Staurosporine Derivatives Generated by Pathway Engineering in a

Article Cite This: J. Nat. Prod. 2018, 81, 1745−1751

pubs.acs.org/jnp

Staurosporine Derivatives Generated by Pathway Engineering in a Heterologous Host and Their Cytotoxic Selectivity Fei Xiao,† Huayue Li,†,‡ Mingyuan Xu,† Tong Li,† Ju Wang,§ Chaomin Sun,‡,§ Kui Hong,⊥ and Wenli Li*,†,‡

J. Nat. Prod. 2018.81:1745-1751. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 08/24/18. For personal use only.

†

Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266000, People’s Republic of China § Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, People’s Republic of China ⊥ Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education of China, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, People’s Republic of China S Supporting Information *

ABSTRACT: Two new staurosporine derivatives, staurosporines M1 and M2 (4 and 5), in addition to five previously reported metabolites (1−3, 6, and 7), were generated by the heterologous expression of engineered spc gene clusters in Streptomyces coelicolor M1146. The structures of these derivatives were determined by a combination of spectroscopic methods and CD measurement. Compounds 1, 2, 4, and 5 showed effective activities against three tumor cell lines (HCT-116, K562, and Huh 7.5), and 3 was active against HCT-116 and K562 cells. In addition, compounds 3 and 5 showed undetectable toxicity up to 100 μM toward the normal hepatic cell line LO2. Based on the IC50 values, their structure and activity relationships are discussed.

I

Several studies on staurosporine and its targets revealed that the sugar moiety is related to its selectivity profiles, mainly due to the effects of conserved hydrogen bonds involving the methylamino nitrogen and the ether oxygen to their surrounding residues in protein kinases.12,21−25 Some amino sugar-modified staurosporine derivatives have been chemically synthesized, and midostaurin (N-benzoylstaurosporine) exhibits a remarkable degree of selectivity for PKC26 and was approved by the FDA in 2017 for the treatment of adult patients with newly diagnosed acute myeloid leukemia.27 Therefore, further modifications on the sugar moiety of staurosporine might be a potential way to produce derivatives with good specificities and low toxicities. In our previous study, the spc gene cluster involved in staurosporine biosynthesis in the marine-derived Streptomyces sanyensis FMA was identified and characterized (Figure 1A), and its heterologous expression resulted in the accumulation of staurosporine.28 The composition and organization of the spc gene cluster are highly conserved with that in Streptomyces sp. TP-A0274.28 The cytochrome P450 SpcN was proposed to be

ndolocarbazoles are a family of natural products that have generated interest due to their structures and biological activities, including antibacterial,1−3 antifungal,4 and antitumor5−9 properties. Staurosporine was the first indolocarbazole compound to be reported. Its cytotoxic activity was based on submicromolar binding to most protein kinases.10,11 However, this broad selectivity also leads to toxicity, and this limits its application in clinics.12 Several staurosporine analogues, including lestaurtinib13 and UCN-01,14 have been chemically synthesized and are used in clinical trials as anticancer agents. Combinatorial biosynthesis approaches are promising alternatives to chemical synthesis to prepare new derivatives.15,16 A number of indolocarbazole compounds have been generated by heterologously expressing parts of, or entire, gene clusters in convenient bacterial hosts.17,18 For example, the isolation of the staurosporine gene cluster enabled the reconstruction of its biosynthetic pathway in Streptomyces albus.19 Based on the flexibility of glycosyltransferase StaG, a series of staurosporine derivatives with effective cytotoxic and protein kinase C (PKC) inhibitory activities were generated,19,20 thus increasing the molecular diversity of indolocarbazoles for drug discovery. © 2018 American Chemical Society and American Society of Pharmacognosy

Received: February 1, 2018 Published: August 14, 2018 1745

DOI: 10.1021/acs.jnatprod.8b00103 J. Nat. Prod. 2018, 81, 1745−1751

Journal of Natural Products

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Figure 1. (A) Genetic organization of the spc gene cluster and (B) chemical structures of 1−7 and staurosporine.

fermentation of the resulting strains S. coelicolor M1146/ pWLI627 and M1146/pWLI628 was carried out, and the accumulated metabolites were analyzed by HPLC. As shown in Figure 2, in both heterologous expression strains (panel ii and iii), a series of compounds with UV spectra similar to that of staurosporine were newly produced in comparison to the control (panel i). Compounds 1−7 were isolated from largescale fermentations of the heterologous expression strains. Compound 4 was isolated as a yellow, amorphous powder. The molecular formula of 4 was assigned as C28H24N4O4 based on the HR-ESIMS data, and it showed 1H and 13C NMR data (Table 1) that were similar to staurosporine. 5 The indolocarbazole core was further confirmed by the combination of the aromatic proton spin systems of H-1 (δH 7.62)/H-2 (δH 7.47)/H-3 (δH 7.28)/H-4 (δH 9.28) and H-8 (δH 8.03)/ H-9 (δH 7.34)/H-10 (δH 7.45)/H-11 (δH 7.99) in COSY, and the HMBC correlations from H-1 to C-4a (δC 122.5), from H4 to C-13a (δC 136.3) and C-4b (δC 114.7), from H-6 (δH 8.57) to C-7a (δC 132.4) and C-4c (δC 119.1), from H-8 to C11a (δC 139.1) and C-7b (δC 113.9), and from H-11 to C-7c (δC 123.9). The proton spin systems of H-1′ (δH 6.88)/H-2′ (δH 2.61, 2.38)/H-3′ (δH 4.25)/7′-NH (δH 7.21) and H-3′/H4′ (δH 4.39)/4′-OH (δH 5.83) in COSY, together with the

responsible for the formation of the second C−N glycosidic linkage (C5′−N12), and the methyltransferases SpcMA and SpcMB were proposed to be involved in the O-methylation and N-methylation of the sugar moiety, respectively. Since the sugar moiety is closely related to the cytotoxic selectivity of staurosporine, we investigated the heterologous expression of spcN deleted and spcMA spcMB doubly deleted spc gene clusters in Streptomyces coelicolor M1146, and this led to the isolation of two new (4 and 5) and five known (1−3, 6, and 7) staurosporine derivatives with modified sugar moieties (Figure 1B). Herein, we describe the structure elucidation and cytotoxic selectivity of these derivatives. Furthermore, the structure−activity relationships (SAR) of 1−7 are discussed.

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RESULTS AND DISCUSSION

The expression cosmids pWLI624 and pWLI626, harboring the engineered spc gene clusters, spcN deletion and spcMA spcMB double-deletion, respectively, were constructed. After confirmation by PCR (Figure S1), the cosmids were equipped with oriT and φC31 attP/int, generating pWLI627 (spcN deletion) and pWLI628 (spcMA spcMB double-deletion). These two plasmids were then introduced into S. coelicolor M1146 as described in the Experimental Section. The 1746



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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for 4 and 5 in DMSO-d6 4 no.

Figure 2. Comparative HPLC analysis of fermentation products of (i) S. coelicolor M1146/pSET152AB, (ii) S. coelicolor M1146/pWLI628, and (iii) S. coelicolor M1146/pWLI627.

HMBC correlations from H-1′ and H-6′ (δH 2.31) to C-5′ (δC 94.2), from H-6′ to C-4′ (δC 71.4), and from H-1′ to C-12b (δC 125.1), assigned a six-membered ristosamine moiety that was attached to the indolocarbazole core. Moreover, the HMBC correlations from the secondary amine 7′-NH and the methyl group H-9′ (δH 1.42) to the carbonyl carbon C-8′ (δC 169.1) assigned an acetyl group attached to N-7′. The CD spectrum of 4 was almost identical to that of staurosporine (Figure 4). Therefore, its absolute configurations were confirmed to be 1′R, 3′R, 4′R, 5′S as reported for staurosporine.29 Thus, 4 was identified as 3′-N-acetyl-4′hydroxylstaurosporine and was named staurosporine M1. Compound 5 was isolated as a yellow, amorphous powder. The molecular formula of 5 was assigned as C32H26N4O4 based on the HR-ESIMS data. The comparison of the NMR data of 5 with 4 (Table 1) indicated that they shared an identical indolocarbazole core and a ristosamine moiety. The COSY correlations of H-8′ (δH 7.10)/H-9′ (δH 6.07) and H-9′/H-10′ (δH 7.20) and the HMBC correlations from the olefinic protons H-8′, H-9′, and H-10′ to C-11′ (δC 129.5) confirmed the formation of a five-membered pyrrole ring. The HMBC correlations from H-13′ (δH 2.50) to C-11′ and the carbonyl carbon C-12′ (δC 188.3) indicated an acetyl group linked to C11′, forming a 1-(1H-pyrrol-2-yl)ethanone group. The HMBC correlation from H-3′ (δH 5.91) to C-8′ (δC 129.3) further confirmed that this substructure was located at C-3′. The CD spectrum of 5 was similar to that of staurosporine but with a few differences (Figure 4), which might be due to the effect of the 1-(1H-pyrrol-2-yl)ethanone group at C-3′. However, the 1 H and 13C chemical shifts and the biosynthesis of 5 confirmed it shared the same absolute configurations as staurosporine.29 Therefore, 5 was identified as a new staurosporine derivative and was named staurosporine M2. Compound 1 was isolated as a yellow, amorphous powder. The molecular formula of 1 was assigned as C26H22N4O3 based on the HR-ESIMS data. As expected, the structural changes in

δC, type

1 2 3 4 4a 4b 4c 5 6 7

108.8, CH 125.6, CH 119.3, CH 125.5, CH 122.5, C 114.7, C 119.1, C 172.0, C

7a 7b 7c 8 9 10 11 11a 12a 12b 13a 1′

132.4, C 113.9, C 123.9, C 121.1, CH 120.0, CH 124.7, CH 114.4, CH 139.1, C 129.4, C 125.1, C 136.3, C 81.1, CH

2′

28.9, CH2

3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 4′OH

45.4, 71.4, 94.2, 29.1,

45.4, CH2

CH CH C CH3

169.1, C 22.4, CH3

5

δH (J in Hz) 7.62, 7.47, 7.28, 9.28,

d (7.8) t (7.8) t (7.8) d (7.8)

8.57, s 4.98, m

8.03, 7.34, 7.45, 7.99,

d (7.8) t (7.8) t (7.8) d (7.8)

6.88, dd (8.4, 5.4) 2.61, m; 2.38, m 4.25, m 4.39, m 2.31, s 7.21, d (6.0) 1.42, s

5.83, d (3.6)

δC, type 108.9, CH 125.3, CH 119.4, CH 125.6, CH 122.6, C 115.1, C 119.1, C 172.0, C 45.4, CH2 132.8, C 114.1, C 123.9, C 120.1, CH 119.4, CH 124.7, CH 114.5, CH 139.1, C 121.3, C 124.9, C 136.3, C 82.1, CH

δH (J in Hz) 7.70, 7.49, 7.30, 9.29,

d (7.8) t (7.8) t (7.8) d (7.8)

8.59, s 4.99, d (18.0); 4.97, d (18.0)

8.04, 7.32, 7.42, 7.86,

d (7.8) t (7.8) t (7.8) d (7.8)

7.11, m

28.2, CH2

2.81, m; 2.67, m

52.6, 72.5, 95.8, 29.1,

5.91, dt (13.2, 3) 4.63, d (7.2)

CH CH C CH3

129.3, CH 108.1, CH 121.1, CH 129.5, C 188.3, C 27.5, CH3

2.34, s 7.10, m 6.07, dd (3.6, 3.0) 7.20, dd (3.6, 1.2)

2.50, s 5.53, d (7.8)

Figure 3. Key 1H−1H COSY and HMBC correlations of 4 and 5.

1 compared to staurosporine5 occurred in the sugar moiety, with the absence of two methyl substitutions at 3′-NH2 and 4′OH, which was consistent with the deletion of the two methyltransferase genes (spcMA and spcMB) in the expression 1747



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Figure 4. CD spectra of 1−7 and staurosporine measured in methanol.

staurosporine29 (Figure 4). Therefore, 3 was identified as 9hydroxyl-3′-N-acetyl-4′-hydroxylstaurosporine, which had been previously reported.33 Compound 6 was isolated as a yellow, amorphous powder. The molecular formula of 6 was assigned as C26H23N3O5 based on the HR-ESIMS data, and it was identified as K252d by comparing the HR-ESIMS data, UV spectrum, and retention time with data we previously reported.28 Compound 7 was isolated as a yellow, amorphous powder. The molecular formula of 7 was assigned as C28H26N4O4 based on the HR-ESIMS data. In the HMBC spectrum of 7, we observed correlations from the secondary amine 12-NH (δH 12.18) to the aromatic carbons C-7b (δC 115.3), C-7c (δC 122.2), C-11a (δC 139.7), and C-12a (δC 127.8). Additionally, in the glycosyl signals, only H-1′ (δH 6.74) showed HMBC correlations with the aglycon part C-12b (δC 124.7) and C-13a (δC 139.0), revealing the connection of the sugar moiety with the aglycon at N-13. Based on the biosynthetic pathway, the origin of the sugar moiety of 7 should be the same as that of staurosporine, which was derived from an L-rhamnose transformed to an aminosugar by a dehydratase and an aminotransferase.34 Thus, the absolute configurations of 7

construct pWLI628. The CD spectrum of 1 was identical to that of staurosporine29 (Figure 4), and thus the absolute configurations of 1 were confirmed to be 1′R, 3′R, 4′R, 5′S. The structure of 1 was identified as 3′-N-demethyl-4′hydroxystaurosporine, which had been previously reported.30 The 1H and 13C NMR data for 1 are listed in Tables S4 and S5. Compound 2 was isolated as a yellow, amorphous powder. The molecular formula of 2 was assigned as C26H24N4O3 based on the HR-ESIMS data. Compound 2 was identified as holyrine A by comparing the 1H NMR data (Table S4) with data previously reported.31,32 Holyrine A is a biosynthesis intermediate of staurosporine. Compound 3 was isolated as a yellow, amorphous powder. The molecular formula of 3 was assigned as C28H24N4O5 based on the HR-ESIMS data. Compound 3 showed NMR data (Tables S4 and S5) that were similar to those of 4, except for the chemical shift changes in the aromatic signals (C-8−C-11). The downfield chemical shift value of C-9 (δC 151.0) suggested that a hydroxy group was located at C-9. The absolute configurations of 3 were confirmed to be 1′R, 3′R, 4′R, 5′S since its CD spectrum was similar to that of 1748



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sugar moiety affected the cytotoxic activity. Notably, the substitution of a 1-(1H-pyrrol-2-yl)ethanone group is proposed to positively contribute to the cytotoxic selectivity since 5 exhibited decreased toxicity on the normal cell line. These results might be related to the alteration of hydrogen bonds from the methylamino nitrogen to protein kinases caused by substitution groups at the 3′-NH2.22,23,25

should be the same as that of staurosporine. Finally, 7 was identified as 3′-N-acetylholyrine A, which had been previously reported.35 The 1H and 13C NMR data for 7 are listed in Tables S4 and S5. The inspection of structures of 1−7 revealed that they all were devoid of methyl groups at 4′-OH and 3′-NH2, which is consistent with SpcMA and SpcMB being the methyltransferases in the last tailoring steps. While the biosynthetic intermediates 1 and 2 and the shunt product 6 accumulated as expected, the generation of 3−5 and 7, with modifications at 3′-NH2, was interesting. The acetyl group in compounds 3, 4, and 7 could be transferred onto 3′-NH2 by an acyltransferase, and the formation of the heterocyclic ring in 5 was rather unexpected, which might have been achieved by the condensation with succinyl semialdehyde-CoA, yielding a Schiff base, followed by condensation with pyruvate and then cyclization and dehydration (Figure S2). As there are no appropriate candidate genes responsible for these modifications within the spc gene cluster, these modifications might be assembled by gene(s) located in the genome of S. coelicolor M1146. In the cytotoxicity evaluation of 1−7 against three human tumor cell lines (colon tumor cell line HCT-116, leukemia cell line K562, and hepatic carcinoma cell line Huh 7.5) and a normal hepatic cell line LO2, compound 1 showed strong cytotoxicity against all the tested cell lines (Table 2).

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Table 2. Cytotoxicity of 1−7 (IC50 in μM) against Tumor (HCT-116, K562, and Huh 7.5) and Normal (LO2) Cell Lines staurosporine 1 2 3 4 5 6 7

HCT-116

K562

Huh 7.5

LO2

0.03 0.2 5.1 2.7 1.0 3.9 9.0 >100

1.7 0.9 7.7 8.8 5.2 10.5 >100 32.4

0.6 0.4 7.0 >100 2.1 2.7 46.0 35.2

9.5 2.7 5.8 >100 24.2 >100 73.8 41.1

EXPERIMENTAL SECTION

General Experimental Procedures. 1D and 2D NMR spectra were recorded on Bruker Avance III 600 or Agilent DD2-500 spectrometers. Chemical shifts were reported with reference to the respective solvent peaks and residual solvent peaks (δH 2.50 and δC 39.5 ppm for DMSO-d6). HR-ESIMS data were obtained on a QTOF Ultima Global GAA076 LC-MS spectrometer. CD spectra were recorded on a JASCO J-715 spectropolarimeter, using MeOH as solvent. UV spectra were recorded on a Beckman DU 640 spectrophotometer. High-performance liquid chromatography (HPLC) was performed on an Agilent 1260 Infinity equipment with a diode array detector. Bacterial Strains and Culture Conditions. Bacterial strains and plasmids used and constructed during this study are listed in Table S1. Escherichia coli DH5α was used as the host for general subcloning.36 E. coli ET12567/pUZ800237 was used as the cosmid donor host for E. coli−Streptomyces intergeneric conjugation. E. coli BW25113/pIJ790 was used for λRED-mediated PCR targeting.38 E. coli strains were grown and manipulated following standard protocols.36−38 S. coelicolor M1146 was used as the surrogate host for heterologous expression. The strains were grown at 30 °C on MS medium for sporulation and were cultured in TSBY medium for genomic DNA preparation. Heterologous Expression of the Engineered spc Gene Clusters. Gene deletion was initially performed using the REDIRECT Technology, according to the literature protocol.38,39 The amplified aac(3)IV resistance gene from pIJ773 was transformed into E. coli BW25113/pIJ790/pWLI615 to replace an internal region of the target genes, resulting in mutant cosmids pWLI624 (ΔspcN) and pWLI625 (ΔspcMA) (Table S2). The apramycin resistant gene was then removed by digestion with XbaI. The amplified aac(3)IV gene was then transformed into E. coli BW25113/pIJ790/pWLI625(AprS) to replace an internal region of spcMB, generating pWLI626. The apramycin resistant gene was further deleted by SpeI digestion. The mutant cosmids pWLI624 and pWLI626 were then equipped with aac(3)IV-oriT-φC31-attP/int, which was derived from pSET152AB, resulting in pWLI627 and pWLI628. The cosmids were passed through E. coli ET12567/pUZ8002 and then introduced into S. coelicolor M1146 via conjugation, according to the established procedure.40 Apramycin-resistant exconjugants were selected to afford S. coelicolor M1146/pWLI627 and S. coelicolor M1146/pWLI628. Isolation and Purification. The fermentation broths (50 mL) of S. coelicolor M1146/pSET152AB, S. coelicolor M1146/pWLI627, and S. coelicolor M1146/pWLI628 were extracted with EtOAc, respectively, and were subsequently subjected to HPLC analysis. Analytical HPLC was performed with a linear gradient from 20% to 60% B/A in 30 min (phase A: H2O + 0.1% HCOOH; phase B: 100% CH3CN + 0.1% HCOOH; YMC-Pack ODS-A column 150 mm × 4.6 mm, i.d. 5 μm; wavelength: 294 nm) to analyze the production changes (Figure 2). The culture broths of S. coelicolor M1146/pWLI627 strain (15 L) and S. coelicolor M1146/pWLI628 strain (15 L) were each extracted with EtOAc at room temperature, which was partitioned between 90% MeOH and n-hexane to remove nonpolar components. Then the MeOH layer was subjected to a stepped-gradient open column (ODSA, 120 Å, S-30/50 mesh) eluting with 20−100% MeOH to yield 13 fractions. Compounds 1 (4 mg) and 4 (15 mg) were obtained by further purification of fraction 8 of the M1146/pWLI628 strain on reversed-phase HPLC (YMC-Pack ODS-A column 250 mm × 10 mm, i.d. 5 μm; wavelength: 294 nm) eluting with 45% CH3CN + 0.1% HCOOH (v/v) (1.5 mL/min). Compound 3 (0.8 mg) was obtained from fraction 7 of the M1146/pWLI628 strain eluting with 45% CH3CN + 0.1% HCOOH (v/v) (1.5 mL/min). Compound 5

Compounds 2 and 4 displayed effective cytotoxic effects on all the tested tumor cell lines. Compound 3 was effective against HCT-116 cells, with an IC50 value of 2.7 μM, and 5 was effective against HCT-116 and Huh 7.5 cells, with IC50 values of 3.9 and 2.7 μM, respectively, while they showed undetectable toxicity up to 100 μM against the normal hepatic cell line LO2. Compounds 6 and 7 showed weak cytotoxicity against all the tested cell lines. The cytotoxic selectivity of these compounds revealed the following SARs. First, the second N-glycosidic linkage (C5′−N12) is important for the cytotoxic activity, since 1 showed an approximately 8−25-fold higher activity than 2, and the same phenomenon was also observed for 4 vs 7. This is consistent with the ether oxygen of the sugar moiety forming hydrogen bonds with the surrounding residues in protein kinases,24 which would be impacted when C5′−N12 is broken. Second, the attachment of a hydroxy group on the aglycon ring may affect the cytotoxic selectivity since 3 was effective against HCT-116 cells but not against Huh 7.5 cells and the normal cell line LO2. Third, the amino group of the sugar moiety is another active group since 6 showed weak cytotoxicity compared to the others. The removal of the methyl groups at the 4′-OH and 3′-NH2 of the 1749



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(0.92 mg) was obtained from fraction 10 of the M1146/pWLI628 strain eluting with 55% CH3CN + 0.1% HCOOH (v/v) (1.5 mL/ min). Compounds 2 (20 mg), 6 (0.5 mg), and 7 (5 mg) were obtained from fraction 8 of the M1146/pWLI627 strain eluting with 45% CH3CN + 0.1% HCOOH (v/v) (1.5 mL/min). The structures of compounds 1−7 are shown in Figure 1. 3′-N-Demethyl-4′-hydroxystaurosporine (1): yellow, amorphous powder; [α]21D +17.8 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 244 (4.02), 284 (4.08), 336 (3.75), 372 (3.52) nm; CD (MeOH) λmax (Δε) 207 (−4.36), 228.5 (+1.04), 238.5 (+0.26), 249 (+1.15), 262.5 (−0.57), 297 (+2.37), 312.5 (+0.18), 333 (+0.48) nm; 1H NMR data, Table S4; 13C NMR data, Table S5; HR-ESIMS m/z 439.1804 [M + H]+ (calcd for C26H22N4O3, 439.1770). Holyrine A (2): yellow, amorphous powder; [α]21D −8.8 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 208 (3.65), 290 (3.96), 334 (2.94) nm; CD (MeOH) λmax (Δε) 209 (+2.63), 227.5 (−1.17), 260.5 (+0.66), 291 (−2.83), 362.5 (+0.74), 380 (−0.23) nm; 1H NMR data, Table S4; HR-ESIMS m/z 441.1938 [M + H]+ (calcd for C26H24N4O3, 441.1927). 9-Hydroxyl-3′-N-acetyl-4′-hydroxylstaurosporine (3): yellow, amorphous powder; [α]21D +28.2 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 224 (3.81), 294 (3.95), 340 (3.26), 380 (2.88) nm; CD (MeOH) λmax (Δε) 212 (−3.51), 236 (+1.07), 247 (+0.21), 254 (+0.88), 268 (−0.17), 300 (+2.60), 336 (+0.57) nm; 1H NMR data, Table S4; 13C NMR data, Table S5; HR-ESIMS m/z 497.1826 [M + H]+ (calcd for C28H24N4O5, 497.1825). Staurosporine M1 (4): yellow, amorphous powder; [α]21D +9.8 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 206 (4.21), 292 (4.38), 336 (3.89), 372 (3.81) nm; CD (MeOH) λmax (Δε) 212 (−6.84), 247 (+1.98), 273 (−0.75), 297.5 (+3.93), 314.5 (+0.28), 329 (+0.67) nm; 1 H and 13C NMR data, Table 1; HR-ESIMS m/z 481.1884 [M + H]+ (calcd for C28H24N4O4, 481.1876). Staurosporine M2 (5): yellow, amorphous powder; [α]21D +198.0 (c 0.025, MeOH); UV (MeOH) λmax (log ε) 244 (4.19), 292 (4.47), 336 (3.78), 372 (3.49) nm; CD (MeOH) λmax (Δε) 208.5 (−10.56), 228.5 (−2.58), 237 (−7.48), 251.5 (+2.21), 264 (−10.16), 298 (+39.59), 327 (+3.84), 332.5 (+4.05) nm; 1H and 13C NMR data, Table 1; HR-ESIMS m/z 531.2039 [M + H]+ (calcd for C32H26N4O4, 531.2032). K252d (6): yellow, amorphous powder; [α]21D −12 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 208 (4.17), 290 (4.31), 336 (3.82), 366 (3.72) nm; CD (MeOH) λmax (Δε) 208 (+5.57), 230.5 (−2.43), 260.5 (+1.60), 291.5 (−1.64), 363 (+1.87) nm; HR-ESIMS m/z 458.1704 [M + H]+ (calcd for C26H23N3O5, 458.1716). 3′-N-Acetylholyrine A (7): yellow, amorphous powder; [α]21D −40.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.40), 290 (4.58), 334 (4.13), 364 (3.99) nm; CD (MeOH) λmax (Δε) 208 (+3.98), 225 (−3.87), 259.5 (+0.89), 277.5 (−2.81), 289 (−0.51), 294.5 (−3.00), 363 (+1.28) nm; 1H NMR data, Table S4; 13C NMR data, Table S5; HR-ESIMS m/z 483.2024 [M + H]+ (calcd for C28H26N4O4, 483.2032). Cell Proliferation Viability Assay. Viabilities of HCT-116, Huh7.5, K562, and LO2 cells were measured by the MTT assay. Briefly, different cells (5 × 104 cells/mL) were seeded into a 96-well plate and cultured at 37 °C overnight. Cells were treated with different concentrations of each compound for 48 h, respectively, and then 30 μL of MTT solution (5 mg/mL) was added into each well. After incubation for 3 h, 100 μL of “Triplex Solution” (10% SDS−5% isobutanol−12 mM HCl) was added to each well for 12 h to dissolve purple crystals of formazan. Absorbance was measured at 570 nm by a multidetection microplate reader (Infinite M1000 Pro, TECAN, Mannedorf, Switzerland). Relative cell viability was presented as a percentage relative to the control group. The IC50 values were determined using triplicate samples and calculated by GraphPad Prism 5.

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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.8b00103. 1 H and 13C NMR data of compounds 1−3 and 7; additional tables and figures; HR-ESIMS and NMR spectra of compounds 3−5 and 7 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +86-532-2803-1813. ORCID

Wenli Li: 0000-0003-1598-3217 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Mervyn J. Bibb (John Innes Centre, UK) for kindly providing us S. coelicolor M1146, and thank Shuangjun Lin (Shanghai Jiaotong University) for helpful discussion. This work was supported by grants from the National Natural Science Foundation of China (41506157 and 31570032) and the NSFC-Shandong Joint Foundation (U1706206 and U1406403).

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REFERENCES

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