₈H₃₁NO₈; [α]_D²⁰ +166.6 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 203 (4.21), 270 (4.04), 304 (4.03) nm; IR (ATR) ν_max 3314, 2963, 2928, 2874, 1698, 1657, 1627, 1567, 1239, 1188, 1108, 1029, 976, 857, 810, 735 cm⁻¹; ¹³C NMR data (150 MHz), Table 1, ¹H NMR data (600 MHz), Table 2; HRESIMS m/z 508.1979 [M–H]⁻ (calcd for C₂₈H₃₀NO₈⁻, 508.1971).

Hygrocin M (7): Light yellow amorphous powder; molecular formula C₂₈H₂₉NO₇; [α]_D²⁰ +166.7 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 201 (4.52), 333 (4.11) nm; IR (ATR) ν_max 3362, 2960, 2930, 2868,1714, 1655, 1626, 1559, 1446, 1346, 1285, 1134, 1044, 860 cm⁻¹; ¹³C NMR data (150 MHz), Table 1, ¹H NMR data (600 MHz), Table 2; HRESIMS m/z 490.1875 [M–H]⁻ (calcd for C₂₈H₂₈NO₇⁻, 490.1866).

Hygrocin N (8): Light yellow amorphous powder; molecular formula C₂₈H₂₉NO₇; [α]_D²⁰ +270.0 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 201 (4.55), 336 (4.26) nm; IR (ATR) ν_max 3275, 2963, 2927, 2877, 1709, 1650, 1617, 1567, 1466, 1338, 1282, 1249, 1132, 1079, 859, 736 cm⁻¹; ¹³C NMR data (150 MHz), Table 1, ¹H NMR data (600 MHz), Table 2; HRESIMS m/z 492.2010 [M+H]⁺ (calcd for C₂₈H₃₀NO₇, 492.2022), 514.1832 [M+Na]⁺ (calcd for C₂₈H₂₉NNaO₇, 514.1842) and 490.1871 [M–H]⁻ (calcd for C₂₈H₂₈NO₇⁻, 490.1866).

Hygrocin O (9): Red amorphous powder; molecular formula C₂₈H₃₁NO₈; [α]_D²⁰ –21.5 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 201 (4.65), 334 (4.33) nm; IR (ATR) ν_max 3257, 2959, 2926, 2872, 1697, 1652, 1600, 1580, 1343, 1257, 1204, 1135, 1022, 978 cm⁻¹; ¹³C NMR data (150 MHz), Table 1, ¹H NMR data (600 MHz), Table 3; HRESIMS m/z 508.1975 [M–H]⁻ (calcd for C₂₈H₃₀NO₈⁻, 508.1971).

Hygrocin P (10): Red amorphous powder; molecular formula C₂₈H₃₁NO₈; [α]_D²⁰ –55.5 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 201 (4.29), 334 (4.07) nm; IR (ATR) ν_max 3229, 2966, 2926, 2875, 1705, 1651, 1622, 1576, 1467, 1339, 1263, 1245, 1120, 1063, 1018, 976, 855, 737 cm⁻¹; ¹³C NMR data (150 MHz), Table 1, ¹H NMR data (600 MHz), Table 3; HRESIMS m/z 508.1974 [M–H]⁻ (calcd for C₂₈H₃₀NO₈⁻, 508.1971).

Hygrocin Q (11): Red amorphous powder; molecular formula C₂₉H₃₃NO₈; [α]_D²⁰ –16.0 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 201 (4.22), 335 (4.10) nm; IR (ATR) ν_max 3345, 2960, 2925, 2875, 1714, 1655, 1629, 1598, 1573, 1435, 1349, 1263, 1188, 1149, 1020, 978, 851, 755 cm⁻¹; ¹³C NMR data (150 MHz), Table 1, ¹H NMR data (600 MHz), Table 3; HRESIMS m/z 522.2125 [M–H]⁻ (calcd for C₂₉H₃₂NO₈⁻, 522.2128).

Hygrocin R (12): Red amorphous powder; molecular formula C₂₉H₃₃NO₈; [α]_D²⁰ –40.0 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 201 (4.14), 335 (4.09) nm; IR (ATR) ν_max 3376, 2959, 2927, 2872, 1696, 1653, 1596, 1578, 1441, 1339, 1261, 1202, 1136, 1064, 976, 851, 802, 728 cm⁻¹; ¹³C NMR data (150 MHz), Table 1, ¹H NMR data (600 MHz), Table 3; HRESIMS m/z 522.2132 [M–H]⁻ (calcd for C₂₉H₃₂NO₈⁻, 522.2128).

Hygrocin S (13): Red amorphous powder; molecular formula C₂₈H₃₁NO₈; [α]_D²⁰ –21.6 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 201 (4.13), 333 (4.06) nm; IR (ATR) ν_max 3240, 2962, 2870, 1709, 1655, 1574, 1350, 1222, 1130, 1068, 976, 855 cm⁻¹; ¹³C NMR data (150 MHz), Table 1, ¹H NMR data (600 MHz), Table 3; HRESIMS m/z 508.1975 [M–H]⁻ (calcd for C₂₈H₃₀NO₈⁻, 508.1971).

Hygrocin T (17): Yellow amorphous powder; molecular formula C₂₇H₃₁NO₇; [α]_D²⁰ –34.0 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 204 (4.35), 211 (4.35), 279 (4.36), 308 (4.10) nm; IR (ATR) ν_max 3324, 2961, 2925, 2874, 1694, 1655, 1567, 1457, 1339, 1261, 1188, 1151, 1012, 973, 806, 735 cm⁻¹; ¹³C NMR (150 MHz) and ¹H NMR (600 MHz) data, Table 4; HRESIMS m/z 480.2024 [M–H]⁻ (calcd for C₂₇H₃₀NO₇⁻, 480.2022).

Hygrocin U (18): Light yellow amorphous powder; molecular formula C₁₆H₁₃NO₄; UV (MeOH) λ_max (log ε) 201 (4.21), 211 (4.12), 280 (4.17), 307 (3.81) nm; IR (ATR) ν_max 3319, 2956, 2920, 2849, 1674, 1651, 1567, 1466, 1327, 1265, 1206, 1146 cm⁻¹; ¹³C NMR (150 MHz) and ¹H NMR (600 MHz) data, Table 5; HRESIMS m/z 282.0770 [M–H]⁻ (calcd for C₁₆H₁₂NO₄⁻, 282.0766).

Streptobenzenepropanamide A (23): White amorphous powder; molecular formula C₁₂H₁₅NO₂; [α]_D²⁰ +12.4 (c 0.10, MeOH); ECD (15 mg/L, MeOH) λ_max (Δε) 215 (+20.14), 244 (–6.93) nm; UV (MeOH) λ_max (log ε) 202 (4.16), 248 (3.91) nm; IR (ATR) ν_max 3333, 2963, 2925, 2852, 1665, 1578, 1447, 1406, 1380, 1261, 1094, 1074, 965, 802, 751 cm⁻¹; ¹³C NMR (150 MHz) and ¹H NMR (600 MHz) data, Table 5; HRESIMS m/z 206.1175 [M+H]⁺ (calcd for C₁₂H₁₆NO₂⁺, 206.1181) and 228.0995 [M+Na]⁺ (calcd for C₁₂H₁₅NNaO₂⁺, 228.1000).

3.6. 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 22.5 min, MeOH/H₂O, 92/8) or (R)-MTPA ester 8r (1.2 mg, t_R 22.5 min, MeOH/H₂O, 92/8).

(S)-MTPA ester 8s: ¹H NMR data (600 MHz, in MeOH-d₄), Table S15; HRESIMS m/z 924.2815 [M+H]⁺ (calcd for C₄₈H₄₄F₆NO₁₁⁺, 924.2819) and 946.2640 [M+Na]⁺ (calcd for C₄₈H₄₃F₆NNaO₁₁⁺, 946.2638).

(R)-MTPA ester 8r: ¹H NMR data (600 MHz, in MeOH-d₄), Table S15; HRESIMS m/z 924.2824 [M+H]⁺ (calcd for C₄₈H₄₄F₆NO₁₁⁺, 924.2819) and 946.2628 [M+Na]⁺ (calcd for C₄₈H₄₃F₆NNaO₁₁⁺, 946.2638).

3.7. ECD Calculations

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

3.8. Sulforhodamine B (SRB) Assay

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

3.9. 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.

4. Conclusions

Marine-derived actinomycetes from the genus Streptomyces continue to be one of the main resources for the discovery of novel bioactive natural products. A chemical investigation of the extract prepared from a scaled-up culture of the marine-derived actinomycete Streptomyces sp. ZZ1956 in GYM liquid medium resulted in the isolation and identification of thirty compounds (1–30), including twelve previously undescribed compounds, namely, hygrocins K–U (5–13, 17, 18) and streptophenylpropanamide A (23). Compounds 1–18 were naphthalenic ansamycin derivatives and a small change in their structures significantly influenced their antiglioma activity. Hygrocins C (1), D (2) and F (4) structurally characterized with ring closing and 3Z-configuration exhibited potent antiproliferative activity against both human glioma U87MG and U251 cells. 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) exhibited antibacterial activity against MRSA and E. coli. The data from this study greatly enrich the chemical and bioactive diversities of the ansamycin antibiotics.