Bioactive Indolocarbazoles from the Marine-Derived Streptomyces sp

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Bioactive Indolocarbazoles from the Marine-Derived Streptomyces sp. DT-A61 Jia-Nan Wang, Hao-Jian Zhang, Jia-Qi Li, Wan-Jing Ding, and Zhong-Jun Ma* Institute of Marine Biology, Ocean College, Zhejiang University, No. 1 Zheda Road, Zhoushan 316021, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new indolocarbazoles (1−9) were isolated from the marine-derived Streptomyces sp. DT-A61. Among them compounds 1−8 featured a hydroxy group at the C-3 or C-9 position. All purified compounds were identified by 1D and 2D NMR and HRESIMS data. The absolute configurations of 4−6, 8, and 9 were determined by electronic circular dichroism spectroscopic data. Compound 7 exhibited significant activity against human prostate PC-3 cancer cells with an IC50 value of 0.16 μM. Compounds 1, 5, 6, and 9 showed moderate inhibition against the same cell line with IC50 values of 8.0, 3.6, 3.1, and 5.6 μM. Compound 2 displayed a notable inhibitory effect against Rho-associated protein kinase (ROCK2) with an IC50 value of 5.7 nM, which was similar to the positive control staurosporine (IC50 7.8 nM). ince the first isolation of the indolocarbazole staurosporine in 1977, this family of compounds has stimulated many research groups.1−6 These compounds exhibit great cytotoxicity to cancer cell lines and have strong inhibition on protein kinases, but many of them have no selectivity especially for staurosporine.7−9 So far, more than 50 different microbial staurosporine derivatives have been reported; among them several molecules have undergone clinical trials such as midostaurin, lestaurtinib, and CEP-1347.10−15 As part our effort to identify new cytotoxic indolocarbazoles from marine-derived actinomycetes, we obtained a series of indolocarbazoles (1−9) featuring a hydroxy group on the C-3 or C-9 position. Herein we describe the isolation, structure elucidation, and biological properties of nine new naturally occurring indolocarbazoles from Streptomyces sp. DT-A61.

S

spectrum (Table 1) showed 18 aromatic carbons, one methylene signal (δC 45.5), and one carbonyl group (δC 172.9). The signals of the 1H and 13C NMR spectra were very similar to the data for the indolocarbazole K252c (staurosporine aglycone) in the literature.16 The disappearance of one aromatic proton indicated the placement of a hydroxy group at C-9, which was confirmed by HMBC correlations of 9-OH with C-8/C-10 and H-11 with C-9 (Figure 1). Compound 1 was therefore identified as 9-hydroxyK252c. Compound 2, a pale brown, amorphous solid, had a molecular formula of C20H13N3O2 according to the HRESIMS data, which was the same as compound 1. The 1H NMR data (Table 1) in DMSO-d6 also showed seven aromatic signals, three NH protons (δH 11.40, δH 11.00, δH 8.40), and one OH signal (δH 8.90), which were similar to the K252c data. The absence of one aromatic proton suggested a substitution on the aromatic ring of K252c. The signal of H-4 (δH 8.60) was shielded compared to that of K252c (δH 9.24), and the coupling constant of H-4 decreased from 7.9 Hz in 1 to 2.4 Hz in 2. This indicated a hydroxy group at C-3, which was supported by HMBC correlations of 3-OH with C-4/C-2 and H-1 with C-3 (Figure 1). Compound 2 was therefore designated as 3-hydroxy-K252c. Compound 3 was also isolated as a pale brown, amorphous solid. The molecular formula was assigned as C21H15N3O3 by HRESIMS. 1H and 13C NMR data (Table 1) were almost identical to those of compound 2. The major difference in the 1H and 13C NMR spectra of compound 3 from compound 2 was the observation of signals for a methoxy group (δH 3.23, δC 51.7) and

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RESULTS AND DISCUSSION The strain was isolated from a sediment sample collected in Dongtou, Zhejiang Province, China. The EtOAc extract from the solid-state fermented products of strain DT-A61 exhibited significant cytotoxicity on human prostate cancer PC-3 cells (81% inhibition at 10 μg/mL) and was separated and purified by Sephadex LH-20 column chromatography and reversed-phase preparative HPLC to give compounds 1−9. All nine compounds had UV absorption bands typical for an indolcarbazole (e.g., 1: λmax 231, 291, 337, 350, 367 nm). Compound 1 was obtained as a pale brown, amorphous solid. The molecular formula was determined to be C20H13N3O2 by HRESIMS. The 1H NMR data (Table 1) revealed the presence of seven aromatic signals, three NH protons (δH 11.26, δH 11.37, δH 8.40), and one OH hydrogen (δH 9.10). The 13C NMR © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 19, 2017

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DOI: 10.1021/acs.jnatprod.7b01058 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

Table 1. 1H 600 MHz and 13C 150 MHz NMR Spectroscopic Data for Compounds 1 and 2 (DMSO-d6) and Compound 3 (CD3OD) 1

2

3

no.

δC, type

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

1 2 3 3-OH 4 4a 4b 4c 5 6 7 7a 7b 7c 8 9 9-OH 10 11 11a 12 12a 12b 13 13a 7-CH3

111.7, CH 125.3, CH 119.3, CH

7.68, d (8.0) 7.40, m 7.22, m

111.0, CH 114.5, CH 149.9, C

7.48, d (8.6) 6.90, dd (8.6, 2.4)

112.9, CH 117.0, CH 152.3, C

7.45, d (8.7) 7.00, dd (8.7, 2.4)

125.6, CH 123.4, C 115.7, C 118.6, C 172.9, C

9.19, d (7.9)

109.3, CH 123.0, C 115.0, C 118.4, C 172.0, C

111.4, CH 125.5, C 117.9, C 120.3, C 175.4, C

8.58, d (2.4)

86.6, CH2 133.1, C 116.9, C 124.5, C 124.1, CH 121.7, CH

6.45, s

127.1, CH 112.8, C 142.0, C

7.40, m 7.63, d (8.2)

45.5, CH2 133.6, C 114.6, C 123.8, C 106.2, CH 151.9, C 114.9, CH 112.8, CH 133.7, C

δH (J in Hz)

8.40, s 4.90, s

7.30, d (2.2) 9.10, s 6.97, dd (8.7, 2.2) 7.58, d (8.7)

44.8, CH2 131.9, C 117.9, C 122.0, C 120.6, CH 119.3, CH 124.4, CH 111.3, CH 138.6, C

11.26, s 129.1, C 125.9, C

8.40, s 4.90, s

8.01, d (7.6) 7.27, m 7.44, m 7.74, d (7.9)

8.28, d (7.7) 7.28, m

11.40, s 133.1, C 122.2, C

129.3, C 130.6, C

11.37, s 139.6, C

8.90 8.60, d (2.4)

11.00, s 132.9, C

136.5, C 51.7, CH3

an sp3 methine group (δH 6.45, δC 86.6). The methoxy group was located at C-7 from the HMBC correlations of CH3O-7 with C-7 and H-7 with C-7a/C-5 (Figure 1). As the specific rotation for compound 3 was zero, it suggested that the C-7 position bearing a methoxy group was racemic. Based on these results, the structure of compound 3 was revealed to be 3-hydroxy-7-

3.23, s

methoxy-K252c. Additionally as MeOH was used in the early stages of the isolation process, it is likely that compound 3 arose as an artifact from a hemiaminal precursor. Compound 4, a pale brown, amorphous solid, gave a molecular formula of C28H26N4O5 by HRESIMS, suggesting a possible sugar modification to the indolocarbazole core. The signals in the B

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Figure 1. COSY and key HMBC correlations for indolocarbazoles 1−9. 1

H and 13C NMR (Table 2) were similar to those of the known compound holyrine A,17 which contains a K252c unit and a sugar moiety. In the 1H NMR data (Table 2), the chemical shifts from δH 7.00 to δH 9.00 were the same as those for compound 1, which indicated the presence of a 9-hydroxy-K252c unit. COSY correlations of H-5′(δH 4.48)/H-4′(δH 3.89)/H-3′(δH 4.68)/ H-2′(δH 2.50)/H-1′(δH 6.69) confirmed the presence of a sugar moiety (Figure 1). The disappearance of one NH signal (δH 11.00) and the HMBC correlation of H-1′ with C-12b (δC 123.7) indicated the connection of the two units only through N-13 and C-1′. In addition to the signals of the holyrine A unit, signals of a methyl group at δH 1.82 (3H, s, COCH3) and one carbonyl group at δC 168.3 (CO) were also observed. An acetyl group was deduced, which was supported by the HMBC correlation of CH3 with CO (Figure 1). The additional acetyl group was bound to the 3′-NH position, which was confirmed by an HMBC correlation of 3′-NH with COCH3 (Figure 1). Comparing the 1 H NMR spectrum with that of holyrine A, along with the NOE correlation (Figure 2), the relative configuration of 4 was identical to that of holyrine A. The absolute configuration of 4 was determined based on electronic circular dichroism (ECD) data analysis. The experimental ECD curve of 4 matched the simulated ECD curve of (1′R,3′R,4′R,5′S)-4 (Figure 3). Compound 4 was therefore established as 9-hydroxy-3′-Nacetylholyrine A. Compound 5 was acquired as a pale brown, amorphous solid. The molecular formula was revealed to be C28H26N4O5 based on HRESIMS. In the 1H NMR data (Table 2), the chemical shifts from δH 7.00 to δH 9.00 were almost identical to those of compound 2, which indicated the presence of a 3-hydroxy-K252c unit. Also in the 1H NMR data, the chemical shifts from δH 1.00 to δH 7.00 were similar to those of compound 4. This revealed the

existence of the sugar unit of holyrine A. An HMBC correlation (Figure 1) of H-1′ with C-12b (δC 126.6) indicated the connection of the two units through N-13 and C-1′. Combining the NOE correlations (Figure 2) and the similar experimental ECD curve of 5 with that of 4 (Figure 3), the absolute configuration of 5 was established as (1′R, 3′R, 4′R, 5′S). Thus, the structure of compound 5 was assigned as 3-hydroxy-3′-Nacetylholyrine A. Compound 6, a pale brown, amorphous solid, had a molecular formula related to 5 of C26H24N4O4 by HRESIMS. Comparing the 13C NMR spectrum (Table 2) with that of compound 5, the data were almost the same except for the disappearance of the acetyl group signals (δC 168.3, δC 22.5). With the 1H data and NOE correlations (Figue 2), the relative configuration of 6 was identical to holyrine A. The absolute configurations of 6 and 4 were revealed to be the same as (1′R, 3′R, 4′R, 5′S) by comparing their experimental ECD data (Figure 3). Based on these results, the structure of compound 6 was assigned to be 3hydroxyholyrine A. Compound 7 was obtained as a white, amorphous solid. Its molecular formula was established as C28H24N4O4 by HRESIMS. The 1H NMR data (Table 3) showed eight aromatic signals and one methylene proton (δH 5.00, 2H, s), which indicated the presence of the K252c unit. COSY correlations (Figure 1) of H3′ (δH 4.13)/H-4′ (δH 4.04)/H-5′ (δH 2.53)/H-6′ (δH 6.7) revealed the presence of a sugar moiety. In addition to the K252c and sugar signals (Table 3), signals of a methyl group at δC 23.1 and one carbonyl group at δC 173.9 (CO) were also observed, indicating an acetyl group, which was confirmed by an HMBC correlation (Figure 1) of CH3 with CO. Comparison of the elementary skeleton with staurosporine showed the addition of an acetyl group and the disappearance of two methyl groups. The C

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Table 2. 1H 600 MHz and 13C 150 MHz NMR Spectroscopic Data for Compound 4 (DMSO-d6) and Compounds 5 and 6 (CD3OD) 4 no. 1 2 3 4 4a 4b 4c 5 6 7 7a 7b 7c 8 9 9-OH 10 11 11a 12 12a 12b 13a 5′ 4′ 3′ 2′ 1′ 5′-CH3 COCH3 COCH3 4′-OH 3′-NH

δC, type 109.2, CH 124.6, CH 119.1, CH 125.1, CH 121.7, C 116.3, C 117.1, C 171.0, C 44.5, CH2 133.8, C 117.8, C 121.9, C 105.0, CH 151.1, C 114.1, CH 111.6, CH 132.8, C

5 δH (J in Hz)

8.01, d (8.4) 7.47, m 7.26, m 9.40, d (7.9)

8.47, s 5.0, m

7.37, d (1.9) 9.16 7.00, dd (8.4, 1.9) 7.50, d (8.4)

δC, type

6 δH (J in Hz)

110.4, CH 116.2, CH 152.3, C 111.7, CH 124.8, C 119.1, C 118.9, C 175.1, C

7.56, d (9.13) 7.04, brd (8.8)

47.0, CH2 135.6, C 116.4, C 123.7, C 122.0, C 121.0, CH

5.00, m

126.0, CH 112.7, CH 141.0, C

8.85, brs

7.98, d (7.6) 7.28, m 7.40, m 7.66, d (8.0)

δC, type 110.3, CH 116.3, CH 152.4, C 111.8, CH 125.2, C 116.5, C 119.4, C 175.8, C 46.9, CH2 135.7, C 119.1, C 123.6, C 122.1, C 121.3, CH 126.5, CH 112.0, CH 140.9, C

δH (J in Hz) 7.57, d (8.6) 7.05, dd (8.6,2.3) 8.86, d (2.3)

5.00, m

8.01, d (7.8) 7.32, m 7.47, m 7.70, d (7.5)

11.84, s 127.0, C 123.7, C 138.3, C 76.3, CH 67.8, CH 43.7, CH 31.6, CH2 75.3, CH 13.7, CH3 168.3, C 22.1, CH3

4.48, q (7.2) 3.89, s 4.68, m 1.74, m 2.50 overlap 6.69, dd (11.3, 2.9) 1.60, d (7.2) 1.82, s 6.88 7.92

129.5, C 126.6, C 135.2, C 78.4, CH 69.0, CH 46.5, CH 33.0, CH2 77.5, CH 14.7, CH3 173.0, C 22.5, CH3

4.62, q (7.3) 4.00, brs 4.70, d (12.5) 2.65, m 1.87, m 6.58, dd (12.3, 2.4) 1.67, d (7.2)

129.4, C 126.4, C 134.9, C 78.3, CH 67.9, CH 48.1, CH 31.9, CH 76.8, CH 14.4, CH3

4.72, q (7.0) 4.15, s 4.20, d (11.5) 2.70, m 2.07, m 6.58, dd (11.2, 2.6) 1.67, d (7.0)

1.67, s

Figure 2. Key NOESY correlations of 4−6.

3′-O-demethyl-4′-N-demethyl-4′-N-acetyl-4′-epi-staurosporine. However, the absolute configuration of 7 was not determined. Compound 8, a pale yellow, amorphous solid, possessed a molecular formula of C26H21N3O5 by HRESIMS. The 1H NMR data (Table 3) showed signals for seven aromatic protons and an sp3 methylene signal (δH 5.00, 2H, s), which were almost

acetyl group was bound to the 4′-NH position, which was confirmed by an HMBC correlation of H-4′ with COCH3. The coupling constant of 11 Hz between H-3′ and H-4′ suggested them to be in a diaxial arrangement. The cofacial orientation of protons 3′ and 5′a was established by NOE signals (Figure 4). From these results, the structure of compound 7 was identified as D

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correlations of H-1′ with C-3′/C-5′/C-12b, H-2′ with C-4′, and CH3-5′ with C-4′ (Figure 1). The two remaining hydroxy groups were deduced through HRESIMS data, and they were connected to C-3′ and C-4′. The coupling constant of 9.9 Hz between H-4′ and H-5′ indicated an anti relationship, and the axial positions of the protons 4′ and 2′a were further confirmed by NOE signals (Figure 4). Comparing to the data for the streptocarbazoles, the relative configuration of 8 was determined (Figure 4). The absolute configuration of 8 was established according to a comparison of the experimental and calculated ECD spectra. The experimental ECD curve of 8 was similar to the predicted ECD curve of (1′R,3′S,4′S,5′S)-8 (Figure 5). Thus, the absolute configuration of 8 was revealed to be (1′R, 3′S, 4′S, 5′S), and compound 8 was named streptocarbazole D. Compound 9, a pale yellow, amorphous solid, had the same molecular formula as that of compound 8. The 1D and 2D NMR data (Table 3, Figure 1) indicated it shared the same planar structure as compound 8. The difference between them was the coupling constant between H-4′ and H-5′, which became very small. The NOESY correlations of H-1′/H-5′, H-2′/H-4′, and H-2′/H-5′ (Figure 4) suggested a syn-oriented relationship among H-1′, H-2′, H-4′, and H-5′, indicating a boat conformation for the sugar ring, which was supported by modeling studies. The ECD spectrum of 9 was also recorded, and it was similar to that of compound 8, which indicated that the absolute configurations of 9 at the 1′ and 3′ positions were the

Figure 3. Measured ECD for compounds 4−6 and calculated ECD spectra for compound 4.

identical to those of compound 2, indicating the presence of a 3hydroxy-K252c unit. Based on the 1H data (δH 1.00−7.00) and 13 C NMR data (δC 37.9−89.7), the existence of a sugar moiety was revealed. Compound 8 was determined to be a streptocarbazole derivative,18 which was confirmed by COSY correlations of H-1′/H-2′ and H-4′/H-5′ and HMBC

Table 3. 1H and 13C NMR Spectroscopic Data for Compound 7 (1H 500 MHz, 13C 125 MHz, CD3OD) and Compounds 8 and 9 (1H 600 MHz, 13C 150 MHz, CD3OD) 7 no.

δC, type

1 2 3 4 4a 4b 4c 5 7 7a 7b 7c 8 9 10 11 11a 12a 12b 13a 2′ 3′

109.6, CH 127.0, CH 121.2, CH 127.5, CH 125.1, C 117.3, C 120.0, C 175.6, C 47.6, CH2 134.6, C 116.5, C 126.3, C 122.1, CH 121.9, CH 126.2, CH 117.7, CH 142.0, C 130.7, C 127.5, C 138.6, C 97.4, C 79.4, C

4′ 5′

45.4, CH 38.0, CH2

6′ 2′-CH3 COCH3 COCH3

83.8, CH 30.1, CH3 173.9, C 23.1, CH3

8 δH (J in Hz)

7.41, overlap 7.48, overlap 7.29, overlap 9.27, d (8.1)

5.0, m

7.96, d (7.7) 7.32, m 7.43, m 8.11, d (8.6)

4.13, d (11.0) 4.04, td (11.0, 3.8) 2.57, ddd (13.8, 3.8, 1.5) 2.53, ddd (13.8, 11.0, 4.5) 6.7, dd (4.5, 1.5) 2.4, s

9

no.

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

1 2 3 4 4a 4b 4c 5 7 7a 7b 7c 8 9 10 11 11a 12a 12b 13a 1′ 2′

110.5, CH 116.5, CH 152.6, C 111.5, CH 125.3, C 116.4, C 119.9, C 175.4, C 47.2, CH2 134.6, C 116.9, C 125.2, C 121.9, CH 122.1, CH 126.0, CH 116.7, CH 141.1, C 129.2, C 128.5, C 135.3, C 78.8, CH 44.2, CH2

7.49, d (8.8) 7.05, dd (8.8, 2.5)

110.6, CH 116.3, CH 152.6, CH 111.5, CH 125.2, C 116.8, C 119.4, C 175.5, C 47.0, CH 134.5, C 117.3, C 124.9, C 121.2, CH 121.5, CH 125.7, CH 119.6, CH 143.5, C 128.7, C 129.3, C 135.5, C 78.4, CH 37.9, CH2

7.48, d (8.9) 7.04, dd (8.9, 2.4)

3′ 4′

89.7, C 81.5, CH

3.9, d (9.9)

87.8, C 73.4, CH

4.03, s

5′ CH3

69.1, CH 18.3, CH3

3.52, dd (9.9, 6.2) 1.13, d (6.2)

68.7, CH 17.4, CH3

3.74, q (6.4) 1.08, d (6.4)

8.70, d (2.5)

5.00, s

8.00, d (7.7) 7.34, m 7.40, m 8.59, d (8.5)

6.55, d (6.1) 3.09, s 3.08, d (6.1)

8.73 d (2.4)

5.01, s

7.94, d (7.7) 7.31, m 7.40, m 8.58, d (8.4)

6.57, d (6.7) 3.27, dd (15.0, 6.7) 2.80, d (15.0)

1.8, s E

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Figure 4. Key NOESY correlations of 7−9.

effects on the various protein kinases were also evaluated (Table 4). Results of the various protein kinase assays revealed that compound 2 displayed significant, selective inhibition against ROCK2, close to the positive control, staurosporine. The other compounds possessed moderate activity on the protein kinases. In conclusion, nine new indolocarbazoles were isolated from the marine-derived Streptomyces sp. DT-A61. Eight of them possessed a hydroxy group at either the C-3 or C-9 position. Of the vast majority of the reported naturally occurring indolocarbazoles, only a small fraction have a hydroxy group only on the C-3 or C-9 position.4,5 Five of the new compounds displayed cytotoxic activity against the PC-3 cancer cell line, and some displayed potent inhibitory activity on protein kinases. This set of new compounds has provided a small cytotoxicity structure−activity relationship, which revealed that when the connection of the sugar moiety to the K252c unit was similar to that of staurosporine, the compound was found to be more potent than those with no sugar moiety or those with only a single attachment of the sugar to the aromatic aglycone.

Figure 5. Measured ECD for compounds 8 and 9 and calculated ECD spectra for compound 8.

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same as those of compound 8 (Figure 5). Therefore, the absolute configuration of 9 was revealed to be (1′R, 3′S, 4′S, 5′R), and compound 9 was named streptocarbazole E. The nine new indolocarbazoles were examined for the cytotoxic activity against human prostate cancer PC-3 cells (Table 4). Compound 7 exhibited strong cytotoxicity with an IC50 value of 0.15 μM, and compounds 1, 5, 6, 9 were also cytotoxic, with IC50 values of 8.0, 3.6, 3.1, and 5.6 μM. Inhibitory

General Experimental Procedures. Optical rotations were obtained with a JASCO P-1030 digital polarimeter in MeOH. The UV spectra were measured by a METASH UV-8000 visible spectrophotometer. ECD spectra were collected on a JASCO J-1500150ST spectrophotometer. IR spectra were collected on a Bruker Vector 22 spectrophotometer. 1D and 2D NMR spectra were recorded using Avance III 600 (1H 600 MHz; 13C 150 MHz, Bruker), Avance III 500 (1H 500 MHz; 13C 125 MHz, Bruker), and Agilent DD2-600 (1H 600 MHz; 13C 150 MHz) spectrometers, where chemical shifts were referenced to the respective solvents (CD3OD, δH 3.31, δC 49.1; DMSO-d6, δH 2.50, δC 39.5). HRESIMS spectra were recorded using an Agilent 6230 TOF LC/MS system mass spectrometer. The HPLC analysis was accomplished using a Shimadzu high-performance liquid chromatography system with an Agilent RP-C18 column (5 μm, 4.6 × 250 mm). The extract was prefractionated on Sephadex LH-20, then separated and purified by preparative HPLC (Beijing Chuangxintongheng LC3000) [Agilent C18 column (5 μm, 21.2 × 250 mm); solvent A: H2O, solvent B: MeOH or MeCN; flow rate: 10 mL/min]. Timeresolved fluorescence resonance energy transfer signals were recorded on a SPARK 10M multifunctional microplate reader. Prostate cancer cell lines was obtained from ATCC, and the protein kinase inhibitor screening assay kit was purchased from Cisbio Bioassays. Artificial sea salt (Zhejiang Province Salt Industry Group Co., Ltd.) was used for preparing seawater. Media, Fermentation, Extraction, Isolation, and Purification. Streptomyces sp.A61 was isolated from a sediment sample collected in Dongtou, Zhejiang Province, China (N 27°87′01.67″ E 121°16′99.45″), and identified by 16S rDNA sequencing analysis and morphological evaluation (GenBank accession number: MG869126). The strain was grown on GS agar plates (soluble starch 20.0 g, KNO3 1.0 g, KH2PO4 0.5 g, MgSO4 0.5 g, NaCl 0.5 g, FeSO4·7H2O 0.01 g, and artificial sea salt

Table 4. Cytotoxicity Assay Results against PC-3 Cells and Enzyme Inhibition of Protein Kinases IC50,a in μM compound

PC-3b

PKC-αc

ROCK2d

ASK1e

1 2 3 4 5 6 7 8 9 STUf

8.0 21 18 ndg 3.6 3.1 0.16 39 5.6 0.039

0.98 3.2 1.4 0.097 0.46 0.079 0.092 2.1 1.4 0.0024

0.34 0.0057 0.91 1.4 0.040 0.15 0.26 0.97 0.34 0.0078

0.74 3.0 >50 >50 6.4 1.1 0.77 4.1 >50 0.00069

EXPERIMENTAL SECTION

a

Concentration inhibiting 50% of PC-3 cell line for a 72 h exposure period of test samples or concentration inhibiting 50% activity of the protein kinases. bHuman prostate cancer cell line. cProtein kinase C alpha. dRho-associated protein kinase 2. eApoptosis signal-regulating kinase 1. fStaurosporine as a positive control. gNot determined. F

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Streptocarbazole D (8): pale yellow, amorphous solid; [α]23 D −30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 222 (4.29), 295 (4.49) 342 (4.03) 358 (3.88) 376 (3.81) nm; IR νmax 3415, 1682, 1459, 1206, 1142, 726 cm−1; 1H and 13C NMR data, Table 3; HRESIMS m/z 456.1558 [M + H]+ (calcd for C26H22N3O5, 456.1559). Streptocarbazole E (9): pale yellow, amorphous solid; [α]23 D −15 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 224 (4.39), 296 (4.6) 342 (4.23) 359 (3.65) 377 (3.09) nm; IR νmax 3407, 1680, 1462, 1353, 1206, 1143, 982, 725 cm−1; 1H and 13C NMR data, Table 3; HRESIMS m/z 456.1551 [M + H]+ (calcd for C26H22N3O5, 456.1559). Cytotoxicity Assay. All compounds (1−9) were tested for their cytotoxic activities in vitro against the PC3 cells using the sulforhodamine B method19 for a single-dose (20 μM) screen and full IC50 determination (0.005−20 μM) in triplicate. Kinase Assay in Vitro. All compounds (1−9) were tested against protein kinases, using the HTRF KinEASE-STK generic method18 to measure the inhibition of protein kinase C alpha (PKC-α), Roh associated protein kinase (ROCK2), and apoptosis signal-regulating kinase-1 (ASK1) for full IC50 determination (0.0005−10 μM) in triplicate.

25.0 g dissolved in 1 L of H2O, pH = 7.0) for 5 days. Then it was inoculated in baffled Erlenmeyer flasks (500 mL) with 250 mL of sterile GS liquid medium as seed broth and cultivated at 28 °C with shaking at 180 rpm for 3 days. After that, seed broth (12 mL) was inoculated in 500 baffled Erlenmeyer flasks (500 mL) with sterile rice solid medium (rice 40.0 g, seawater 60.0 mL). Then the medium was cultured under static conditions at 28 °C for about two months. The whole fermented cultures were extracted three times with EtOAc at room temperature overnight. The EtOAc extract was subsequently evaporated in vacuo to afford 30 g of an oily extract, then dissolved in 90% MeOH/H2O and extracted with petroleum ether (3 × 1 L) to afford the 90% MeOH/H2O fraction (20 g), which was submitted to Sephadex LH-20 eluting with MeOH/ H2O (20%, 40%, 60%, 80%, 100%) to give 26 fractions (A−Z). Fraction Z (40 mg) was separated by preparative HPLC (23−30%, 40 min MeCN/H2O, flow rate 10 mL/min) to give 6 (1.5 mg, tR = 16 min). Fraction S (90 mg) was separated by preparative HPLC (50−80%, 50 min, MeOH/H2O, flow rate 10 mL/min) to give five fractions (S-1 to S5). Fraction S-3 (8 mg) was separated by preparative HPLC (38−42%, 40 min, MeCN/H2O, flow rate 10 mL/min) to give 3 (1.5 mg, tR = 20 min). Fraction T (120 mg) was separated by preparative HPLC (30− 50%, 50 min, MeCN/H2O, flow rate 10 mL/min) to give six fractions (T-1 to T-7). Fraction T-6 (9 mg) was separated by preparative HPLC (35−40%, 40 min, MeCN/H2O, flow rate 10 mL/min) to give 2 (1.6 mg, tR = 16 min). Fraction T-7 (7 mg) was separated by preparative HPLC (35−40%, 40 min, MeCN/H2O, flow rate 10 mL/min) to give 7 (1.9 mg, tR = 20 min). Fraction Q (180 mg) was separated by preparative HPLC (50−80%, 50 min, MeOH/H2O, flow rate 10 mL/min) to give six fractions (Q-1 to Q-7). Fraction Q-2 (15 mg) was separated by preparative HPLC (35−40%, 40 min, MeCN/H2O, flow rate 10 mL/ min) to give 4 (2.0 mg, tR = 16 min), 5 (1.5 mg, tR = 16.7 min), and 1 (1.6 mg, tR = 22 min). Fraction O (80 mg) was separated by preparative HPLC (40−65%, 60 min, MeCN/H2O, flow rate 10 mL/min) to give six fractions (O-1 to O-6). Fraction O-2 (12 mg) was separated by preparative HPLC (30−40%, 30 min, MeCN/H2O, flow rate 10 mL/ min) to give 8 (1.2 mg, tR = 18 min) and 9 (1.5 mg, tR = 20 min). 9-Hydroxy-K252c (1): pale brown, amorphous solid; λmax (log ε) 226 (3.89), 292 (4.13) 340 (3.59) 370 (3.23) nm; IR νmax 3416, 2926, 1648, 1617, 1460, 1400, 1244, 736 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 328.1080 [M + H]+ (calcd for C20H14N3O2, 328.1086). 3-Hydroxy-K252c (2): pale brown, amorphous solid; λmax (log ε) 231 (4.4), 291 (4.64) 337 (4.13) 350 (4.04) nm; IR νmax 3341, 1647, 1584, 1457, 1410, 842, 796 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 328.1076 [M + H]+ (calcd for C20H14N3O2, 328.1086). 3-Hydroxy-7-methoxy-K252c (3): pale brown, amorphous solid; [α]23 D 0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 230 (3.3), 294 (3.45) 304 (3.45) 372 (2.5) nm; IR νmax 3331, 2929, 1684, 1584, 1404, 1209, 799 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 358.1188 [M + H]+ (calcd for C21H16N3O3, 358.1192). 9-Hydroxy-3′-N-acetylholyrine A (4): pale brown, amorphous solid; [α]23 D −74 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 234 (4.3), 293 (4.56) 326 (3.94) 339 (4.01) 356 (3.88) nm; IR νmax 3337, 1644, 1559, 1460, 1250, 1206, 747 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 499.1980 [M + H]+ (calcd for C28H26N4O5, 498.1981). 3-Hydroxy-3′-N-acetylholyrine A (5): pale brown, amorphous solid; [α]23 D −62 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (4.03), 295 (4.28) 341 (3.75) 375 (3.47) nm; IR νmax 3361, 1677, 1458, 1394, 1207, 1142, 799, 727 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 499.1966 [M + H]+ (calcd for C28H27N4O5, 498.1981). 3-Hydroxyholyrine A (6): pale brown, amorphous solid; [α]23 D −76 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 222 (3.8), 269 (3.74) 295 (4.04) 341 (3.53) 374 (3.28) nm; IR νmax 3381, 1684, 1456, 1206, 1142, 725 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 457.1873 [M + H]+ (calcd for C26H25N4O4, 457.1876). 3′-O-Demethyl-4′-N-demethyl-4′-N-acetyl-4′-epi-staurosporine (7): white, amorphous solid; [α]23 D +66 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 237 (4.29), 291 (4.6) 334 (4.06) 353 (3.9) 371 (3.96) nm; IR νmax 3411, 1665, 1586, 1458, 1348, 1136, 838 cm−1; 1H and 13C NMR data, Table 3; HRESIMS m/z 481.1866 [M + H]+ (calcd for C28H25N4O5, 481.1876).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01058. ECD method for compounds 4 and 8; NMR (1D, 2D), UV, HRESIMS, and IR spectra of compounds 1−9 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-0580-2092306. Fax: +86-0580-2092891. E-mail: [email protected]. ORCID

Zhong-Jun Ma: 0000-0002-5825-5095 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Zhejiang University Cross Researching Fund [grant number JCZZ-2013021]. We appreciate Mrs. J. Pan at Pharmaceutical Informatics Institute of Zhejiang University, for performing the NMR spectroscopy.

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REFERENCES

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